Salvia Divinorum Scientific Papers

AEDMP Asociación para el Estudio y la Divulgación de la Medicina Psicodélica

Salvia Divinorum Scientific Papers -2014Asociación para el Estudio y la Divulgación de la Medicina Psicodélica. Castellarnau, 11 2º 1ª 43004 Tarragona Spain Tel. 675 55 33 44 Email: [email protected] www.medicinapsicodelica.org Research conducted by: Ana Elda Genís Oña

Content _____________________________________ 1. What is Salvia Divinorum? 2. Scientific papers about Salvia arranged chronologically (1982-2014) -

A. Ortega et al., (1982). Salvinorin, a New trans-Neoclerodane Diterpene from Salvia divinorum (Labiatae)

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D. J. Siebert (1994). Salvia Divinorum and Salvinorin A: New Pharmacological Findings

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C. Giroud et al., (2000). Salvia Divinorum: An Hallucinogenic Mint Which Might Become a New Recreational Drug in Switzerland

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B. L. Roth et al., (2002). Salvinorin A: A Potent Naturally Occurring Nonnitrogenous k Opioid Selective Agonist

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D. Sheffler & B. Roth (2003). Salvinorin A: The “Magic Mint” Hallucinogen Finds a Molecular Target in the Kappa Opioid Receptor

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E. Butelman et al., (2003). The Plant-derived Hallucinogen, Salvinorin A, Produces k-opioid Agonist-like Discriminative Effects in Rhesus Monkeys

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C. Chavkin et al., (2004). Salvinorin A, an Active Component of the Hallucinogenic Sage Salvia Divinorum is a Highly Efficacious k-Opioid Receptor Agonist: Structural and Functional Considerations

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R. Bücheler et al., (2004). Use of Nonprohibited Hallucinogenic Plants: Increasing Relevance for Public Health?

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T. E. Prisinzano (2005). Psychopharmacology of the Hallucinogenic Sage Salvia Divinorum

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M. Schmidt et al., (2005). Pharmacokinetics of the Plant-derived k-Opioid Hallucinogen Salvinorin A in Nonhuman Primates

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F. Yan et al., (2005). Identification of the Molecular Mechanisms by Which the Diterpenoid Salvinorin A Binds to k-Opioid Receptors

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M. A. Ansonoff et al., (2006). Antinociceptive and Hypothermic Effects of Salvinorin A are Abolished in a Novel Strain of k-Opioid Receptor-1 Knockout Mice

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W. A. Carlezon et al., (2006). Depressive-Like Effects of the k-Opioid Receptor Agonist Salvinorin A on Behavior and Neurochemistry in Rats

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D. González et al., (2006). Pattern of Use and Subjective Effects of Salvia Divinorum Among Recreational Users

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T. A. Vortherms & B. L. Roth (2006). Salvinorin A. From Natural Product to Human Therapeutics

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D. Braida et al., (2007). Hallucinatory and Rewarding Effect of Salvinorin A in Zebrafish: k-Opioid and CB1-cannabinoid Receptor Involvement

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E. Butelman et al., (2007). Effects of Salvinorin A, a k-Opioid Hallucinogen, on a Neuroendocrine Biomarker Assay in Nonhuman Primates with High k-Receptor Homology to Humans

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O. Grundmann et al., (2007). Salvia Divinorum and Salvinorin A: An Update on Pharmacology and Analytical Methodology

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R. Rothman et al., (2007). Salvinorin A: Allosteric Interactions at the µ-Opioid Receptor

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C. B. Willmore-Fordham et al., (2007). The Hallucinogen Derived from Salvia Divinorum, Salvinorin A, has k-Opioid Agonist Discriminative Stimulus Effects in Rats

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J. Hooker et al., (2008). Pharmacokinetics of the Potent Hallucinogen, Salvinorin A in Primates Parallels the Rapid Onset and Short Duration of Effects in Humans

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K. M. Babu et al., (2008). Opioid Receptors and Legal Highs: Salvia Divinorum and Kratom

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Z. S. Teksin et al., (2009). Evaluation of the Transport, In Vitro Metabolism and Pharmacokinetics of Salvinorin A, a Potent Hallucinogen

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J. Hooker et al., (2009). Salvinorin A and Derivates: Protection from Metabolism does not Prolong Short-term, Whole-brain Residence

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M. Grilli et al., (2009). Salvinorin A Exerts Opposite Presynaptic Controls on Neurotransmitter Exocytosis From Mouse Brain Nerve Terminals

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M. W. Johnson et al., (2010). Human Psychopharmacology and Dose-effects of Salvinorin A, a Kappa Opioid Agonist Hallucinogen Present in the Plant Salvia Divinorum

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J. E. Lange et al., (2010). Salvia Divinorum: Effects and Use Among Youtube Users

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E. R. Butelman et al., (2010). The Discriminative Effects of the k-Opioid Hallucinogen Salvinorin A in Nonhumans Primates: Dissociation from Classic Hallucinogen Effects

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M. J. Baggott et al., (2010). Use Patterns and Self-reported Effects of Salvia Divinorum: An Internet-based Survey

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J. E. Mendelson et al., (2011). Lack of Effect of Sublingual Salvinorin A, a Naturally Occurring kOpioid, in Humans: A Placebo-controlled Trial

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D. Su et al., (2011). Salvinorin A Produces Cerebrovasodilation Through Activation of Nitric Oxide Synthase, κ-Receptor, and Adenosine Triphosphate–sensitive Potassium Channel

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C. W. Cunningham et al., (2011). Neuropharmacology of the Naturally Occurring k-Opioid Hallucinogen Salvinorin A

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J. Listos et al., (2011). Pharmacological Activity of Salvinorin A, the Major Component of Salvia

Divinorum -

K. M. Prevatt-Smith et al., (2011). Potential Drug Abuse Therapeutics Derived from the Hallucinogenic Natural Product Salvinorin A

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H. R. Sumnall et al., (2011). Salvia Divinorum Use and Phenomenology: Results from an Online Survey

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G. Aviello et al., (2011). Ultrapotent Effects of Salvinorin A, a Hallucinogenic Compound from

Salvia Divinorum, on LPS-stimulated Murine Macrophages and its Anti-inflammatory Action in Vivo -

C. R. Travis et al., (2012). A report of Nausea and Vomiting with Discontinuation of Chronic Use of

Salvia Divinorum -

P. Addy (2012). Acute and Post-acute Behavioral and Psychological Effects of Salvinorin A in Humans

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M. Ranganathan et al., (2012). Dose-related Behavioral, Subjective, Endocrine and Psychophysiological Effects of the Kappa Opioid Agonist Salvinorin A in Humans

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K. MacLean et al., (2012). Dose-related Effects of Salvinorin A in Humans: Dissociative, Hallucinogenic, and Memory Effects

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J. Stogner et al., (2012). Regulating a Novel Drug: An Evaluation of Changes in Use of Salvia

Divinorum in the First Year of Florida’s Ban -

E. Meyer & B. Writer (2012). Case Reports. Salvia Divinorum

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Z. Wang et al., (2012). Salvinorin A Administration after Global Cerebral Hypoxia/Ischemia Preserves Cerebrovascular Autoregulation via Kappa Opioid Receptor in Piglets

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M. Paudel et al., (2013). Development of an Enzyme Immunoassay Using a Monoclonal Antibody against the Psychoactive Diterpenoid Salvinorin A

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J. L. Díaz (2013). Salvia Divinorum: A Psychopharmacological Riddle and a Mind-body Prospect

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J. Zawilska & J. Wojcieszak (2013). Salvia Divinorum: From Mazatec Medicinal and Hallucinogenic Plant to Emerging Recreational Drug

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F. Moreira et al., (2014). Analytical Investigation of Legal High Products Containing Salvia

Divinorum Traded in Smartshops and Internet -

V. Serra et al., (2014). Behavioral and Neurochemical Assessment of Salvinorin A Abuse Potential in the Rat

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I. Casselman et al., (2014). From Local to Global-Fifty Years of Research on Salvia Divinorum

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P. Polepally et al., (2014). Michael Acceptor Approach to the Design of New Salvinorin A-based High Affinity Ligands for the Kappa-opioid Receptor

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B. Kivell et al., (2014). Salvinorin A Regulates Dopamine Transporter Function via a Kappa Opioid Receptor and ERK1/2-dependent Mechanism

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T. Vasiljevik et al., (2014). Studies Toward the Development of Antiproliferative Neoclerodanes from Salvinorin A

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K. Stiefel et al., (2014). The Claustrum’s Proposed Role in Consciousness is Supported by the Effect and Target Localization of Salvia Divinorum

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K. Sufka et al., (2014). The Effect of Salvia Divinorum and Mitragyna Speciosa Extracts, Fraction and Major Constituents on Place Aversion and Place Preference in Rats

What is Salvia Divinorum? ______________________________________ Salvia Divinorum is a perennial herb member of the mint family with psychoactive and dissociative properties. For hundreds of years, it has been used in religious and healing ceremonies by the Mazatec Indians, who live in the province of Oaxaca, in Mexico. Neuropsychological effects of Salvia include alterations of mood, behavior, and cognition. It possesses potent hallucinogenic effects in humans according to descriptive studies and self-reports. Salvinorin A, a terpene compound found in the leaves of S. divinorum, is the active ingredient and one of the most potent naturally occurring hallucinogen known to date. Some of the subjective effects of salvinorin A are distinct from other hallucinogens, possibly because it possesses a peculiar pharmacological profile, which includes potent and selective binding to the κ-opioid receptor.

Scientific papers about Salvia arranged chronologically (1982-2014)

J. CHEM. SOC. PERKIN TRANS. I

2505

1982

Salvinorin, a New trans-Neoclerodane Diterpene from Sa/via divinorurn (Labiatae) By Alfred0 Ortega." lnstituto de Quimica, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria, Mexico 20, D.F., Mexico John F. Blount and Percy S. Manchand," Chemical Research Department, Hoffmann-La Roche Inc., Nutley, New Jersey 071 10, U.S.A. Salvinorin, isolated from Salvia divinorum, has been shown by spectroscopic and X-ray-crystallographic methods to be a trans-neoclerodane diterpene of structure (1). Crystals of compound (1)are orthorhombic, space group P212121 with a = 6.368(2),b = 11.338(3),c = 30.710(6)A, and Z = 4. The structure was refined by leastsquares to R 0.052and R' 0.056.

THE essential oils produced by certain members of the widespread genus Salvia (Labiatae) are used extensively in the food and cosmetic industries. Examples are Dalmatian sage oil from S. ojicinalis (used to flavour certain foods) and Clary sage oil from S. sclarea (used in perfumery).l S . divinorum (' hojas de la Pastora ', possibly identical with ' pipilzintzintli ') is a relatively rare plant that is used by the Mazatec Indians of Mexico in their divination rites,2 but no previous chemical studies have been reported for it. However, various biand tri-cyclic diterpenes have been isolated from other Salvia species., Extraction of the leaves of S. divinorum has now yielded a novel bicyclic diterpene, salvinorin (1), C,,H,,O,, whose structural eludication forms the subject of this paper. Although the i.r. spectrum (CHCl,), of salvinorin (1) showed only one peak in the carbonyl region (vmax. 1 735 cm-l), the 13C n.m.r. spectrum (CDC1,; G/p.p.m.) revealed carbons due to four carbonyl groups : one of the ketone type (singlet at 202.04) and three of the ester type (singlets at 171.57, 171.15, and 169.94). Other salient features in the 13C n.m.r. spectrum of compound (1) included absorptions due to a @-substituted furan (singlet at 125.25 and doublets at 143.66, 139.46, and 108.41), four methyl carbons (quartets at 51.90, 20.56, 16.36, and 15.19), and two methine carbons bearing

18

i9

4\ 0 OMe

0

4

23

oxygen (doublets at 75.03 and 72.00, these are assigned to C-2 and C-12, respectively). There were also absorptions due to three methine carbons a to carbonyl groups (doublets a t 63.90, 53.47, and 51.26), four unassigned methylene carbons (triplets at 43.23, 38.08, 30.75, and 18.11), and two quaternary carbons (singlets at 42.06 and 35.41). The lH n.m.r. spectrum (CDC1,) had absorptions due to two tertiary methyl groups (singlets at 1.11 and 1.45), a methyl ester (singlet at 3.74) and the @-substituted furan (1 H-multiplet at 6.38 and 2 H-multiplet

FIGUREAn ORTEP stereoscopic drawing of salvinorin (1)

2506

J. CHEM. SOC. PERKIN TRANS. I

at 7.41). Absorption due to the acetate appeared at 2.16; that the acetate was a secondary one was evident from the presence of a one-proton triplet (& 5.14, J 10 Hz). A one-proton doublet of doublets (6H 5.51, J 12 and 6 Hz) is assigned to the 12-H. Final proof of the stereostructure of salvinorin (1)was obtained from a single-crystal X-ray analysis using direct methods4 Details of the X-ray analysis are given in the Experimental section, and listings of final atomic TABLE1 Final atomic parameters for salvinorin (1) (standard deviations in parentheses) XIQ

0.281 4(6) 0.199 O(6) 0.346 2(9) 0.760 3(9) 0.409 2(11) 0.935 8(6) 1.020 6(6) 1.023 l(8) 0.255 l(39) 0.413 3(8) 0.393 0(9) 0.389 6(10) 0.674 3(10) 0.580 O(8) 0.776 7(8) 0.830 8(9) 0.868 8(8) 0.669 2(8) 0.602 6(7) 0.727 9(8) 0.804 8(9) 0.929 0(9) 1.126 7(10) 1.177 l(12) 0.874 l(10) 0.938 9(8) 0.666 7(16) 0.379 8(9) 0.486 9(8) 0.194 6(12) -0.026 3(12) 0.767 Q(18) 0.613 0.264 0.397 0.706 0.899 0.763 0.716 0.966 0.971 0.726 0.606 0.844 0.681 1.208 1.309 0.738 0.383 0.263 0.376 0.369 0.446 0.638 -0.095 -0.013 -0.102 0.916 0.692 0.694

Y/ b 0.479 6(3) 0.642 l(3) 0.366 2(4) 0.961 l(4) 0.961 3(4) 0.406 9(3) 0.674 6(3) 0.039 6(3) 0.131 2(20) 0.542 7(6) 0.684 2(5) 0.718 6(5) 0.771 8(5) 0.728 O(4) 0.779 3(5) 0.719 9(5) 0.687 9(6) 0.627 4(4) 0.690 6(4) 0.400 8(6) 0.333 6(6) 0.227 6(6) 0.221 l(6) 0.109 3(6) 0.114 9(6) 0.626 O(6) 0.904 2(6) 0.770 3(6) 0.626 7(6) 0.425 9(7) 0.387 3(7) 1.079 3(6) 0.654 0.746 0.742 0.744 0.768 0.864 0.736 0.766 0.678 0.677 0.367 0.403 0.308 0.290 0.078 0.090 0.743 0.738 0.869 0.486 0.610 0.485 0.360 0.318 0.462 1.109 1.113 1.111

B 4c 0.369 5( 1) 0.288 2(1) 0.279 6(2) 0.326 9(1) 0.323 9(2) 0.478 2(1) 0.607 8(1) 0.430 6(1) 0.271 l(7) 14.0 (11) 0.353 l(2) 0.306 3(2) 0.304 6(2) 0.329 6(2) 0.378 6(2) 0.401 2(2) 0.444 6(2) 0.437 7(1) 0.419 6(1) 0.376 6(1) 0.409 3(2) 0.449 4(2) 0.439 O(2) 0.417 6(2) 0.414 O(2) 0.446 6(2) 0.477 6(2) 0.326 9(2) 0.402 2(2) 0.464 8(2) 0.279 O(2) 0.268 2(2) 0.324 8(2) 0.289 4.6 0.318 5.0 0.274 6.0 0.316 4.0 0.381 4.0 0.406 4.0 0.465 4.0 0.455 4.0 0.415 3.6 0.367 3.0 0.397 4.0 0.387 4.0 0.467 4.0 0.407 6.0 0.401 7.0 0.460 6.0 0.433 6.0 0.388 6.0 0.402 6.0 0.443 4.0 0.462 4.0 0.481 4.0 0.296 8.0 0.247 8.0 0.264 8.0 0.326 12.0 0.362 12.0 0.299 12.0

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1982

TABLE2 Bond lengths (A) in salvinorin (1) (standard deviations in parentheses) 1.213(6) 1.437(7) 1.348(9) 1.186(9) 1.343(11) 1.455(8) 1.195( 11) 1.468(6) 1.362(7) 1.199 (6) 1.368(8) 1.357(8) 1.518(7) 1.604(7) 1.624(8) 1.531(8) 1.682(7)

C (4)-C( 18)

c(5)-C(6)

C(5)-C ( 10) C(5)-C( 19) C(6)-C(7) C(7)-C(8) C(8)--C(9) C(8)-C (17) c(9)-c ( 10) c(9)-c ( 11) C(9)-C( 20) C( 11)-C( 12) C( 12)-C ( 13) C( 13)-C (14) C(13)-C( 16) C(14)-C(15) C(21)-C(22)

TABLE3 Bond angles (") in salvinorin (1) (standard deviations in parentheses) C(2)-0(2)-C(2 1) C(18)-0 (4)-C( 23) C(12)-O( 6)-C( 17) C (16)-0 (8)-C (16) O(1)-C( 1)-C(2)

o(l)-c(l)-c(lo) c(2)-C( 1)-c(10)

O(2)-C( 2)-C( 1) O(2)-C(2)-C( 3) c(l)-C(2)-C (3) C ( W C(3)-c (4) C(3)-c (4)-C (5) C(3)-C(4)-C(18) C( 5)-C (4)-C ( 18) c(4)-c (6)-C(6) C (4)-C (6)-C (10) C(4)-C( 6)-C (19) c (6)-C (5)-C ( 10) C(6)-C( 6)-C( 19) c( 10)-c (5)-C ( 19) C(5)-C (6)-C( 7) C (6)-C( 7)-C (8) C(7)-C (8)-C (9) C(7)-C (8)+ (17) C(9)-C (8)-C (17) C(8)-C( 9)-C( 10) C(8)-C(9)-C( 11) C(8)-C (9)-C(20) c( 10)-c (9)-c (11) c( 10)-c (9)-c (20) C(11)-c(9)-C( 20) c 1)-c( 10)-c ( 5 ) c( 1)-c(10)-c (9) c(5)-C (10)-c (9) C(9)-C( 11)-C( 12) O(6)-C( 12)-C( 11) O(6)-C( 12)-C( 13) c( 11)-C( 12)-C( 13) C(12)-C( 13)-C( 14) C(12)-C( 13)-C( 16) C( 14)-C ( 13 ) s( 16) c(13)-C( 14)-c (15) O(8)-C (16)-C (14) 0(8)-C( 16)-C ( 13) 0(6)-C ( 17)-0 (7) O(6)-C( 17)-C( 8) O(7)-C (17)-C( 8) 0(4)-C ( 18)-0 ( 5 ) 0(4)-C ( 18)-C (4) 0(5)-C(18)-C(4) O(2)-C( 2 1)-O( 3) O(2)-C( 2 1)-C(22) O(3)-C(2 1)-C( 22)

115.1(6) 115.2(7) 123.9(4) 105.5(6) 121.1(5) 124.7 (4) 114.1(4) 109.7(4) 107.8(4) 110.3(4) 111.3(5) 111.9(4) 109.3( 5 ) 112.7(4) 109.2(4) 106.1(4) 109.3(4) 108.6(4) 109.7(4) 113.8(4) 114.3(4) 109.9 (4) 112.9(4) 112.9(4) 111.3(4) 107.0(4) 104.8(4) 109.8(4) 108.9(4) 115.7(4) 110.1 (4) 107.7(4) 115.3(4) 116.6(4) 113.1(4) 112.9(4) 106.4 (4) 113.8(4) 128.2(6) 127.2(6) 104.5( 5 ) 108.0(6) 110.6(6) 111.4(5) 117.O(6) 118.4(4) 124.4 (5) 123.8(6) 111.4(7) 124.8(8) 123.1(7) 110.5(6) 126.3(7)

1.507(8) 1.541(7) 1.566(7) 1.544(7) 1.531(8) 1.622(7) 1.648(7) 1.600(7) 1.648(6) 1.534(7) 1.544(7) 1.528(7) 1.473(8) 1.417(8) 1.340(8) 1.313(9) 1.504(11)

J. CHEM. SOC. PERKIN TRANS. I

2507

1982

Parameters, bond lengths, bond angles, and torsion angles are given in Tables 1 4 . An ORTEP stereoscopic drawing of compound (l),as determined from the X-raycrystallographic analysis, is displayed in the Figure. This figure also represents the absolute stereochemistry of salvinorin, which was deduced from the negative c.d. curve (294 nm, E -5 600 in dioxan) due the keto-group at C-1 , in accord with that reported for isofructicolone.5 TABLE4 Torsion angles (") in salvinorin (1) (standard deviations in parentheses) C (10)-c ( 1)-c(2)-c (3) C(1)-C( 2)-c (3)-C(4) C( 2)-c(3)-c (4)-c (5) C(3)-c (4)-c (5)-C ( 10) c(4)-c (5)-C ( 10)-C ( 1) c(5)-C ( 10)-C( 1)-C( 2) C ( 10)-C (5)-C (6)-C (7) C(5)-C(S)-C(7)-C(S) C (6)-C (7)-C (8)-C (9) c(7)-c M)-C 19)-C ( 10) C(8)-c (9)-c ( 10)-c(5) C (9)-C ( 10)-C (5)-C (6) C( 17)-c (8)-C (9)-c ( 1 1) c (8)-c (9)-c ( 11)-c( 12) C(9)-C( 1 1)-C( 12)-O( 6) C( 1 1)-C( 12)-O( 6)-C( 17) C ( 12)-0 (6)-C ( 17)-C(8) O(6)-C( 17)-C( 8)-C( 9) C(l6)-C(13)-C(14)-C(15) c(13)-C ( 14)-C (15)-0 (8) C( 14)-C (15)- 0 (8)-C( 16) C ( 15)-0 ( 8)-C ( 16)-C (13) O(8)-C( 16)-C( 13)-C( 14) 0(6)-C ( 1 2)-C ( 1 3)-C ( 14) O(6)-C( 12)-C( 13)-C (16) C( 1 1)-C (12)-C( 13)-C( 16) C(ll)-C( 12)-C( 13)-C( 14)

-56.5(6) 5 1.3(6) - 55.9 (6) 60.1 (5) -- 61.1(5) 62.3(5) - 50.0(5) 56.1(6) - 59.7(5) 57.0(5) - 53.2(5) 50.2(5) -59.3(5) 61.1(5) - 41.2(6) 18.9(6) - 19.3(7) 40.5(6) - 1.7(7) 1.9(8) - 1.3(7) 0.1(6) 1.0(6) - 60.8(6) 123.3(5) --111.8(6) 64.1 (7)

Salvinorin (1) thus belongs to the neoclerodane class of diterpenes, a group of compounds that has attracted considerable interest because of problems associated with their stereochemistry and because of the diverse biological activities shown by some members (e.g. insect antifeedant, antitumour, and antifungal properties) .' Except for differences in the substituents and the stereochemistry at C-8 and C-12, salvinorin (1) is structurally similar to salviarin (2) and splendidin (3),*compounds which were recently isolated from S. splendens by Hanson and his collaborators. EXPERIMENTAL

The map. was determined in a capillary tube. 1.r. and n.m.r. spectra were determined in chloroform and deuteriochloroform, respectively. The lH and 13C n.m.r. spectra were determined a t 200 and 50.8 MHz, respectively. Chemical shifts are expressed in p.p.m. downfield from tetramethylsilane as internal reference, with coupling constants ( J ) in Hz. The mass spectrum was recorded a t 70 eV; m/z values are given with relative intensities (yo) in parentheses. Thin-layer chromatography (t.1.c.) was

*

performed on silica (PF254, Merck) plates and spots were made visible by spraying with 10% phosphomolybdic acid in propan-2-01, followed by heating. Column chromatography was carried out using ' Tonsil ' as adsorbent. Isolation of Salvinorin (1).-Dried, milled leaves (200 g) of Salvia divinorurn, collected a t Huautla, Oaxaca (Mexico) in November 1980, were extracted with boiling chloroform. Evaporation of the solvent gave a green residue (27 g) which was purified by chromatography on ' Tonsil ' (200 g) with chloroform as eluant. Thirteen fractions of 50.0 ml were collected, the sixth and seventh of which contained compound (1) as ascertained by t.1.c. (45% ethyl acetate in hexane as developer; R , 0.7). Crystallization from methanol yielded salvinorin (1) as colourless crystals, m.p. 238240°C; [ol]D25-41" (c, 1 inCHC1,); vmax. 1735 cm-l; 8~ 1.11 (3 H, s, Me), 1.45 (3 H, s , Me), 2.1 6(3 H, s , COMe), 3.74 (3 H, s,CO,Me), 5.14 (1 H, t, J 10, 2-H), 5.51 (1 H, dd, J 12 and 6, 12-H), 6.38 (1 H, m, 14-H), and 7.41 (2 H , m, 15- and 16-H); 80 15.19 (9, C-19), 16.36 (9, C-ZO), 18.11 (t, CH,), 20.56 (q, C-22), 30.75 (t, CH,), 35.41 (s, C-9), 38.08 (t, C-11), 42.06 (s, C-5), 43.23 (t, CHJ, 51.26 (d, C-8), 51.90 (9,C-23), 53.47 (d, C-4), 63.90 (d, C-lo), 72.00 (d, C-12), 75.03 (d, C-2), 108.41 (d, C-14), 125.25 (s, C-13), 139.46 (d, C-16), 143.66 (d, C-15), 169.94 (s, C-21), 171.15 (s, C-18), 171.57 ( s , C-17), and 202.04 p.p.m. ( s , C-1) (assignments are ' tentative and are based on chemical shifts and off-resonance decoupled spectra); m/z 432 (M+, 20), 404 (15), 359 ( 5 ) , 318 (20), 273 (30), and 94 (100) (Found: C, 63.5; H, 6.3. C2,H,,08 requires C, 63.88; H, 6.53%). X - R a y Crystallographic Analysis of Salvinorin (1).C2,Hz80e, M = 432.47. Orthorhombic, space group P2,2,Z1, a = 6.368(2), b = 11.338(3), G = 30.710(6) A ; 2 = 4; D, = 1.295 g cm-,; ~(CU-K,)= 8.3 cm-l. The intensity data, uncorrected for absorption, were measured on a fully automated Hilger-Watts diffractometer (Nifiltered Cu-K, radiation ; 8-20 scans ; pulse-height discrimination) using a crystal of dimensions ca. 0.08 x 0.20 x 0.6 mm grown from methanol. Of 1763 independent reflections for 8 < 57", 1 518 were considered to be observed [ I > 2 . 5 0 ( 1 ) ] . The structure and relative stereochemistry of compound (1) were solved by a multiplesolution procedure and refined by full-matrix least-squares. I n the final refinement the non-hydrogen atoms were refined anisotropically, except for the oxygen atom of a molecule of water, which was refined isotropically. The occupancy factor of the oxygen atom of the water molecule was included in the refinement and was found to be 0 . 3 2 ( 1 ) . The hydrogen atoms were included in the structure-factor calculations but their parameters were not refined. The final discrepancy indices were R 0.052, R' 0.056 for the 1 518 observed reflections. The final difference map had no peaks Listings of final atomic parameters, greater than 0.2 e Hi-,. bond lengths, bond angles, and torsion angles are given in Tables 1-4. Observed and calculated structure factors and atomic thermal parameters are given in Supplementary Publication No. S U P 23371 (8 pp.).? We thank Mr. Louis Todaro and Ms. Ann-Marie Chiu for their assistance with the X-ray-crystallographic work. [2/375 Received, 3rd March, 19821

' Tonsil ' is a cominercially available bentonitic earth with

the following composition: 50, (72.5y0),A1,0, (13y0), Fe,O, ( 5 % ) , MgO (1.5%), CaO (7.2%), and H,O (8.5%). and has pH 3. t For details see Notice to Authors No. 7 in J . Chem. Soc., Perkzn Trans. 1, 1981, Index issue.

REFERENCES 1

A. F. Halim and R . P. Collins, J . Agyic. Food Chenz., 1975,

23, 506; W. H. Lewis and M. P. F. Elvin-Lewis, 'Medical Botany,' Wiley, New York, 1977, p. 338.

2508

J. CHEM. SOC. PERKIN TRANS. I

J: M. Watt in ' Plants in the Development of Modern Medicine, ed. T. Swain, Harvard University Press, 1972, p. 67. G . Savona, M. P. Paternostro, F. Piozzi, J . R . Hanson, P. B. Hitchcock, and S. A . Thomas, J . Chem. Soc., Perkin Trans. 1 , 1978, 643, and references cited. G. Germain, P. Main, and M. M. Woolfson, Acta Crystallo,gr., Sect. A , 1971, 27, 368. M Martinez-Ripoll, J. Fayos, B. Rodriguez, M. Paternostro, and J . R . Hanson, J . Chem. Soc., Perkin Trans. 1, 1981, 1186. D. Rogers, G. G. Unal, D. J . Williams, S. V. Ley, G. A . Sim,

0 Copyright

1982

B. S. Joshi, and K . R. Ravindranath, J . Chem. Soc., Chem. Commun., 1979, 97; G. Trivedi, H. Komura, I. Kubo, K . Nakanishi, and B. S. Joshi, ibid., 1979, 8 8 5 ; I . Kubo, M. Kido, and Y. Fukuyama, ibid., 1980, 897; F. Piozzi, Heterocycles, 1981, 15, 1489. For reviews see J. R. Hanson in ' Terpenoids and Steroids,' Specialist Periodical Reports, The Chemical Societv, London, 1981, vol. 10 and preceding volumes. * G. Savona, M. P. Paternostro, F. Piozzi, and J . R. Hansoii, J . Chem. Soc., Perkin Trany. 1, 1979, 533.

1982 by The Royal Society of Chemistry

~

Journal of

ETHNOPHARMACOLOGY E LSEVI E R

Journal of Ethnopharmacology43 (1994) 53-56

Salvia divinorum and Salvinorin A: new pharmacologic findings Daniel J. Siebert P.O. Box 661552, Los Angeles, CA 90066, USA

Received 18 August 1993; revision received 28 December 1993: accepted 24 March 1994

Abstract

The diterpene salvinorin A from Salvia divinorum (Epling and Jativa-M), in doses of 200-500 #g produces effects which are subjectively identical to those experienced when the whole herb is ingested. Salvinorin A is effectively deactivated by the gastrointestinal system, so alternative routes of absorption must be used to maintain its activity. Traditionally the herb is consumed either by chewing the fresh leaves or by drinking the juices of freshly crushed leaves. The effects of the herb when consumed this way depend on absorption of salvinorin A through the oral mucosa before the herb is swallowed. Keywords: Salvia divinorum; Salvinorin A; Psychoactive plants; Psychoactive compounds

1. Introduction

Salvia divinorum is used by the Mazatec Indians of northeastern Oaxaca, Mexico primarily for its psychoactive effects which aid in ritual divination (Wasson, 1962, 1963). It is also employed remedially to treat various health conditions (Valdes et al., 1983). The first live specimens of S. divinorum were given to Carl Epling by R. Gordon Wasson in 1962 and were cultivated at the University of California in Los Angeles (Wasson, 1962). Cuttings of this original clone were distributed to other botanical collections over the years and most of the plants in cultivation in the USA today originated from this original clone (Valdes et al., * Corresponding author.

1987). Recently, other clones have been appearing in collections. As of this writing there are at least four different clones present in public and private botanical collections in the USA. The chemistry of this plant has been investigated several times. The diterpenes salvinorin A and salvinorin B have been identified and characterized. Salvinorin A has been shown to be active in mice while salvinorin B was inactive. No human studies with these compounds have previously been reported (Ortega et al., 1982, Valdes et al., 1984). Trace amounts of other diterpenes have been detected but have not yet been characterized (Valdes et al., 1984). There are two methods of ingestion traditionally employed: either the fresh whole leaves are masticated and swallowed or, alternatively, the leaves are crushed to extract the juices which are

0378-8741/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD! 0378-8741(94)01133-K

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D.J. Siebert /J. Ethnopharmacol. 43 (1994) 53-56

then drunk. Of these two methods, chewing of the leaves is most reliable and requires a smaller quantity of leaves. The liquid preparation is often ineffective and when it does produce effects they are usually much milder than those reported for chewing, even when substantially larger quantities of leaves are used in the preparation. When the leaves are chewed whole they must first be chewed well enough to be easily swallowed and so spend quite some time in contact with the oral mucosa. When the leaf juice preparation is consumed it can be swallowed fairly quickly and consequently spends relatively little time in contact with the oral mucosa. The level of effects reported relates quite closely to the length of time the material spends in the mouth before being swallowed. This presentation describes the effects of salvinorin A in humans, its deactivation by the gastrointestinal system and the essential role of the oral mucosa as an absorption site for salvinorin A from orally ingested leaves. 2. Materials and methods

All plant material used in this study was propagated from the clone originally brought into the USA by R. Gordon Wasson in 1962.

2.1. Salvia divinorum leaves In order to investigate the relative importance of the oral mucosa as an absorption site for the active principals in S. divinorum leaves, the following experiments were carried out by six volunteers using ten large fresh leaves each (approximately 30 g total) which had been homogenized with 100 ml water using a blender. Each experiment was separated by several days. (A) The material was swallowed as quickly as possible with the intention of quickly bypassing the oral mucosa; then the mouth was immediately rinsed with water to wash away any residual material that might be clinging to the oral mucosa. None of the volunteers reported any noticeable effects when the material was ingested in this manner. (B) The material was held in the mouth for 10 min without swallowing; then the entire contents were spit out. This method proved consistently el'-

fective with all of the volunteers reporting very definite psychoactive effects.

2.2. Saivinorin A Salvinorin A was isolated following the method of Valdes (Valdes et al., 1984). The identity of this material was verified by comparison with an authentic sample of salvinorin A using TLC, melting point and NMR. Salvinorin A has previously been shown to be active in mice but it has remained uncertain whether this compound is responsible for the psychoactive effects produced in humans. In order to determine this, salvinorin A was administered to a group of 20 volunteers. When salvinorin A was encapsulated and swallowed in doses as high as 10 mg there was no detectable activity. Experiments with the leaves indicate that the active principle of the plant is deactivated by the gastrointestinal system. To test for activity of salvinorin A, alternative routes of ingestion were attempted. Salvinorin A is not water soluble so injection was not attempted. Absorption through the oral mucosa. A 2-mg quantity of salvinorin A was dissolved in 1 ml anhydrous ethyl alcohol then sprayed on the inner surfaces of the mouth using an aspirator. The material proved to be active; however only a small percentage is absorbed this way before it gets dispersed by salivary flow. Consequently this method was inefficient and results were inconsistent. Inhalation of the vaporized compound. The material was placed on a piece of aluminum foil. A butane micro torch was then held beneath the foil until the material was seen to vaporize. As soon as this began, the vapors were inhaled through a 15mm glass tube. Inhalation of the vapors produced by heating salvinorin A proved to be the most efficient method of ingestion tested. When 200-500 /~g of salvinorin A is vaporized and inhaled the subjective effects produced are identical to those typically produced by the fresh herb. Doses up to 2.6 mg were tested in this manner. Typically threshold effects are noted at about 200/~g. 2.3. Effects When salvinorin A is absorbed through the oral

D.J. Siebert / J. Ethnopharmacol. 43 (1994) 53-56

55

mucosa the first effects are usually experienced in 5-10 min. The strength of the effects builds very quickly over a few minutes, maintaining a plateau for about 1 h. The effects gradually subside over another 1-h period. The evolution of effects over time is identical to that of orally ingested S. divinorum leaves. When salvinorin A is vaporized and inhaled the full effects are experienced in about 30 s. There is almost no transition period experienced. The strongest effects last 5-10 min and then gradually subside over about 20-30 min. As dosage increases above 1 mg the duration of the effects are somewhat increased. A similar evolution of effects is reported for smoked S. divinorum leaves. The oral mucosa apparently acts as a time release buffer, slowly diffusing salvinorin A into the blood stream; hence when consumed orally, the effects begin more gradually, last longer and subside over a longer period of time than when the material is vaporized and inhaled. Although variable in duration, the effects experienced have the same overall characteristics regardless of the route of absorption used. The nature of the effects experienced depends on many factors including dose, set and setting. Frequently people report having seen visions of people, objects, and places. With doses above 1 mg, out of body experiences are frequent. Occasionally individuals get up and move about with no apparent awareness of their movements or behavior. Some individuals speak gibberish during the most intense phase of the experience, others laugh hysterically. Certain themes are common to many of the visions and sensations described. The following is a listing of some of the more common themes:

(7)

(1)

When S. divinorum leaves are consumed, either by chewing the fresh leaves or by retaining the leaf juices in the mouth, enough of the highly active compound salvinorin A is absorbed through the oral mucosa and into the blood stream to produce a psychoactive effect. Swallowing of the herb is unnecessary and its effects are increased by lengthening the amount of time that the herb remains in the mouth. When the leaf juices are quickly swallowed, minimizing contact with the oral mucosa, the only route of absorption is

(2) (3) (4) (5) (6)

Becoming objects (yellow plaid French fries, fresh paint, a drawer, a pant leg, a Ferris wheel, etc.). Visions of various two dimensional surfaces, films and membranes. Revisiting places from the past, especially childhood. Loss of the body and/or identity. Various sensations of motion, or being pulled or twisted by forces of some kind. Uncontrollable hysterical laughter.

Overlapping realities. The perception that one is in several locations at once.

Some of the effects appear to parallel those of other hallucinogens (i.e. the depersonalization experienced with ketamine, the rapid onset of effects and short duration of smoked DMT). The volunteers who were experienced with other hallucinogens all agreed that despite some similarities, the content of the visions and the overall character of the experience is quite unique. 2.4. Receptor Site Screening and MAO Inhibition A sample of salvinorin A was submitted to NovaScreen TM for receptor site screening. At screening concentrations of 10-5 M there was no significant inhibition (i.e. 50% or less) for the following sites. Neurotransmitters: Adenosine, alpha 1, alpha 2, beta, dopamine 1, dopamine 2, GABAA, GABAB, serotonin 1, serotonin 2, muscarinic 3, NMDA, kainate, quisqualate, glycine (stry sens.). Regulatory sites: Benzodiazepine(centrl), glycine (stry insens.), PCP, MK-801. Brain~gut peptides: angiotensin Ty2, argvasopressin V1, bombesin, CCK central, CCK peripheral, substance P, substance K, NPY, neurotensin, somatostatin, VIP. Growth factors and peptides: ANF1, EGF, NGF. Ion channels: Calcium (type N), calcium (type T and L), chloride, potassium (low conduct). Second messengers: Forskolin, phorbol ester, inositol triphosphate. Monoamine oxidase inhibition: Monoamine oxidase A, monoamine oxidase B. 3. Discussion and conclusions

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D.J. Siebert /J. Ethnopharmacol. 43 (1994) 53-56

through the gastrointestinal system where salvinorin A is deactivated before entering the blood stream. When pure salvinorin A is encapsulated and swallowed it is inactive even at relatively large doses, but when absorbed through the oral mucosa or vaporized and inhaled is extremely active. It is likely that if salvinorin A were administered by injection, it would prove to be active at even lower doses than those described in this paper. Salvinorin A is the first entheogenic diterpene reported and is active in humans at extraordinarily low doses. It does not appear to affect any of the receptor sites affected by other hallucinogens. Further research into the methods of action and possible medicinal values of this and similar compounds may prove to be quite rewarding.

Acknowledgments I am grateful to Dr Leander Valdes III for supplying a reference sample of salvinorin A, and Dr David Nichols for his role in the receptor site screening of salvinorin A through his NIMHfunded research program.

References Ortega, A., Blount, J.F. and Manchand, P.S. (1982) Salvinorin, a new trans-neoclerodane diterpene from Salvia divinorum (Labiatae). Journal of the Chemical Society, Perkin Transactions I: Organic and Bio-Organic Chemistry 2505-2508. Valdes, L.J. IlI., Diaz J.L. and Ara G. Paul. (1983)Ethnopharmacology of Ska Maria Pastora (Salvia divinorum Epling and Jativa-M.). Journal of Ethnopharmacology 7, 287-312. Valdes, L. J. Ill., Butler, W.M., Haffield, G.M., Paul, A.G. and Koreeda, M. (1984) Divinorin A, a psychotropic terpenoid, and divinorin B from the hallucinogenic mint Salvia divinorum. Journal of Organic Chemistry 49, 4716-4720. Valdes, L.J. lIl., Hatfield, G.M., Paul, A.G.and Kol~eda M. (1987) Studies of Salvia divinorum (Lamiacea¢), an hallucinogenic mint from the Sierra Mazateca in Oaxaca, Central Mexico. Economic Botany 4|(2), 283-291. Wasson, R.G. (1962) A new Mexican psychotropic drug from the mint family. Botanical Museum Leaflets, Harvard University 20, 77-84. Wasson, R.G. 0963) Notes on the present status of ololuiqui and the other hallucinogens of Mexico. Botanical Museum Leaflets, Harvard University 20, 161-193.

Forensic Science International 112 (2000) 143–150 www.elsevier.com / locate / forsciint

Salvia divinorum: an hallucinogenic mint which might become a new recreational drug in Switzerland a, b,d a,c a C. Giroud *, F. Felber , M. Augsburger , B. Horisberger , L. Rivier a , P. Mangin a a

´ ´ Legale , rue du Bugnon 21, Laboratoire de Toxicologie Analytique, Institut Universitaire de Medecine 1005 Lausanne, Switzerland b ˆ , Switzerland Institut de Botanique de l’ Universite´ , Neuchatel c Center for Human Toxicology, University of Utah, Salt Lake City, UT, USA d ˆ , Switzerland Jardin Botanique de l’ Universite´ et de la Ville, Neuchatel Received 30 December 1999; accepted 24 March 2000

Abstract Salvia divinorum Epling & Jativa is an hallucinogenic mint traditionally used for curing and divination by the Mazatec Indians of Oaxaca, Mexico. Young people from Mexican cities were reported to smoke dried leaves of S. divinorum as a marijuana substitute. Recently, two S. divinorum specimens were seized in a large-scale illicit in-door and out-door hemp plantation. Salvinorin A also called divinorin A, a trans-neoclerodane diterpene, was identified in several organic solvent extracts by gas chromatography–mass spectrometry. The botanical identity of the plant was confirmed by comparing it to an authentic herbarium specimen. More plants were then discovered in Swiss horticulturists greenhouses. All these data taken together suggest that many attempts exist in Switzerland to use S. divinorum as a recreational drug. This phenomenon may be enhanced because neither the magic mint, nor its active compound are banned substances listed in the Swiss narcotic law.  2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hallucinogenic plants; Salvia divinorum; Salvinorin A; GC–MS

1. Introduction During a recent fire of a private home, two unknown coleus-like shrubs at the *Corresponding author. Tel.: 141-21-314-7086; fax: 141-21-314-7090. E-mail address: [email protected] (C. Giroud). 0379-0738 / 00 / $ – see front matter  2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 00 )00180-8

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C. Giroud et al. / Forensic Science International 112 (2000) 143 – 150

vegetative stage were found together with an illicit, indoor, large-scale hemp cultivation. A plant sample was collected and subjected to botanical and chemical investigations.

2. Botany The unknown plants were about 0.5 m in height. The leaves were about 15 cm long, ovate, dentate and acuminate, opposite and decussated. The fleshy stems were quadrangular with characteristic flanged angles. Their morphology was typical of plants belonging to the Lamiaceae family.

3. Toxicological analyses All chemicals and solvents used were analytical reagent grade.

3.1. Equipment and chromatographic conditions For screening investigations, a Hewlett-Packard (HP) Series 5890 series II plus gas chromatograph was used in combination with a HP MSD Series 5971 mass spectrometer, a HP 7673 Series injector and a HP Vectra XM Series 4 workstation (GC–MS). The GC conditions were as follows: splitless injection mode (purge time: 1 min); HP Ultra-2, 5% phenyl-methyl-silicone capillary column (25 m 3 0.2 mm I.D., 0.33 mm film thickness); column temperature, programmed from 708C (initial time: 3 min) to 1908C (rate: 208C / min) and then to 3058C (rate: 108C / min, final time: 11.0 min); carrier gas: helium, constant flow-rate: 1.2 ml / min; injection port temperature: 2608C. The MS conditions were the following: scan mode (40–400 a.m.u., threshold: 50, 1.3 scan / s from 3 to 7 min and 40 to 550 a.m.u., threshold 50, 0.9 scans / s from 7 to 31.5 min); ionization energy, 70 eV; MS interface and MS temperatures 280 and 1808C, respectively; EM offset: 200 V above the tune value. Data were automatically processed with macro-programs which comprise peak search and tentative identification with the Pfleger, Aafsdrug, Nbs75k, Wiley138 and our own spectra libraries.

3.2. Extractions 3.2.1. Fresh material About 100 mg of fresh leaves were ground with a mortar and a pestle in the presence of 2 ml saturated ammonia buffer (pH 9.5). After addition of 2 ml of chloroform / isopropanol (9:1, v / v), the mixture was shaken for 30 min. Phase separation was achieved by centrifugation. The organic phase was collected and evaporated under N 2 at 408C. The dried residue was acetylated with 100 ml acetic anhydride / pyridine (3:2, v / v) for 30 min at 608C. The solvent was taken to dryness and the plant extract dissolved into 100 ml ethyl acetate. One ml was subjected to gas chromatography–mass spectrometry analysis (GC–MS).

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3.2.2. Dried material The plant material was air-dried under pressure for several days. Then, 100 mg of dried leaves were crushed, mixed with 3 ml methanol and sonicated for 0.5 min. The powder was then extracted for 30 min on a horizontal shaker. After paper-filtration (Schleicher & Schuell 595 round filters) on a Buchner funnel, rinsing with 2 ml of fresh methanol, the extract was taken to dryness with N 2 at 408C and dissolved in 100 ml acetonitrile. One ml was subjected to GC–MS.

4. Results Beside sugars, fatty acids, vitamins and plant sterols, one mass spectrum from the total ion chromatogram of the acetylated basic extract of the fresh plant was tentatively identified as divinorin with the Wiley database. This compound turned out to be a furanolactone neoclerodane diterpene already known as the main active drug of Salvia divinorum, a plant belonging to the Lamiaceae family. Its chemical structure is shown in Fig. 1. Divinorin [1] is also called divinorin A, its chemical structure is identical to salvinorin A, a molecule which was previously described by Ortega et al. [2]. Divinorin B or salvinorin B refer to their desacetyl analog [1]. Neophytadiene, alcanes, tocopherol, stigmasterol, fatty acids and salvinorin A were detected in the methanolic extract of the dried plant. The total ion chromatogram and the mass spectrum of salvinorin A are shown in Figs. 2 and 3, respectively. Salvinorin A occurred as a major peak at the end of the chromatogram (retention time: 23.5 min). The base peak of its mass spectrum was m /z594 and the putative molecular ion m /z5432 corresponding to the raw structure C 23 O 8 H 28 . Other significant ions were m /z555, 107, 121, 166, 220, 273, 318, 359 and 404. This mass spectrum is similar to those reported for salvinorin A isolated from the pharmacologically active fractions of purified S. divinorum extracts or by chromatography of a hot chloroform extract [1,2]. Salvinorin A was also detected by GC–MS in the methanolic extract of a plant cultivated by a horticulturist as S. divinorum (results not shown). The detection of salvinorin A (which is acetylated on its diterpenic nucleus) in both the acetylated basic extract and the methanolic extract indicate that the seized plant

Fig. 1. Chemical structure of salvinorin A (5divinorin A).

146 C. Giroud et al. / Forensic Science International 112 (2000) 143 – 150

Fig. 2. Total ion chromatogram of a methanolic extract of a Lamiaceae identified as Salvia divinorum Epling & Jativa.

C. Giroud et al. / Forensic Science International 112 (2000) 143 – 150

Fig. 3. The 70-eV mass spectrum of salvinorin A or divinorin A.

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C. Giroud et al. / Forensic Science International 112 (2000) 143 – 150

should contain significant amounts of the psychoactive acetylated salvinorin A molecule. A mass spectrum which could correspond to its desacetyl analog (salvinorin B) was detected in the methanolic extract only. Its putative molecular ion was m /z5390 and the majors ions occurred at m /z543, 94 and 291. Due to the lack of a commercially available authentic standard of salvinorin A, no quantification could be performed and the pharmacological activity of the seized plant could not be assessed. Because salvinorin A has thus far been attributed uniquely to S. divinorum and that, as far as we know, no other natural source for this compound has been identified up to now nor synthetically produced, it was assumed that the unknown plant was S. divinorum.

4.1. Botanical identification The dried plant was then compared and found to be identical to an authentic specimen of S. divinorum (G 340168) stored at the herbarium of the Conservatoire et Jardin ` Botaniques de la Ville de Geneve, Switzerland. In particular, the specific characteristics of the stem and the typical morphology of the leaves confirmed that the specimens belonged to that species.

5. Discussion Salvia divinorum is employed as a shamanic inebriant by the Mazatec Indians of the Mexican state of Oaxaca [3]. This plant is known from the Indians as the ‘leaves of Mary, the Shepherdess’. They believe it allows them to travel to heaven and talk to God and the Saints about divination, diagnosis and healing [4]. Interesting enough, even though this species was known to the Mazatec Indians for several centuries, it was described only recently, in 1962 [3]. Both human and animal testing of salvinorin A indicated it had psychoactivity and potency similar to that of mescaline [4]. Vaporizing and inhaling 200–500 mg of pure salvinorin A induces profound hallucinations. Levels in leaves were found to range from 0.89 to 3.70 mg / g dry weight [5], a concentration which is very likely sufficient enough to induce psychoactive effects. In order to assess salvinorin A levels in plant organs, a reference standard was purified from S. divinorum leaf extracts and authenticated by NMR. Salvinorin A was quantified in plant tissues by reversed-phase high-performance liquid chromatography [5]. The leaves of S. divinorum are prepared in various manners for use as a psychotropic agent. The dried leaves may be smoked like marijuana joints. Traditionally, the fresh whole leaves are masticated and swallowed or, alternatively, the leaves are crushed to extract the juices which are then drunk [6]. The oral mucosa seems to play an essential role as an absorption site for salvinorin A from orally ingested leaves. The leaves may be crushed in water to prepare an infusion. Taken in small doses (4–5 pairs of fresh or dried leaves), the plant acts as a tonic or panacea as well as for magical healing. When prepared with large doses (20–60 pairs of fresh leaves), the infusion acts as a mild but

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effective hallucinogen [7]. It was reported that ingestion of the infusion resulted in an astounding visual, oral / aural, and tactile hallucination [8]. Until recently, ignorance of drug addicts in the existence of this mint, its bitter taste, and a misunderstanding of its psychotropic effects have kept it from becoming a recreational drug. Nevertheless, it was reported that young people from Mexican cities travel to the Sierra Mazateca and purchase dried leaves of S. divinorum to make into cigarettes and smoke as a marijuana substitute. The effect is reportedly milder than that of Cannabis [5]. Since discovering this first specimen of Salvia divinorum, other plants have been found in the greenhouses of several horticulturists suggesting that interest for this mint is growing in Switzerland. Here it is important to point out that neither S. divinorum nor its active compound salvinorin A are listed in the Swiss narcotic law. In California and other parts of the US, it is reported to be employed as a legal hallucinogen [4]. Many web sites are dedicated to S. divinorum. For instance, data concerning the botany, ethnobotany, biochemistry and pharmacology of S. divinorum can be found on http: / / salvia.lycaeum.org / ott.html. Another relevant web site is http: / / sabia.com / salvia /.

6. Conclusions In conclusion, forensic toxicologists are facing a growing amount of new psychotropic drugs, some of them are difficult to detect and quantify because no reference standard can be purchased and only scarce data are available about their metabolism, pharmacokinetic and toxicology. In this regard, Salvia divinorum and salvinorin A are good examples.

Acknowledgements Laurence Mauron is thanked for her technical assistance.

References ` W.M. Butler, G.M. Hatfield, A.G. Paul, M. Koreeda, Divinorin A, a psychotropic terpenoid, [1] L.J. Valdes, and divinorin B from the hallucinogenic Mexican mint Salvia divinorum, J. Org. Chem. 49 (1984) 4716–4720. [2] A. Ortega, J.F. Blount, P.S. Manchand, Salvinorin, a new trans-neoclerodane diterpene from Salvia divinorum (Labiatae), J. Chem. Soc., Perkins Trans. I (1982) 2505–2508. ´ [3] C. Epling, C.D. Jativa-M, A new species of Salvia from Mexico, Botanical Museum Leaflets, Harvard University 20 (1962) 75–84. ´ Salvia divinorum and the unique diterpene hallucinogen, salvinorin (divinorin) A, J. [4] L.J. Valdes, Psychoactive Drugs 26 (1994) 277–283.

150

C. Giroud et al. / Forensic Science International 112 (2000) 143 – 150

[5] J.W. Gruber, D.J. Siebert, A.H. der Marderosian, R.S. Hock, High-performance liquid chromatographic quantification of salvinorin A from tissues of Salvia divinorum Epling & Jativa, Phytochem. Anal. 10 (1999) 22–25. [6] D.J. Siebert, Salvia divinorum and salvinorin A: new pharmacologic findings, J. Ethnopharmacol. 43 (1994) 53–56. ` G.M. Hatfield, M. Koreeda, A.G. Paul, Studies of Salvia divinorum (Lamiaceae), an [7] L.J. Valdes, hallucinogenic mint from the Sierra Mazateca in Oaxaca, Central Mexico, Econ. Bot. 41 (1987) 283–291. ` J.L. Diaz, A.G. Paul, Ethnopharmacology of Ska Maria Pastora ( Salvia divinorum Epling [8] L.J. Valdes, ´ and Jativa-M), J. Ethnopharmacol. 7 (1983) 287–312.

Salvinorin A: A potent naturally occurring nonnitrogenous ␬ opioid selective agonist Bryan L. Roth*†‡§¶, Karen Baner*, Richard Westkaemper㥋, Daniel Siebert**, Kenner C. Rice††, SeAnna Steinberg*, Paul Ernsberger*‡‡, and Richard B. Rothman§§ *National Institute of Mental Health Psychoactive Drug Screening Program, and Departments of †Biochemistry, ‡Psychiatry, §Neurosciences, and ‡‡Pharmacology and Nutrition, Case Western Reserve University Medical School, Cleveland, OH 44106; §§Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224; 㛳Department of Medicinal Chemistry, Medical College of Virginia, Richmond, VA 23298; **The Salvia divinorum Research and Information Center, Malibu, CA 90263; and ††Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Edited by Erminio Costa, University of Illinois, Chicago, IL, and approved July 9, 2002 (received for review April 18, 2002)

Salvia divinorum, whose main active ingredient is the neoclerodane diterpene Salvinorin A, is a hallucinogenic plant in the mint family that has been used in traditional spiritual practices for its psychoactive properties by the Mazatecs of Oaxaca, Mexico. More recently, S. divinorum extracts and Salvinorin A have become more widely used in the U.S. as legal hallucinogens. We discovered that Salvinorin A potently and selectively inhibited 3H-bremazocine binding to cloned ␬ opioid receptors. Salvinorin A had no significant activity against a battery of 50 receptors, transporters, and ion channels and showed a distinctive profile compared with the prototypic hallucinogen lysergic acid diethylamide. Functional studies demonstrated that Salvinorin A is a potent ␬ opioid agonist at cloned ␬ opioid receptors expressed in human embryonic kidney-293 cells and at native ␬ opioid receptors expressed in guinea pig brain. Importantly, Salvinorin A had no actions at the 5-HT2A serotonin receptor, the principal molecular target responsible for the actions of classical hallucinogens. Salvinorin A thus represents, to our knowledge, the first naturally occurring nonnitrogenous opioid-receptor subtype-selective agonist. Because Salvinorin A is a psychotomimetic selective for ␬ opioid receptors, ␬ opioid-selective antagonists may represent novel psychotherapeutic compounds for diseases manifested by perceptual distortions (e.g., schizophrenia, dementia, and bipolar disorders). Additionally, these results suggest that ␬ opioid receptors play a prominent role in the modulation of human perception.

alvia divinorum, a member of the mint family, is a psychoactive plant that has been used in traditional spiritual practices by the Mazatec people of Oaxaca, Mexico for many centuries (1). S. divinorum also grows in California and has been used as a legal hallucinogen for several years (2). Traditionally, S. divinorum is ingested as a quid or smoked for its psychoactive properties (1) and has been reported to have potent hallucinatory actions (1, 3). The main active ingredient of S. divinorum is Salvinorin A (Fig. 1), a novel neoclerodane diterpene of known absolute configuration (4) whose structure was determined by single-crystal x-ray analysis in two independent studies (5, 6). Salvinorin A is structurally distinct from the naturally occurring hallucinogens N,N-dimethyltryptamine, psilocybin, and mescaline and synthetic hallucinogens such as lysergic acid diethylamide (LSD), 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB), and ketamine. Salvinorin A has been reported to be the most potent naturally occurring hallucinogen, with an effective dose in humans in the 200- to 1,000-␮g range when smoked (1, 3). Salvinorin A thus rivals the synthetic hallucinogens LSD and DOB in potency. Salvinorin A has been reported to induce an intense hallucinatory experience in humans, with a typical duration of action being several minutes to an hour or so (1). Several prior investigations attempted unsuccessfully to identify the molecular and cellular targets responsible for the actions of Salvinorin A (1, 3) by using mainly nonhuman molecular targets. Since then, it has become widely recognized that the

S

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pharmacological properties of rodent and human molecular targets are frequently distinct (7), and that tissue-based radioligand binding assays frequently yield inaccurate estimates of drug potency and selectivity. Accordingly, we reexamined the molecular pharmacological profile of the novel diterpene Salvinorin A at a large number of cloned human G protein-coupled receptors (GPCRs), channels, and transporters. We report here that Salvinorin A is a potent and selective ␬ opioid receptor (KOR) agonist and represents, to our knowledge, the first nonalkaloid opioid subtype-selective drug. We suggest that because the KOR has long been recognized as a target for psychotomimetic agents, KOR antagonists may represent a novel class of psychotherapeutic compounds. Our results also suggest that the KOR兾dynorphin peptide system functions to modulate human perception. Materials and Methods Materials. Two sources of Salvinorin A were used for the studies

described here: Biosearch and the Salvia divinorum Research and Information Center, Malibu, CA; both samples were identical by thin-layer chromatography and mass spectroscopy and showed the expected molecular ion in the mass spectrum. In addition, the Biosearch sample showed the reported melting point (6), and the Varian 300 MHz NMR spectrum was identical with that reported. The coding region of the KOR was cloned via PCR-amplification of ‘‘Quick-Clone’’ cDNA (CLONTECH) and subcloned into the eukaryotic expression vector pIRESNEO via NotI adaptors to yield pIRESNEO-KOR. The entire insert was verified by automated double-stranded DNA sequencing (Cleveland Genomics, Cleveland). A stable human embryonic kidney-293 cell line expressing the KOR was also constructed (KOR-293) and was used for radioligand-binding and functional assays. GF-62 cells, a stable cell line expressing the 5-HT2A receptor (8), was used for functional studies of 5-HT2A receptors. All other receptors were obtained as previously described (9, 10) as part of the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) resource. Frozen guinea pig brains and rat brains were purchased from Harlan Bioproducts for Science (Indianapolis). [D-Ala-2MePhe4,Gly-ol5]enkephalin (DAMGO), D-Phe-Cys-Tyr-D-TrpArg-Thr-Pen-Thr-NH2 (CTAP), and H-Tyr-Tic-Phe-Phe-OH (TIPP) were obtained from Multiple Peptide Systems (San Diego) through arrangement with Paul Hillery of the Research Technology Branch, National Institute on Drug Abuse. SNC-80 was obtained from K.C.R. (⫺)-Nor-binaltorphine 2HCl (NorBNI) and (⫹)-U69593 were obtained from Research BiochemiThis paper was submitted directly (Track II) to the PNAS office. Abbreviations: KOR, ␬ opioid receptor; MOR, ␮ opioid receptor; DOR, ␦ opioid receptor; LSD, lysergic acid diethylamide; NIMH-PDSP, National Institute of Mental Health Psychoactive Drug Screening Program; GPCR, G protein-coupled receptor. ¶To

whom reprint requests should be addressed. E-mail: [email protected].

www.pnas.org兾cgi兾doi兾10.1073兾pnas.182234399

cals (Natick, MA). [35S]Guanosine 5⬘-(␥-thio)-triphosphate ([35S]-GTP[␥S], 45 TBq兾mmol) was purchased from DuPont兾 NEN. BSA, naloxone, GDP, and GTP[␥S] were purchased from Sigma. Radioligand-Binding Assays. Radioligand-binding assays at human cloned GPCRs, ion channels, and transporters were performed as previously detailed (9, 10) by using the resources of the NIMH-PDSP. Detailed on-line protocols are available for all assays at the NIMH-PDSP web site (http:兾兾pdsp.cwru.edu). ␬ opioid radioligand-binding assays in situ in guinea pig brain and ␮ opioid receptor (MOR)- and ␦ opioid receptor (DOR)-binding assays in rat brain were performed as previously detailed (11). Initial screening assays were performed by using 10 ␮M Salvinorin A or 10 ␮M LSD by using quadruplicate determinations and the percent inhibition of specific binding determined. Where Roth et al.

10 ␮M test compound inhibited ⬎50% of specific binding, Ki determinations were performed by using six concentrations of unlabeled ligand spanning a 10,000-fold dose range. Kis were calculated by using GRAPHPAD PRIZM and represent the mean ⫾ SEM of quadruplicate determinations. Functional Assays. Phosphoinositide hydrolysis assays at 5-HT2A receptors were performed as previously described (8, 12). ␬ opioid agonist-dependent inhibition of adenylate cyclase was performed by using the KOR-cell line. Briefly, cells were split into polylysine-coated 24-well plates and then incubated overnight in serum-free medium. The next day, medium was replaced with Hanks’ F12 medium containing 100 ␮M isobutylmethylxanthine and 100 ␮M forskolin together with various concentrations of test agent. After incubation at 37°C for 15 min, the reaction was terminated and cAMP content determined as PNAS 兩 September 3, 2002 兩 vol. 99 兩 no. 18 兩 11935

NEUROBIOLOGY

Fig. 1. Molecular modeling predicts Salvinorin A is a structurally novel ␬ opioid ligand. A shows the structure of Salvinorin A, enadoline, U69593 and LSD whereas B shows a superimposition of the structures of Salvinorin A and U69593. C shows potential residues on the KOR identified by molecular modeling, which might interact with Salvinorin A, and D shows a model of Salvinorin A’s interactions with the KOR (see supporting information for further details).

Table 1. Salvinorin A is a potent and selective ␬ opioid ligand

Cloned receptors Brain receptors

␮ Ki

␦ Ki

␬ Ki (pKi ⫾ SEM)

⬎10,000 nM ⬎5,000 nM

⬎10,000 nM ⬎5,000 nM

16 (7.2 ⫹兾⫺ 0.06) 4.3 (8.6 ⫹兾⫺ 0.05)

Shown are mean Ki values for Salvinorin A at cloned receptors expressed in human embryonic kidney-293 cells or opioid receptors expressed in situ in rat (␮, ␦) or guinea pig brain (␬). Data represent mean ⫾ SEM of computer-derived estimates of Ki and pKi values for n ⬎ 3 separate experiments.

described previously (13). All data reported here represent the results of at least four separate experiments with EC50 and Emax values calculated by using GRAPHPAD PRIZM (Graph PAD, San Diego). The 35S-GTP[␥S]-binding assay proceeded with modifications of the methods described previously (14, 15). Guinea pig caudate membranes, prepared as described previously (16) (10–20 ␮g of protein in 300 ␮l of 50 mM Tris䡠HCl, pH 7.4, with 1.67 mM DTT and 0.15% BSA) were added to polystyrene 96-well plates filled with 200 ␮l of a reaction buffer containing 50 mM Tris䡠HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 100 ␮M GDP, 0.1% BSA, 0.05–0.01 nM 35S-GTP[␥S], and varying concentrations of drugs. The reaction mixture was incubated for 3 h at 22°C (equilibrium). The reaction was terminated by the addition of 0.5 ml of ice-cold Tris䡠HCl, pH 7.4 (4°C) followed by rapid vacuum filtration through Whatman GF兾B filters previously soaked in ice-cold Tris䡠HCl, pH 7.4 (4°C). The filters were washed twice with 0.5 ml of ice-cold distilled H2O (4°C). Bound radioactivity was counted at an efficiency of 98% by liquid scintillation spectroscopy. Nonspecific binding was determined in the presence of 10 ␮M GTP[␥S]. Molecular Modeling. Molecular modeling investigations were conducted by using the SYBL molecular modeling package (Ver. 6.7, 2001, Tripos Associates, St. Louis). Molecular mechanics minimizations of receptor models and complexes were performed after the addition of hydrogen atoms by using the Tripos force field with Gasteiger–Hu ¨ckel charges (distance-dependent dielectric constant, nonbonded cutoff ⫽ 8 Å) without constraints and were terminated at an energy gradient of 0.05 kcal兾mol兾Å, essentially as previously described (17–19) The UNITY program within SYBL was used to perform the three-dimensional database searches. Full details of the modeling methods and results are published as supporting information (Tables 3–5) on the PNAS web site, www.pnas.org.

Results Salvinorin A Selectively Inhibits KOR Binding. To identify Salvinorin

A’s molecular target, we screened Salvinorin A (10 ␮M) at a large panel of mainly cloned human GPCRs, transporters, and ligand-gated ion channels by using the resources of the NIMHPDSP. For comparison, we screened the same molecular targets with the prototypic hallucinogen LSD, also at 10 ␮M. As shown in Fig. 1 and in supporting information, Salvinorin A inhibited only [3H]-bremazocine-labeled KORs and did not significantly inhibit binding to cloned human ␮ (MOR) or ␦ opioid (DOR) receptors or any of the 48 other molecular targets screened. Ki determinations (Table 1) showed that Salvinorin A was a potent agonist of KOR and guinea pig (gp)KOR. Additionally, Salvinorin A had Ki values ⬎5,000 nM at the gpMORs and gpDORs (Table 1). These results indicate that Salvinorin A is, to our knowledge, the first naturally occurring ␬ opioid selective ligand. By comparison, LSD potently inhibits the binding of a large number of biogenic amine receptors (Fig. 1) with Kis ⬍50 nM for several GPCRs (data not shown). Interestingly, Salvinorin A had no detectable affinity for the 5-HT2A serotonin receptor and did not activate 5-HT2A receptors (not shown), which represent the 11936 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.182234399

main molecular target responsible for the classical hallucinogens such as LSD, N,N⬘-dimethyltryptamine, psilocybin, mescaline, and 4-bromo-2,5-dimethoxyphenylisopropylamine (20, 21). Salvinorin A Represents a Structurally Novel KOR Ligand. Because Salvinorin A represents a structurally novel hallucinogen, we next performed molecular modeling studies to provide insights into how this compound might interact with KORs. A previously reported model of the KOR complexed with the KOR-selective agonist U69593 was used as a starting point (22). This model has the advantage that it was derived from a set of distance constraints between potential hydrogen bond-forming pairs unique to the opioid receptor sequences themselves. The result thus does not depend directly on any direct experimental structural data for rhodopsins. Although this model was constructed before the publication of the crystal structure of rhodopsin (23), it is remarkable that the overall configurations are quite similar (rms deviation ⫽ 4 ⌬ by fitting the helix C-␣ atoms of identical residues in both sequences). The U69593 KOR complex places the arylacetamide portion of the ligand in a position analogous to the tyramine moiety with the carbonyl hydrogen bonded to Y139 (22). The only structural similarity between U69593 and Salvinorin A (Fig. 2A) is the presence of an aromatic ring and the amide and ester carbonyl groups separated by a short linkage. Because of this similarity, and the nearly complete lack of similarity of salvinorin and any known KOR ligand, the salvinorin crystal structure (5) was initially docked by superimposition of aromatic centroids and the carbonyl atoms of salvinorin with those of bound U69593. The role of the carbonyl functionality for arylacetamide ligands as a hydrogen bond acceptor has been demonstrated experimentally (24) and indirectly supports the proposed role of Y139 and its interaction with the lactone carbonyl of salvinorin. Multiple sterically allowed complexes were generated by using a systematic conformational search about all rotatable Y139 bonds, a dummy bond between a Y139 OH hydrogen atom, and salvinorin carbonyl, following a previously described method (18). Candidate complexes were evaluated interactively for steric fit and hydrogen bond donating properties of the receptor cavity visualized as a Connolly channel plot color coded for hydrogen-bonding potential. Only one family of complexes allowed simultaneous hydrogen bond formation between the receptor side chains and ligand features shown in Fig. 2C (see Table 3 for additional modeling results and details of modeling procedures). In this orientation, the furan substituent of Salvinorin A pointed toward TM1 and TM2, the 4-methoxycarbonyl toward TM5 and TM6, with the A and C rings toward the extra- and intracellular sides, respectively (Fig. 2D and supporting information). Not unexpectedly, there is very little atom-by-atom correspondence between bound U69593 and Salvinorin A, although both occupy a similar space (Fig. 2B). Docking of salvinorin into hydrogen bond potentialcoded Connolly channels defining the binding sites of the MOR and DOR models (22) indicates that salvinorin is sterically compatible with each in slightly different binding modes but could not as readily accommodate the four-point hydrogen bond donor兾acceptor scheme (Fig. 2D) seen with the KOR receptor (e.g., the KOR models could accommodate the furan oxygen and 4-methoxycarbonyl functionality but not the 2-acetoxy group). Residues potentially forming the salvinorin-binding site of the KOR receptor model are listed in Table 4. The identities of 11 of these are conserved in both the MOR and DOR, whereas the remaining seven are variable. The variable residues cause significant alterations in the steric and electronic characteristics of MOR and DOR in the regions analogous to the salvinorinbinding site of the KOR. The substantial differences in the region of the salvinorin-binding site between the KOR and MOR兾DOR receptors are consistent with the observed KOR selectivity of salvinorin. Roth et al.

NEUROBIOLOGY

Fig. 2. Large-scale screening of human cloned GPCRs reveals Salvinorin A is selective for KOR. Shown is the mean percent inhibition of radioligand binding or functional activity (metabotropic glutamate receptors only) to 50 receptors and transporters for LSD (yellow bars) and Salvinorin A (red bars) tested at 10 ␮M. With the exception of the rat ␤1 and ␤2 adrenergic and bovine dopamine transporter (DAT) all of the assays were performed with cloned human receptors heterologously expressed (see Materials and Methods and supporting information on the PNAS web site for details). As can be seen (arrow), Salvinorin A inhibited only KOR binding at 10 ␮M. See Table 5 for details. SERT, serotonin transporter; NET, norepinephrine transporter; DAT, dopamine transporter; rGABAA, rat GABA-A receptor.

The proposed KOR salvinorin-binding site model is also consistent with what little is known about the structural features of salvinorin required for psychotropic activity. For example, the Roth et al.

one-position carbonyl of salvinorin is not able to form specific donor兾acceptor contacts with residues in the receptor model, partially because of its sterically hindered environment, and is PNAS 兩 September 3, 2002 兩 vol. 99 兩 no. 18 兩 11937

stably expressed in human embryonic kidney-293 cells and gpKOR expressed in situ in guinea pig brain. As shown in Fig. 3, Salvinorin A was a potent KOR agonist with an EC50 for inhibition of adenylate cyclase of 1.05 nM as compared with an EC50 for the KOR agonist U69593 of 1.2 nM (Table 2). Salvinorin A was also a potent agonist at gpKOR expressed in situ with an EC50 for [35S]GTP[␥S] binding of 235 nM with U69593 having an EC50 of 377 nM (Table 2). Taken together, these results indicate that Salvinorin A represents, to our knowledge, the first nonnitrogenous KOR-selective agonist.

Fig. 3. Salvinorin A is a potent KOR agonist. A shows that Salvinorin A potently inhibits 3H-bremazocine binding to cloned KORs, whereas B shows the ability of Salvinorin A to inhibit forskolin-stimulated adenylate cyclase in KOR-393 cells. Data represent the mean ⫾ SD of triplicate determinations from a representative experiment that has been replicated three times. For the inhibition of forskolin-stimulated cyclase activity, an EC50 value of 1 ⫾ 0.5 nM was calculated for Salvinorin A, compared with an EC50 value of 1.2 ⫾ 0.6 nM for U69593.

not essential for psychotropic activity (25). The 2-acetoxy group of salvinorin does make specific donor兾acceptor contacts in the model and is required for activity (5). Interestingly, a threedimensional search of the National Cancer Society Database using the pharmacophore features and geometries derived from salvinorin docked with the KOR model produced splendidin (26) and deoxydeoxygedunin (27) (not shown). Splendidin was originally isolated from Salvia splendens, a species distinct from S. divinorum and from which salvinorin is derived. S. splendens has been reported to have psychotropic activity. Salvinorin A Is a Potent KOR Agonist at Recombinant KORs and KORs Expressed in Situ. We next examined the agonist兾antagonist

properties of Salvinorin A by using two model systems: KOR

Discussion The main finding of this paper is that Salvinorin A, the active ingredient of the hallucinogenic plant S. divinorum, is a potent and selective KOR agonist. Salvinorin A is a novel nonalkaloid diterpene that has no structural resemblance to any known hallucinogens but does have modest structural homology to enadoline, a selective KOR agonist (Fig. 1 A and C). Salvinorin A thus represents a class of hallucinogens with potent actions at KORs. Because KOR agonists have long been known to have psychotomimetic actions (28), these results imply that the actions of Salvinorin A in humans are mediated, at least in part, via activation of KORs. Additionally, these results imply that KORselective antagonists could conceivably represent treatments for diseases in which hallucinations are prominent, including schizophrenia, depression with psychotic features, and the hallucinosis associated with certain dementias (Alzheimer’s, Huntington’s, and Pick diseases) and certain types of drug abuse (e.g., amphetamine and cocaine psychosis) (29, 30). Previous studies evaluating naltrexone, which is a nonselective opioid antagonist, for the treatment of schizophrenia have yielded equivocal results (31, 32). Dynorphin was discovered in 1979 by Goldstein et al. (33) and was demonstrated to be an extraordinarily potent endogenous peptide with selectivity for the KOR (34), a GPCR in the opioid receptor family (35). Before the cloning of the KOR, a large amount of behavioral (36, 37), developmental (38, 39), and biochemical (40, 41) data suggested the existence of distinct KORs. Although dynorphin and related peptides represent, in some cases, potent and relatively selective endogenous ligands for the KOR, other types of naturally occurring ligands have heretofore not been identified. The discovery that Salvinorin A is a potent naturally occurring nonalkaloid agonist for the KOR is thus unexpected.

Table 2. Salvinorin A is a potent ␬-opioid agonist: [35S]GTP-␥-S studies using guinea pig brain caudate membranes Drug Percent stimulation DAMGO SNC80 U69,593

Unblocked

CTAP, 200 nM

TIPP, 20 nM

Nor-BNI, 0.2 nM

414 ⫾ 47 0.96 ⫾ 0.02 758 ⫾ 131 0.91 ⫾ 0.04 377 ⫾ 39 1.70 ⫾ 0.04

11,124 ⫾ 2,126 0.99 ⫾ 0.09 987 ⫾ 120 0.94 ⫾ 0.03 540 ⫾ 57 1.70 ⫾ 0.04

494 ⫾ 56 0.97 ⫾ 0.02 9,565 ⫾ 4,115 0.90 ⫾ 0.19 442 ⫾ 76 1.60 ⫾ 0.06

355 ⫾ 61 0.92 ⫾ 0.03 855 ⫾ 119 0.86 ⫾ 0.3 1,554 ⫾ 168 1.60 ⫾ 0.05

259 ⫾ 40 0.81 ⫾ 0.06

643 ⫾ 128 0.89 ⫾ 0.10

Percent of maximal stimulation produced by 10 ␮M U69,593 Salvinorin A 235 ⫾ 26 204 ⫾ 20 0.79 ⫾ 0.04 0.83 ⫾ 0.03

[35S]-GTP-␥-S-binding assays were conducted as described in Materials and Methods. Agonist dose–response curves were generated by using 8 –10 drug concentrations in the absence and presence of fixed concentrations of selective antagonists: CTAP to block ␮ receptors, TIPP to block ␦ receptors, and nor-BNI to block ␬ receptors. The concentrations were chosen on the basis of previous studies to selectively block the targeted receptor. Values in parentheses are the maximal percent stimulation. For Salvinorin-A, the value is reported as a percent of the stimulation produced by 10 ␮M U69,593. Each value is ⫾ SD (n ⫽ 3). DAMGO, [D-Ala-2-MePhe4,Gly-ol5]enkephalin; CTAP, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2; TIPP, H-Tyr-Tic-Phe-Phe-OH. 11938 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.182234399

Roth et al.

We gratefully acknowledge Christina M. Dersch, Beth Popadok, and Sandra Hufesein for superb technical assistance. This work was supported in part by a Research Scientist Development Award KO2MH01366 (B.L.R.) and by the NIMH-PDSP NO2MH80004 (B.L.R.).

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yielded conflicting results (46–48), whereas one study examining affective disorder was negative (47). On the other hand, two well-controlled studies have demonstrated an up-regulation of KORs in Alzheimer’s disease (49, 50), whereas MORs and DORs were down-regulated (50) or unchanged (49). In conclusion, we report the discovery that Salvinorin A is a potent selective KOR agonist. Salvinorin A thus represents a unique structural class of nonnitrogenous opioid subtypeselective agonists. Additionally, these results suggest that KORs play a prominent role in the regulation of human perception and suggest that KOR antagonists could represent a novel drug class with specific activity in diseases in which alterations in perception are predominant. Finally, these results imply that the KOR兾dynorphinergic system functions to modulate human perception and cognition.

It is now well established that the activation of KORs induces a large number of behavioral effects that include analgesia, sedation, and perceptual distortions. In the past, studies on the precise role of KORs in humans were hampered by the lack of selective agonists, although studies with compounds such as cyclazocine and ketocyclazocine suggested that KOR agonists were psychotomimetic (28). More recently, human studies with the highly selective KOR agonist enadoline (42) indicated that KOR activation induced visual distortions, feelings of unreality, and depersonalization. These effects of enadoline are reminiscent of those previously reported for Salvinorin A (2, 3). Taken together, these results suggest that the KOR兾dynorphinergic system functions to modulate human perception and cognition, as might be inferred from detailed anatomical studies of dynorphin peptide distribution studies (43–45). One of the implications of these results is that KORs or KOR signaling may also be important in the pathogenesis of diseases characterized by perceptual distortions. The most obvious diseases implicated are schizophrenia, dementia, and bipolar disorders, because all are characterized by hallucinations and delusions. Prior studies evaluating KORs in schizophrenia have

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Salvinorin A: the ‘magic mint’ hallucinogen finds a molecular target in the kappa opioid receptor Douglas J. Sheffler1 and Bryan L. Roth2 1

Department of Biochemistry, Case Western Reserve University Medical School, 10900 Euclid Avenue, Cleveland, OH 44106, USA National Institute of Mental Health Psychoactive Drug Screening Program and Departments of Biochemistry, Psychiatry, and Neurosciences, Case Western Reserve University Medical School, 10900 Euclid Avenue, Cleveland, OH 44106, USA 2

Salvinorin A, a neoclerodane diterpene, is the most potent naturally occurring hallucinogen known and rivals the synthetic hallucinogen lysergic acid diethylamide in potency. Recently, the molecular target of salvinorin A was identified as the kappa opioid receptor (KOR). Salvinorin A represents the only known non-nitrogenous KOR selective agonist. Based on the selectivity of salvinorin A for the KOR, this receptor represents a potential molecular target for the development of drugs to treat disorders characterized by alterations in perception, including schizophrenia, Alzheimer’s disease and bipolar disorder. Salvia divinorum (Diviner’s sage) (Fig. 1a) is a rare member of the mint family that has been used for many centuries by the Mazatec people of Oaxaca, Mexico in traditional spiritual practices (Box 1). More recently, S. divinorum (also known as ‘magic mint’) has been used as a marijuana substitute by Mexican youths [1]. Furthermore, a large number of S. divinorum plants were discovered recently in Swiss horticulturists’ greenhouses and a few more were seized at a large-scale plantation in Switzerland, implicating their increasing use as a recreational drug in Europe [2]. For several years, S. divinorum has been cultivated in California for use as a legal hallucinogen. At present, neither Swiss nor US laws for controlled substances ban the use of S. divinorum or its active compounds. In traditional spiritual practices, S. divinorum is typically ingested by one of three routes: (1) mastication and swallowing of the leaves; (2) crushing the leaves to extract the juices and then swallowing the extract; or (3) smoking the leaves [3]. The hallucinatory effect that results has been reported to be potent and intense, lasting for up to an hour [1,3]. The presumed main active ingredient of S. divinorum salvinorin A (Fig. 1b) was first identified by Alfredo Ortega in 1982 and independently isolated by Leander Valdes soon thereafter [1,4]. Salvinorin A is a neoclerodane diterpine of known absolute stereochemistry whose structure has been determined using 1H nuclear magnetic resonance (NMR) and by two independent single-crystal X-ray studies (Fig. 1c) [4 –6]. Salvinorin A represents the only known psychoactive terpenoid [7] and is chemically Corresponding author: Bryan L. Roth ([email protected]). http://tips.trends.com

unique. Additionally, salvinorin A has been reported to be the most potent naturally occurring hallucinogen, with an effective dose, when smoked, of 200– 1000 mg in humans. Thus, it is similar in potency to the synthetic hallucinogens lysergic acid diethylamide [LSD (the typical human dose used in abuse is 50 –250 mg)] and 4-bromo-2,5-dimethoxyphenylisopropylamine [DOB (the typical human dose used in abuse is 500– 1000 mg)] [1,3]. The molecular target of salvinorin A Previous attempts to determine the proximal molecular target(s) of salvinorin A have been unsuccessful. Shortly after the recognition of the psychoactive properties of salvinorin A (by Daniel Siebert in 1994), salvinorin A was submitted for screening to NovaScreene to discover its molecular target [3]. Unfortunately, salvinorin A showed no significant inhibition of radioligand binding at any of the receptors, including various biogenic amine receptors, cannabinoid receptors and sigma receptors, screened by the NovaScreene process [3]. Recently, the pharmacological profile of salvinorin A at a variety of human G-protein-coupled receptors (GPCRs), ligand-gated

(a)

(b)

(c)

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Fig. 1. (a) Salvia divinorum is a rare member of the Lamiaceae (mint) family. It has been used in traditional spiritual practices and as a recreational drug. (b) The twodimensional structure of salvinorin A, the presumed main active ingredient of S. divinorum. (c) The three-dimensional structure of salvinorin A.

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Box 1. The ethnopharmacology of Salvia divinorum Salvia divinorum is known by many names including ska Maria, ska Pastora, hojas de Maria, hojas de la Pastora, hierba Maria, and la Maria. These names reflect the Mazatec belief that S. divinorum is the incarnation of the Virgin Mary; hence, the plant is highly prized and respected [16– 18]. Despite its many names, S. divinorum was not identified until 1962 when Wasson and Hofmann collected a member of the Lamiaceae (mint) family that was known by the Mazatec people of Oaxaca, Mexico to have psychoactive properties [19]. This mint only grows in forest ravines and in other moist areas of the Sierra Mazateca between 750 m and 1500 m altitude [16]. Interestingly, S. divinorum flowers rarely, blossoming with white corollas and purple chalyces [16]. Seed production is even more rare because S. divinorum has never been reported to produce seeds in its native habitat of the Sierra Mazateca. Indeed, only

ion channels, and transporters was re-examined via the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) (http://pdsp.cwru.edu) [8]. Salvinorin A was discovered to be a potent and selective kappa opioid receptor (KOR) agonist (importantly, KOR binding was not assayed using NovaScreene [3]). None of the other 48 tested molecular targets that were screened using the NIMH-PDSP [8], including the mu opioid receptor (MOR) and delta opioid receptor (DOR), showed significant inhibition of radioligand binding by salvinorin A, which demonstrates that salvinorin A is apparently selective for the KOR. Opioid receptors are heptahelical GPCRs that have an extracellular N-terminus and an intracellular C-terminus. These opioid receptors are subdivided into MORs, DORs and KORs, each of which has receptor subtypes. The KORs, MORs and DORs are coupled to the G-protein subfamily Gi/Go, and thus opioid receptor activation elicits analgesic (DORs, MORs and KORs) and psychotomimetic (KORs) [9] effects that are expected to involve inhibition of adenylyl cyclases, the activation of inward rectifying Kþ channels, and the inhibition of N-, P-, Q- and R-type voltage-activated Ca2þ channels. Radioligand binding studies [8] disclosed that salvinorin A had high affinity at both cloned (Ki ¼ 16 nM ) and naturally occurring (guinea-pig brain; Ki ¼ 4.3 nM ) KORs. Interestingly, salvinorin A did not have detectable affinity for 5-HT2A receptors, nor did it activate 5-HT2A receptors, which represent the primary target for classical hallucinogens [8,10]. In functional studies, salvinorin A was found to be a potent agonist of the human KOR expressed in human embryonic kidney 293 (HEK293) cells with an EC50 of 1.05 nM for the inhibition of adenylyl cyclase activity, compared with an EC50 of 1.2 nM for the KOR agonist U69593 (see Chemical name) [8]. Recently, the actions of salvinorin A on tail-flick latency (a measure of

Chemical name U69593: (þ)-(5a,7a,8b)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzeneacetamide

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Valdes and Siebert have reported the identification of seeds. S. divinorum primarily reproduces by vegetative growth from a shoot that is stuck into the ground, and can be readily grown from cuttings. Some believe that all S. divinorum strains present in the USA are one of seven vegetatively propagated clones, originating from the Sierra Mazateca in Oaxaca, Mexico. Most clones in the USA are derived from the original clone identified by Wasson in 1962 [18,20]. Because S. divinorum grows easily from cuttings, there is great potential for its increased use as a recreational drug; a yield of even a gram of salvinorin A per kilogram of dried leaves would provide 2000 doses of salvinorin A [21]. For additional information on S. divinorum and salvinorin A, see ‘The Salvia divinorum Research and Information Center’ (http://www. sagewisdom.org), an online resource maintained by Daniel Siebert.

analgesia) observed in wild-type mice have been shown to be abrogated in KOR knockout animals (J. Pintar, pers. commun). The uniqueness of salvinorin A is underscored by the fact that it is: (1) the first non-nitrogenous, naturally occurring, KOR selective agonist; (2) the first nonnitrogenous, KOR selective ligand; and (3) the only known non-alkaloidal hallucinogen. Thus, from a medicinal chemistry standpoint, salvinorin A represents a structurally novel class of KOR selective compounds. Molecular modeling performed in the original study [8] resulted in a docked structure of salvinorin A to the KOR (Fig. 2a) and implicated residues that might be involved in the binding of salvinorin A (Fig. 2b) [8]. Mutagenesis studies of these residues to determine their potential contribution to the binding of salvinorin A to the KOR will provide a wealth of insight for selective KOR drug development. KOR antagonists as therapeutic agents? KOR agonists have been shown previously to be psychotomimetic [9] and the psychoactive properties of salvinorin A in humans are probably mediated by KORs. Indeed, there is one anecdotal report that the hallucinogenic effects of salvinorin A are blocked by pretreatment with naloxone, a nonspecific opioid receptor antagonist with modest affinity for KORs (D. Siebert, pers. commun). Thus, overdoses of salvinorin A are likely to respond to naloxone (Narcanw) or naltrexone administration. There are numerous diseases characterized by hallucinations, including schizophrenia, depression with psychotic features, and the hallucinosis associated with certain dementias such as Alzheimer’s, Huntington’s and Pick diseases. Diseases characterized by hallucinosis could, conceivably, reflect alterations in KOR number or KOR signal transduction; thus, KOR selective antagonists might represent novel therapeutic targets for diseases in which hallucinations are prominent [11]. However, there are few examples in the literature to corroborate this hypothesis. For example, administration of naloxone and naltrexone to schizophrenics showed only modest results [12]. Clearly, further studies using KOR selective antagonists are warranted, although it is

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(e.g. Alzheimer’s disease, schizophrenia, bipolar disorder and dementia). Previous development of antipsychotic drugs has primarily focused on the 5-HT2A and the D2 dopamine receptor [15]. Knowledge that the KOR mediates the hallucinatory effects of salvinorin A provides a novel molecular candidate for the development of antipsychotic drugs. References

Fig. 2. (a) A model of the interactions of salvinorin A (blue) with the kappa opioid receptor (KOR; purple). (b) A close-up view of residues on the KOR, identified by molecular modeling, that might interact with salvinorin A.

possible that KOR selective antagonists will have no efficacy in diseases manifested by psychosis. Intriguingly, two independent post-mortem studies of Alzheimer’s disease have reported an upregulation of KOR expression in the amygdala, putamen and cerebellar cortex [13,14]. In these same studies, the expression of MORs and DORs remained unchanged or was downregulated in these regions [13,14]. Concluding remarks Salvinorin A represents a new class of non-nitrogenous KOR selective agonists, and KORs and/or KOR signaling probably play a role in the modulation of human cognition and perception. Because salvinorin A, a KOR selective agonist, has psychoactive properties, KOR selective antagonists could conceivably prove useful in the treatment of certain psychiatric disorders http://tips.trends.com

1 Valdes, L.J. III (1994) Salvia divinorum and the unique diterpene hallucinogen, salvinorin (divinorin) A. J. Psychoactive Drugs 26, 277 – 283 2 Giroud, C. et al. (2000) Salvia divinorum: an hallucinogenic mint which might become a new recreational drug in Switzerland. Forensic Sci. Int. 112, 143– 150 3 Siebert, D.J. (1994) Salvia divinorum and salvinorin A: new pharmacologic findings. J. Ethnopharmacol. 43, 53 – 56 4 Ortega, A. et al. (1982) Salvinorin, a new trans-neoclerodane diterpene from Salvia divinorum (Labiatae). J. Chem. Soc. Perkins Trans. 1, 2505– 2508 5 Valdes, L.J. et al. (1984) Divinorin A, a psychotropic terpenoid, and divinorin B from the hallucinogenic Mexican mint Salvia divinorum. J. Org. Chem. 49, 4716– 4720 6 Koreeda, M. et al. (1990) The absolute stereochemistry of salvinorins. Chem. Lett., 2015– 2018 7 Abramov, M.M. and Yaparova, S.A. (1963) J. Appl. Chem (USSR) 36, 2471 8 Roth, B.L. et al. (2002) Salvinorin A: a potent naturally occurring nonnitrogenous k opioid selective agonist. Proc. Nat. Acad. Sci. U. S. A. 99, 11934 – 11939 9 Roth, B.L. et al. (1998) 5-Hydroxytryptamine2-family receptors (5-hydroxytryptamine2A, 5-hydroxytryptamine2B, 5-hydroxytryptamine2C): where structure meets function. Pharmacol. Ther. 79, 231 – 257 10 Pfeiffer, A. et al. (1986) Psychotomimesis mediated by k opiate receptors. Science 233, 774 – 776 11 Ni, Q. et al. (1995) Opioid peptide receptor studies. 3. Interaction of opioid peptides and other drugs with four subtypes of the k2 receptor in guinea pig brain. Peptides 16, 1083 – 1095 12 Marchesi, G.F. et al. (1995) The therapeutic role of naltrexone in negative symptom schizophrenia. Prog. Neuro-Psychopharmacol. Biol. Psychiat. 19, 1239 – 1249 13 Mathieu-Kia, A. et al. (2001) m-, d-, and k-opioid receptor populations are differentially altered in distinct areas of postmortem brains of Alzheimer’s disease patients. Brain Res. 893, 121– 134 14 Bard, J. et al. (1993) Opioid receptor density in Alzheimer amygdala and putamen. Brain Res. 632, 209 – 215 15 Roth, B.L. et al. (1999) Activation is hallucinogenic and antagonism is therapeutic: role of 5-HT2A receptors in atypical antipsychotic drug actions. The Neuroscientist 5, 254– 262 16 Valdes, L.J. et al. (1982) Ethnopharmacology of Ska Maria Pastora (Salvia divinorum, Epling and Javita-M.). J. Ethnopharmacology 7, 287– 312 17 Valdes, L.J. (2001) The early history of Salvia divinorum. The Entheogen Review X, pp. 73– 75 18 Valdes, L.J. et al. (1987) Studies of Salvia divinorum (Lamiaceae), an hallucinogenic mint from the Sierra Mazateca in Oaxaca, Central Mexico. Economic Botany 41, 283 – 291 19 Wasson, W.G. (1962) A new Mexican psychotropic drug from the mint family. In Botanical Museum Leaflets, Harvard University Press 20, pp. 77– 84 20 Siebert, D.J. (1994) Salvia divinorum and salvinorin A: new pharmacologic findings. J. Ethnopharmacol. 43, 53 – 56 21 Valdes, L.J. (1994) Salvia divinorum and the unique diterpine hallucinogen Salvinorin (Divinorin) A. J. Psychoactive Drugs 26, 277– 283 0165-6147/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0165-6147(03)00027-0

Psychopharmacology (2004) 172:220–224 DOI 10.1007/s00213-003-1638-0

ORIGINAL INVESTIGATION

Eduardo R. Butelman · Todd J. Harris · Mary Jeanne Kreek

The plant-derived hallucinogen, salvinorin A, produces k-opioid agonist-like discriminative effects in rhesus monkeys Received: 28 May 2003 / Accepted: 20 August 2003 / Published online: 30 October 2003
Springer-Verlag 2003

Abstract Rationale: Salvinorin A is the active component of the hallucinogenic plant Salvia divinorum. The potential mode of action of this hallucinogen was unknown until recently. A recent in vitro study detected high affinity and efficacy of salvinorin A at k-opioid receptors. It was postulated that salvinorin A would produce discriminative stimulus effects similar to those of a high efficacy k-agonist (U69,593) in rhesus monkeys. Methods: Monkeys were previously trained to discriminate U69,593 (0.0056 or 0.013 mg/kg; SC) from vehicle in a food-reinforced FR20 (fixed ratio 20) operant conditioning procedure (n=3). The ability of salvinorin A to cause generalization (90% U69,593-appropriate responding) was examined in time course and cumulative dose-effect curve studies. Results: All subjects dose-dependently emitted full U69,593-appropriate responding after salvinorin A (0.001–0.032 mg/kg, SC). Salvinorin A-induced generalization started 5–15 min after injection, and dissipated by 120 min. The opioid antagonist quadazocine (0.32 mg/kg) fully blocked the effects of salvinorin A. The k-selective antagonist GNTI (1 mg/kg; 24 h pretreatment) did not cause significant antagonism of the effects of salvinorin A (GNTI, under these conditions, was only effective as an antagonist in two of three monkeys). The NMDA antagonist ketamine (0.1–3.2 mg/kg) was not generalized by any subject, indicating that not all compounds that produce hallucinogenic or psychotomimetic effects in humans are generalized by subjects trained to discriminate U69,593. Conclusions: The naturally occurring hallucinogen salvinorin A produces discriminative stimulus effects similar to those of a high efficacy k-agonist in non-human primates. Keywords k-Opioid · Salvinorin A · Drug discrimination · Hallucinogen E. R. Butelman ()) · T. J. Harris · M. J. Kreek The Rockefeller University, 1230 York Avenue, Box 171, New York, NY, 10021, USA e-mail: [email protected] Fax: +1-212-3278574

Introduction Salvinorin A is the main pharmacologically active component of the ethnopharmacological hallucinogenic plant, Salvia divinorum (Ortega et al. 1982; Valdes 1994). Salvia divinorum was administered in traditional ethnopharmacological practice by the Mazatec indigenous people of Oaxaca, Mexico (Valdes et al. 1993; Valdes 1994). More recently, there have been reports of Salvia divinorum self-administration in non-ethnopharmacological settings, presumably due to its hallucinogenic effects. Salvia divinorum leaf preparations (occasionally fortified with extracted salvinorin A) are widely available in Western Europe and the USA, notably on Internet sites (Drug Enforcement Administration 2002; Observateur franais des drogues et des toxicomanies 2002). The potential mode of action of salvinorin A was unknown until recently, since salvinorin A does not bind to receptor sites known to be the targets of many psychotomimetic/hallucinogenic compounds (e.g. serotonergic, NMDA, PCP sites) (Siebert 1994; Roth et al. 2002). A recent pivotal study reported that salvinorin A was a selective, high efficacy k-agonist in cloned human k-receptors, and in guinea pig brain (Roth et al. 2002). Synthetic k-agonists administered systemically to humans are known to produce a variety of dose-dependent and reversible subjective/interoceptive effects, including sedation, dysphoria and “psychotomimetic” effects (Pfeiffer et al. 1986; Ur et al. 1997; Walsh et al. 2001). Interestingly, potential k-agonist effects of Salvia divinorum may underlie its voluntary use (self-administration) as a hallucinogen in non-ethnopharmacological practice in humans (Giroud et al. 2000; Drug Enforcement Administration 2002; Sheffler and Roth 2003). There is at present no available evidence that focuses on the potential k-agonist effects of salvinorin A in primates. In the present studies, we therefore characterized the effects of systemically administered salvinorin A in rhesus monkeys trained to discriminate the highefficacy, centrally penetrating synthetic k-agonist, U69,593 (Remmers et al. 1999; Butelman et al. 2002).

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Focused antagonist studies with quadazocine and with the k-selective antagonist GNTI were instituted, in order to study the receptor mediation of the discriminative effects of salvinorin A (Butelman et al. 1999; Negus et al. 2002). The effectiveness of ketamine (a non-competitive NMDA antagonist) was also studied in this assay, in order to determine whether pharmacologically diverse hallucinogenic psychotomimetic compounds may produce U69,593-like discriminative effects (Krystal et al. 1994; Bowdle et al. 1998).

points (e.g. 5–120 min) after a single SC injection. In cumulative dosing tests, doses of the compounds were increased in 0.5 log unit steps (each cycle commenced with a 15-min time-out period, followed by a 5-min response period, as described above). Agonist doses were increased in each subject until either a) generalization (i.e. at least 90% U69,593-appropriate responding), b) rates of responding were decreased by more than 50%, or c) in the presence of effects which interfered with behavioral testing. Tests in the same subject were separated by at least 2 consecutive training days at criterion performance (i.e. at least 90% injection-appropriate responding and no more than 20 responses on the incorrect lever before the first food presentation in any cycle, with responses rates of at least 0.8 responses/s).

Materials and methods

Design

Subjects

Salvinorin A (0.001–0.032 mg/kg), its vehicle (0.16 ml/kg) and U69,593 (0.001–0.01 mg/kg) were compared in time course and cumulative dose-effect curve studies. Selected time course experiments for salvinorin A were re-determined 60 min following pretreatment with quadazocine (0.32 mg/kg). A quadazocine control study (i.e. for the effects of quadazocine alone over the relevant time period) was also completed. The cumulative salvinorin A dose-effect curve was re-determined after the same quadazocine pretreatment, and after GNTI (1 mg/kg; 24 h) pretreatment. Due to the long duration of action of GNTI, training and testing was suspended for 3 weeks after the GNTI test (Negus et al. 2002). A cumulative dose-effect curve with ketamine (0.1– 3.2 mg/kg) was also completed. Each of the above experiments was carried out as one or two determinations per subject.

Intact, adult captive-bred rhesus monkeys (M. mulatta; one male and two females) were singly housed in a room maintained at 20– 22oC with controlled humidity, and a 12:12 hour light: dark cycle (lights on at 0730 hours). They were fed approximately 10 jumbo primate chow biscuits (PMI Feeds, Brentwood, MO, USA) daily, supplemented by fruit, nuts and multivitamins. Water was available ad libitum in home cages. These studies were reviewed by the Institutional Animal Care and Use Committee of the Rockefeller University, and were in accordance with Guidelines of the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Health Council (Department of Health, Education and Welfare, Publication ISBN 0-309-05377-3, revised 1996).

Data analysis Procedures Three subjects were previously trained to discriminate U69,593 (0.0056 or 0.013 mg/kg, SC) from vehicle in a two-key (fixed ratio) FR 20 task, in operant conditioning chambers (Med Associates, St Albans, Vt., USA). The chambers were connected to a computer, via a Med Associates interface. Subjects were not exposed to chronic drug administration at any time and had received principally opioid compounds intermittently as part of discrimination training and testing. Training Subjects were trained in multiple-cycle sessions; each cycle consisted of an initial SC injection followed by a 15-min time out period (time-out responses had no consequence). At the end of the time-out period, a 5-min response period started (signaled by stimulus lights above each lever), during which monkeys could receive up to 10 sucrose pellets (300 mg; PJ Noyes/Research Diets; New Brunswick, N.J., USA), by responding on the injectionappropriate lever. A maximum of 4 cycles occurred within each training session. When multiple training cycles occurred, the first cycle was always a vehicle cycle. If drug (U69,593) was administered, it would always be on the last cycle within a training session. Monkeys were previously trained and had attained criterion (e.g. at least 90% injection-appropriate responses in four consecutive sessions, and no more than 20 responses on the incorrect lever before the first food presentation in any cycle, with response rates of at least 0.8 responses/s). Testing Test sessions were identical to that described above, except that 20 responses on either lever resulted in pellet delivery. Tests followed either a time course or a cumulative dosing procedure. In time course tests, a response period would commence at standard time

The main dependent variable was percent drug (U69)-appropriate responding (%DAR; presented graphically as mean€SEM). The subjects were considered to fully generalize if they emitted at least 90% of the responses within a response period on the U69,593appropriate lever. Cumulative dose-effect curves for salvinorin A (0.001–0.032 mg/kg) alone or after quadazocine (0.32 mg/kg) or GNTI (1 mg/kg) pretreatment were analyzed in a two-way (salvinorin A dosepretreatment condition) repeated measures ANOVA, followed by Duncan’s test for pretreatment condition (Sigmastat- SPSS, Chicago, Ill., USA). In two individual cases in which 100% U69,593-appropriate responding occurred at a nonmaximal salvinorin A dose (e.g. 0.01 mg/kg), a value of 100% was assigned for the maximal dose for this analysis. Significance (a) was set at the 0.05 level. Drugs U69,593 (Pharmacia, Kalamazoo, Mich., USA) and quadazocine mesylate (Sanofi-Winthrop, Malvern, Pa., USA) were dissolved in sterile water with the addition of 2 drops of lactic acid (pH approximately 6; further dilutions were made with sterile water). GNTI dihydrochloride (5’-guanidinonaltrindole; Tocris, Ellisville, Mo., USA) was dissolved in sterile water. Salvinorin A (Biosearch, Novato, Calif., USA) was dissolved in ethanol: Tween 80: sterile water vehicle (1:1:8 proportion, by volume). Ketamine hydrochloride (Fort Dodge, Iowa, USA) was diluted with sterile water, from stock solution (100 mg/ml). All drugs were injected SC in volumes of 0.05–0.16 ml/kg. Drug doses are expressed as the salt or base forms of each compound described above.

222 Fig. 1 Time-course effects of SC U69,593 (left panel) and salvinorin A (right panel) in monkeys (n=3) trained to discriminate U69,593 from vehicle. Abscissae (all panels): time (min) from injection. Ordinates (all panels): percent drug-appropriate responding (%DAR). All doses are in mg/kg

Results Baseline performance Monkeys were previously trained in the discrimination and had achieved criterion performance prior to the beginning of these studies. Stimulus control was stable in these subjects in the period prior to these studies. Thus, in the 5 consecutive training days before the first salvinorin A test, the following mean training values were obtained: vehicle training cycles mean (SEM): %DAR=0.67% (SEM 0.6); rate of responding=2.76 responses/s (SEM 0.84); U69,593 training cycles mean (SEM): %DAR= 99.8% (SEM 0.17); rate of responding=2.62 responses/s (SEM 0.12). None of the experimental conditions studied with salvinorin A or U69,593 resulted in robust changes in rates of responding; rate of responding data are therefore not presented herein. Time course studies The vehicle solution used for salvinorin A resulted in vehicle-appropriate responding over the course a 60-min time course test in all subjects (Fig. 1). U69,593 produced dose-and time-dependent generalization in all subjects (0.001–0.01 mg/kg). When tested under identical conditions, salvinorin A (0.001–0.032 mg/kg) also dosedependently generalized in all subjects (one subject, JA, generalized initially at 0.0032–0.01 mg/kg, the two other subjects, FL and VE, generalized at 0.032 mg/kg). At the active doses, both U69,593 and salvinorin A displayed rapid onsets following bolus SC administration (i.e. within 5–15 min). At these doses, the two compounds did not cause a robust rate-decreasing effect (not shown). Quadazocine (0.32 mg/kg) was administered 60 min before the lowest dose of salvinorin A, which produced robust generalization in each subject (e.g. 0.01 mg/kg for subject JA and 0.032 mg/kg for the other two subjects). In each case, quadazocine fully blocked the discriminative effects of salvinorin A in each 60-min time course test. That is, all salvinorin A-treated subjects emitted less than

Fig. 2 Cumulative dose-effect curve studies of U69,593, salvinorin A and ketamine in monkeys trained to discriminate U69,593 from vehicle. Abscissa: dose (mg/kg); ordinate: percent drug-appropriate responding. The U69,593 dose-effect curve was re-determined after 60 min pretreatment with quadazocine (quad; 0.32 mg/kg; error bars for this data set omitted for clarity). The salvinorin A doseeffect curve was also re-determined after 60 min pretreatment with quadazocine (0.32 mg/kg), or 24 h pretreatment with 5’-guanidinonaltrindole (GNTI; 1 mg/kg). Other details as in Fig. 1

2% DAR in all time points, in the presence of quadazocine (not shown). Quadazocine (0.32 mg/kg) alone, tested over this same time period (i.e. 60–120 min, in the presence of bolus vehicle administration) produced 0% DAR in all subjects, throughout (not shown). Cumulative dosing studies A “cumulative” vehicle test (4 cycles) resulted in vehicleappropriate responding in all subjects (i.e. 0% DAR was obtained in all subjects, throughout). Cumulative U69,593 and salvinorin A were dose-dependently generalized by all subjects, within a similar dose range (Fig. 2). Rates of responding were not robustly decreased over the studied dose ranges for U69,593 and salvinorin A (not shown). Quadazocine pretreatment (0.32 mg/kg; 60 min before the test) fully blocked the effects of salvinorin A (0.001– 0.032 mg/kg) in all subjects in these cumulative dosing determinations. For comparison, quadazocine (0.32 mg/ kg; under identical conditions) also caused a robust antagonism of the effects of U69,593 (0.001–0.032 mg/ kg). Complete surmountability experiments were not

223

attempted, due to solubility limits for salvinorin A in the present vehicle. The long-lasting k-selective antagonist GNTI (1 mg/kg) was studied as a 24-h pretreatment to the cumulative salvinorin A dose-effect curve. GNTI caused a shift in the salvinorin A mean dose-effect curve, and this was not fully surmounted at the largest salvinorin A dose tested herein. However, it should be noted that GNTI did not block the salvinorin A dose-effect curve for subject JA, but did block those for subjects FL and VE. Cumulative dose-effect curves for salvinorin A (0.001–0.032 mg/kg) alone or after quadazocine (0.32 mg/kg) or GNTI (1 mg/kg) pretreatment were analyzed using a two-way (salvinorin A dosepretreatment condition) repeated measures ANOVA. This ANOVA was significant for salvinorin A dose [F(3,6)=7.32] and for pretreatment condition [F(2,4)=7.41], as well as for their interaction [F(6,12)=3.16]. A Duncan’s test for pretreatment condition revealed that the quadazocine (q=5.38), but not the GNTI (q=1.96) pretreatment condition was different from salvinorin A alone. The ketamine cumulative dose-effect curve (0.1– 3.2 mg/kg), was determined under identical conditions. Up to ketamine doses that suppressed response rates by more than 50% from mean vehicle baseline values, none of the subjects generalized ketamine.

Discussion Salvinorin A produces discriminative stimulus effects similar to those of the centrally penetrating k-agonist U69,593, following SC administration. Salvinorin A was approximately equipotent with U69,593 in the present studies. Salvinorin A is also approximately equipotent with U69,593 at k-receptors in vitro (Roth et al. 2002). That is, EC50 values in the adenylyl cyclase and GTPgS assays were within 0.25 log units of each other, for these two agonists. Time course studies with the larger doses of SC salvinorin A revealed a fast onset and moderate duration of action (e.g. 5 and 60 min, respectively). A rapid onset for interoceptive effects of smoked salvinorin A has been reported in humans (Siebert 1994); no data are available for any systemic route in humans, to our knowledge. The largest salvinorin A dose used herein (0.032 mg/ kg; SC) caused only slight overt behavioral effects (sedation-like) over the 60 min following administration (pilot observational studies). This is consistent with previous studies indicating that discriminable doses of k-agonists in this procedure (e.g. U69,593 0.01 mg/kg) are accompanied by only slight sedative effects (Butelman and Kreek 2001; Butelman et al. 2003). All subjects returned to baseline behavior after the present salvinorin A tests. No emesis or motor effects (e.g. tremors) were observed during or after any of the present salvinorin A tests. The discriminative effects of salvinorin A were blocked by quadazocine (0.32 mg/kg) in all subjects. This quadazocine dose is sufficient to block the behav-

ioral and neuroendocrine effects of the selective k-agonist U69,593 in this species (Butelman et al. 1999). The longlasting k-selective antagonist GNTI (Negus et al. 2002) antagonized the effects of cumulative salvinorin A in only two of the three subjects, under the present conditions. In recent studies, these U69,593-discriminating subjects generalized structurally diverse k-agonists, but not m- or d-opioid agonists (Butelman et al. 2002). These findings, taken together with salvinorin A’s high and selective affinity at k-receptors in vitro (Roth et al. 2002), indicate that salvinorin A produces k-agonist-like discriminative effects following SC administration in non-human primates. In a control study, the NMDA antagonist ketamine was not generalized by any of the subjects, up to doses that produced robust rate-decreasing effects. The presently studied ketamine dose range (0.1–3.2 mg/kg) includes doses that produce discriminative effects in ketaminetrained monkeys (France et al. 1989). Ketamine has also been reported to produce psychotomimetic or hallucinatory effects in humans (Krystal et al. 1994; Bowdle et al. 1998). The present ketamine experiment therefore suggests that not all compounds with psychotomimetic or hallucinatory effects in humans produce U69,593-like effects in rhesus monkeys. These are the first studies on the behavioral effects of salvinorin A in non-human primates, and the first drug discrimination studies with salvinorin A in any species, to our knowledge. The findings are consistent with the in vitro characterization of salvinorin A as a k-agonist (Roth et al. 2002). The present studies are also consistent with the hypothesis that Salvia divinorum‘s self-administration in humans (presumably for its interoceptive/hallucinogenic effects) is due to the k-agonist effects of salvinorin A. Acknowledgement The present studies were supported by National Institutes of Health/NIDA grants DA11113 (E.R.B.), DA05130 and DA00049 (M.J.K.).

References Bowdle TA, Radant AD, Cowley DS, Kharasch ED, Strassman RJ, Roy-Byrne PP (1998) Psychedelic effects of ketamine in healthy volunteers: relationship to steady-state plasma levels. Anesthesiology 88:82–88 Butelman ER, Kreek MJ (2001) Kappa-opioid receptor agonistinduced prolactin release in primates is blocked by dopamine D(2)-like receptor agonists. Eur J Pharmacol 423:243–249 Butelman ER, Harris TJ, Perez A, Kreek MJ (1999) Effects of systemically administered dynorphin A(1–17) in rhesus monkeys. J Pharmacol Exp Ther 290:678–686 Butelman ER, Ball JW, Kreek MJ (2002) Comparison of the discriminative and neuroendocrine effects of centrally-penetrating kappa-opioid agonists in rhesus monkeys. Psychopharmacology 164:115–120 Butelman ER, Ball JW, Kreek MJ (2003) Peripheral selectivity and apparent efficacy of dynorphins: comparison to non-peptidic kappa-opioid agonists in rhesus monkeys. Psychoneuroendocrinology (in press)

224 Drug Enforcement Administration (2002) List of drugs and chemicals of concern. Internet website: www deadiversion usdoj gov/drugs_concern/salvia_d/summary.htm France CP, Snyder AM, Woods JH (1989) Analgesic effects of phencyclidine-like drugs in rhesus monkeys. J Pharmacol Exp Ther 250:197–201 Giroud C, Felber F, Augsburger M, Horisberger B, Rivier L, Mangin P (2000) Salvia divinorum: an hallucinogenic mint which might become a new recreational drug in Switzerland. Forens Sci Int 112:143–150 Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD, Heninger GR, Bowers MB Jr, Charney DS (1994) Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen Psychiatry 51:199– 214 Negus SS, Mello NK, Linsenmayer DC, Jones RC, Portoghese (2002) Kappa antagonist effects of the novel kappa antagonist 5’-guanidinonaltrindole (GNTI) in an assay of schedule controlled behavior. Psychopharmacology 163:412–419 Observateur francais des drogues et des toxicomanies (2002) Premier identification du principle actif de la salvia divinorum dans SINTES. Internet website: www.drogues.gouv fr/fr/professionels/info_rapides_trend/info19_07_2002.html Ortega A, Blount JF, Marchand P (1982) Salvinorin, a new transneoclerodane diterpene from Salvia divinorum (Labiatae). J Chem Soc Perkins Transact 1:2505–2508 Pfeiffer A, Brantl V, Herz A, Emrich HM (1986) Psychotomimesis mediated by kappa opiate receptors. Science 233:774–776

Remmers AE, Clark MJ, Mansour A, Akil H, Woods JH, Medzihradsky F (1999) Opioid efficacy in a C6 glioma cell line stably expressing the human kappa opioid receptor. J Pharmacol Exp Ther 288:827–833 Roth BL, Baner K, Westkaemper R, Siebert D, Rice KC, Steinberg S, Ernsberger P, Rothman RB (2002) Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist . Proc Natl Acad Sci USA 99:11934–11939 Sheffler DJ, Roth BL (2003) Salvinorin A: the “magic mint” hallucinogen finds a molecular target in the kappa opioid receptor. Trends Pharmacol Sci 24:107–109 Siebert DJ (1994) Salvia divinorum and salvinorin A: new pharmacologic findings. J Ethnopharmacol 43:53–56 Ur E, Wright DM, Bouloux PM, Grossman A (1997) The effects of spiradoline (U-62066E), a kappa-opioid receptor agonist, on neuroendocrine function in man. Br J Pharmacol 120:781–784 Valdes LJ (1994) Salvia divinorum and the unique diterpene hallucinogen, salvinorin (divinorin) A. J Psychoact Drugs 26:277–283 Valdes LJ, Diaz JL, Paul AG (1993) Ethnopharmacology of ska Maria Pastora (Salvia divinorum, Epling and Jativa-M.). J Ethnopharmacol 7:287–312 Walsh SL, Strain EC, Abreu ME, Bigelow GE (2001) Enadoline, a selective kappa opioid agonist: comparison with butorphanol and hydromorphone in humans. Psychopharmacology 157:151– 162

0022-3565/04/3083-1197–1203$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics JPET 308:1197–1203, 2004

Vol. 308, No. 3 59394/1130181 Printed in U.S.A.

Salvinorin A, an Active Component of the Hallucinogenic Sage Salvia divinorum Is a Highly Efficacious ␬-Opioid Receptor Agonist: Structural and Functional Considerations Charles Chavkin, Sumit Sud, Wenzhen Jin, Jeremy Stewart, Jordan K. Zjawiony, Daniel J. Siebert, Beth Ann Toth, Sandra J. Hufeisen, and Bryan L. Roth

Received August 31, 2003; accepted November 7, 2003

ABSTRACT The diterpene salvinorin A from Salvia divinorum has recently been reported to be a high-affinity and selective ␬-opioid receptor agonist (Roth et al., 2002). Salvinorin A and selected derivatives were found to be potent and efficacious agonists in several measures of agonist activity using cloned human ␬-opioid receptors expressed in human embryonic kidney-293 cells. Thus, salvinorin A, salvinorinyl-2-propionate, and salvinorinyl2-heptanoate were found to be either full (salvinorin A) or partial (2-propionate, 2-heptanoate) agonists for inhibition of forskolinstimulated cAMP production. Additional studies of agonist potency and efficacy of salvinorin A, performed by cotransfecting either the chimeric G proteins Gaq-i5 or the universal G protein Ga16 and quantification of agonist-evoked intracellular calcium mobilization, affirmed that salvinorin A was a potent and effective ␬-opioid agonist. Results from structure-function studies suggested that the nature of the substituent at the 2-position of

Salvia divinorum, a member of the Lamiaceae family, has been used by the Mazatec Indians of northeastern Oaxaca, Mexico, primarily for its psychoactive effects (Wasson, 1962, 1963) for many hundreds of years (for reviews, see Valdes et al., 1983; Sheffler and Roth, 2003). The active ingredient of S. divinorum is salvinorin A, a non-nitrogenous neoclerodane diterpene that represents the most potent naturally occurring hallucinogen known (Valdes et al., 1984; Siebert, 1994).

The work was supported by U.S. Public Health Service Grant RO1 DA04123 from National Institute on Drug Abuse (to C.C.) and by the National Institute of Mental Health Psychoactive Drug Screening Program and KO2MH01366 and RO1DA017204 (to B.L.R.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.059394.

salvinorin A was critical for ␬-opioid receptor binding and activation. Because issues of receptor reserve complicate estimates of agonist efficacy and potency, we also examined the agonist actions of salvinorin A by measuring potassium conductance through G protein-gated K⫹ channels coexpressed in Xenopus oocytes, a system in which receptor reserve is minimal. Salvinorin A was found to be a full agonist, being significantly more efficacious than (trans)-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulfonate hydrate (U50488) or (trans)-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulfonate hydrate (U69593) (two standard ␬-opioid agonists) and similar in efficacy to dynorphin A (the naturally occurring peptide ligand for ␬-opioid receptors). Salvinorin A thus represents the first known naturally occurring non-nitrogenous full agonist at ␬-opioid receptors.

Salvinorin A induces an intense, short-lived hallucinogenic experience qualitatively distinct from that induced by the classical hallucinogens lysergic acid diethylamide, psilocybin, and mescaline (Siebert, 1994). Both S. divinorum and salvinorin A have been used recreationally for their hallucinogenic properties (Giroud et al., 2000). Intriguingly, an anecdotal case report has suggested that S. divinorum may have antidepressant properties as well (Hanes, 2001). Quite recently, we discovered that salvinorin A has high affinity and selectivity for the cloned ␬-opioid receptor (KOR) and suggested, based on limited functional studies, that salvinorin A was a KOR agonist (Roth et al., 2002). We now present a detailed report on the agonist properties of salvinorin A and selected derivatives. We discovered that salvinorin A is an extraordinarily efficacious and potent ␬-opioid

ABBREVIATIONS: KOR, ␬-opioid receptor; hKOR, human ␬-opioid receptor; nor-BNI, nor-binaltorphimine; U50488, (trans)-3,4-dichloro-Nmethyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate; U69593, (⫹)-(5␣,7␣,8␤)-N-methyl-N-[7-(1-pyrrolidinyl)-1oxaspiro[4.5]dec-8-yl]-benzeneacetamide. 1197

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Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C., S.S., W.J.); Salvia divinorum Research and Information Center, Los Angeles, California (D.S.); Department of Pharmacognosy, University of Mississippi, Oxford, Mississippi (J.S., J.K.Z.); and National Institute of Mental Health Psychoactive Drug Screening Program and Department of Biochemistry, Case Western Reserve University Medical School, Cleveland, Ohio (B.A.T., S.J.H., B.L.R.)

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agonist. We also found, based on structure-function studies, that the nature of the substituent on the 2-position of salvinorin profoundly affects functional activity. Together, these results support the hypothesis that the unique effects of salvinorin A on human perception are due to selective activation of KOR.

Materials and Methods

TABLE 1 Calculated molecular weights were obtained using ChemDraw software Yields and Masses of Salvinorinyl Esters

1) 2) 3) 4) 5) 6) 7) 8) 9)

Propionate Heptanoate Pivalate p-Bromobenzoate 2,2,2-Trichloroethylcarbonate Ethylcarbonate Piperonylate 1-Naphthoate Cyclopropanecarboxylate

9.0 mg, 78.5% 10.5 mg, 81.6% 11.1 mg, 91.4% 12.4 mg, 84.4% 11.5 mg, 79.4% 9.8 mg, 82.7% 1.6 mg, 11.6% 2.1 mg, 15.1% 10.5 mg, 89.4%

Calculated

Found(M ⫹ 23)for

446.1941 502.2567 474.2254 572.1046 564.0721 462.1890 538.1839 544.2097 458.1941

469.1917 525.2566 497.2215 595.1009 587.0689 485.1833 561.1834 567.2087 481.1952

sodium

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Materials. U50488, U69593, dynorphin A, norbinaltorphimine (nor-BNI) were obtained from Sigma-Aldrich (St. Louis, MO). [3H]Bremazocine was from PerkinElmer Life Sciences (Boston, MA). Complementary DNA Clones and cRNA Synthesis for Oocyte Studies. The rat KOR was obtained from Dr. David Grandy (GenBank accession no. D16829). The human KOR cDNA was obtained from the Guthrie Research Foundation (GenBank accession no. NM000912) and subcloned into the eukaryotic expression vector pIRESNEO (Invitrogen, Carlsbad, CA); cDNAs for KIR3.1 (accession no. U01071) and KIR3.2 (accession no. U11859) were obtained from Drs. Cesar Lebarca and Henry Lester, respectively. The chimeric G protein Gq-i5 was obtained from Bruce Conklin (University of California, San Francisco), whereas G␣16 was obtained from the Guthrie Research Foundation; both constructs were verified by automated dsDNA sequencing (Cleveland Genomics, Inc., Cleveland, OH) before use. Plasmid templates for all constructs were linearized before cRNA synthesis, and the mMESSAGE MACHINE kit (Ambion, Austin, TX) was used to generate capped cRNA. Cell Lines and Maintenance. A stable line expressing the human KOR (hKOR-293) was obtained by transfecting an hKOR expression vector (hKOR-pIRESNEO) into human embryonic kidney293 cells (maintained and transfected as previously detailed; Roth et al., 2002) and selecting in 600 ␮g/ml G418. Surviving clones were expanded and characterized with one (hKOR-293) that expressed high levels of hKOR (ca. 1 pmol/mg) used for further studies. Oocyte Maintenance and Injection. Healthy stage V and VI oocytes were harvested from mature anesthetized Xenopus laevis (Nasco, Ft. Atkinson, WI) and defolliculated enzymatically as described previously (Snutch, 1988). The oocytes were maintained at 18°C in standard oocyte buffer, ND96 (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.5), supplemented with 2.5 mM sodium pyruvate and 50 ␮g/ml gentamicin (SigmaAldrich). One day after harvest, cRNAs were injected (50 nl/oocyte) with a Drummond microinjector. Each oocyte was injected with 0.5 ng of KOR cRNA and 0.1 ng of KIR3.1 and KIR3.2 cRNA. Recordings were made at least 48 h after injection. Electrophysiological Studies. An Axon Geneclamp 500 amplifier was used for standard two-electrode voltage-clamp experiments. The FETCHEX program (Axon Instruments, Foster City, CA) and recorded data traces were used for data acquisition and analysis. Oocytes were then removed from incubation medium, placed in the recording chamber containing ND96 medium, and clamped at – 80 mV. Recordings were made in hK buffer (72.5 mM NaCl, 24 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.5). To facilitate the recording of inward K⫹ currents through the KIR3 channels, the

normal oocyte saline buffer was modified to increase the KCl concentration to 24 mM K⫹. Microelectrodes were filled with 3 M KCl and had resistances of 0.4 to 2.0 M⍀. Radioligand Binding and Functional Studies. Radioligand binding studies were performed as described previously (Roth et al., 2002) with the exception that 150 mM NaCl was added to the standard binding buffer to mimic physiological sodium concentrations. In brief, membranes (10 –50 ␮g) were incubated together with [3H]bremazocine in a final volume of 0.5 ml with a buffer of the following composition: 50 mM Tris-HCl, 150 mM NaCl, pH 7.40 along with test agents for 90 min at room temperature. Incubations were terminated by rapid filtration and collection on GF/C glass fiber filters and washing with ice-cold binding buffer. Dried filters were put into sample vials, scintillation fluid was added, and dpm were measured by liquid scintillation spectroscopy. Measurements of the ability of KOR agonists to inhibit forksolin-stimulated adenylate cyclase activity were performed as detailed previously (Roth et al., 2002). For studies involving measurements of intracellular calcium mobilization, a Molecular Devices FLEXSTATION was used as recently detailed (Rothman et al., 2003). For these studies, hKOR were cotransfected with the chimeric G protein Gaq-i5 (Conklin et al., 1993) or the “universal” G protein Ga16 (Offermanns and Simon, 1995). Measurements of intracellular calcium mobilization and quantification of agonist efficacy and potency were performed as described in Rothman et al. (2003). Data Analysis. EC50 values and curve fitting were determined using Nfit software (Island Products, Galveston, TX) or GraphPad Prism (GraphPad Software, Inc., San Diego, CA). Student’s t test was used for comparison of independent means, with values reported as two-tailed p values. Chemistry. Salvinorin A was isolated from dried leaves of S. divinorum by the method reported previously (Valdes et al., 1984). Salvinorin A was hydrolyzed using potassium carbonate in methanol to yield salvinorin B. The reported esters were formed using salvinorin B, dimethylaminopyridine, and the corresponding acid chloride in methylene chloride. Salvinorin B was characterized by 1H NMR, 13C NMR, and highresolution mass spectrometry (HRMS) and found to be authentic by comparison with literature values (Valdes et al., 1984). The reported esters were purified by high-performance liquid chromatography and characterized by HRMS. 1H and 13C NMR spectra were recorded on a Bruker AMX 500 MHz NMR spectrometer in CDCl3. The HRMS were measured using a Bioapex FT mass spectrometer with electrospray ionization. High-performance liquid chromatography was conducted on a Waters Deltaprep 4000 system using a Waters Xterra RP18, 5 ␮m, 4.6 ⫻ 150-mm column, with mobile phase H2O/acetonitrile (1:1). Thin layer chromatography analyses were carried out on precoated Si gel G254, 250-␮m plates, with the developing system hexane/ethyl acetate (2:1) and visualized with vanillin/H2SO4 in ethanol. Preparation of Esters. Salvinorin B (10 mg, 26 nmol) and 4-dimethylaminopyridine (catalytic amount) were dissolved in methylene chloride (3 ml). The corresponding acid chloride (130 nmol) was added, and the reaction stirred at room temperature overnight. The

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Fig. 1. Structures of salvinorin A, B, and 2-salvinorinyl esters. Shown are the structures of the compounds used in this study.

mixture was quenched with methanol, loaded onto silica, and purified by vacuum liquid chromatography using Si gel (230 – 400-mesh) with hexane/ethyl acetate (3:1) solvent system. Calculated molecular weights were obtained using ChemDraw software (Table 1).

Results In initial studies, we examined the abilities of salvinorin A and selected derivatives (see Fig. 1 for structures) for their ability to bind to hKORs. As can be seen, the synthetic derivatives of salvinorin A differ solely in the nature of the substituent in the 2-position. As is shown in Table 2, salvinorinyl-2-propionate was the only derivative with submicromolar affinity for hKORs; also of note is that salvinorin B was inactive at hKORs. A screen of a number of other receptor subtypes showed that the salvinorin A derivatives tested had

no significant activity at other receptors, including various serotonergic, dopaminergic, muscarinic, adrenergic, cannabinoid, and ␴ receptors (see Table 2 for details) We next evaluated the ability of salvinorin A and the propionate and heptanoate derivatives to activate hKORs by measuring the ability to inhibit forskolin-stimulated cAMP production using U69593 as the comparator. As shown in Table 2, salvinorin A and salvinorinyl-2-propionate were potent and full agonists compared with U69593, whereas salvinorinyl-2-heptanoate was a partial agonist. We also evaluated the ability of U69593, dynorphin A, salvinorin A, and the propionate derivative of salvinorin A to activate hKORs using a fluorescent-microplate-reader (FLEXSTATION) wherein hKORs were cotransfected with either the chimeric G protein Gqi5 or the universal G protein

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TABLE 2 Effect of salvinorin A derivatives on KOR binding and inhibition of forskolin-stimulated adenylate cyclase in KOR-293 cells Shown are the mean values ⫾ S.D. from n ⫽ 2– 4 separate experiments in which Ki values for inhibition of [3H]bremazocine binding and EC50 and Emax values for inhibition of adenylate cyclase in KOR-293 cells were performed as detailed under Materials and Methods with the response induced by U69593 defined as 100%. The salvinorin A derivatives listed above were also screened at a large number of cloned receptors and found to have no significant activity, when tested at 10 ␮M at the following receptors: serotonin (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5a, 5-HT6, 5-HT7), dopamine (D1, D2, D3, D4, D5), muscarinic (m1, m2, m3, m4, m5), ␮, ␦, and ORL-1 opioid receptors, ␴1, ␴2, ␣1adrenergic (1a, 1b, 1d), ␣2-adrenergic (2A, 2B, 2C) ␤2-adrenergic, and CB-1 cannabinoid receptors [assayed as previously detailed (Shi et al., 2003)]. EC50 in nM (pEC50 ⫾ S.E.M.)

Emax

18.74 ⫾ 3.38 32.63 ⫾ 15.7 3199 ⫾ 961.2 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000

0.63 (⫺0.2 ⫾ 0.07) 4.7 (0.7 ⫾ 0.3) 40 (1.6 ⫾ 0.4) NA NA NA NA NA NA NA NA

100 100 34 ⫾ 11 NA NA NA NA NA NA NA NA

NA, not active at 10,000 nM.

Ga16 as detailed previously (Rothman et al., 2003). Figure 2 shows representative results for U69593 and salvinorin A using either G␣16 (A and B) or Gq-i5 (C and D). No responses were seen in untransfected cells or in cells transfected with hKOR alone (data not shown). Figure 2 also shows a representative dose-response study using Gq-i5 as the chimeric G protein. Because both methods seemed to yield equivalent results, further studies were performed with Gq-i5. Table 3 shows representative EC50 and Emax values for a variety of KOR agonists using Gq-i5. In these studies, salvinorin A was more potent than any other of the tested KOR agonists (Table 3). In terms of maximal response, all of the active compounds gave similar responses. It is well known that overexpression systems tend to provide inaccurate estimates of agonist potencies and efficacies because of issues of receptor reserve (Kenakin, 2002). As well, it has been well described that unnatural expression systems wherein chimeric or “universal” G proteins are used also lead to misleading estimates of agonist potencies and maximal responses (Woolf et al., 2001; Kenakin, 2002). Accordingly, we next determined the maximal agonist responses (Emax) and potencies (EC50 values) of selected compounds using a system without receptor reserve. Salvinorin A Is a Full agonist. For these studies, Xenopus oocytes were coinjected with inwardly rectifying K⫹ channels and KORs. In the experiment shown, a representative oocyte voltage clamped at – 80 mV was first perfused with hK buffer (containing 24 mM KCl) to shift the reversal potential of potassium and facilitate K⫹ current through Kir3 (Fig. 3). Perfusion with 1 ␮M salvinorin A significantly increased the inward current, and the activation was reversed by 100 nM nor-BNI. Similarly, 1 ␮M U69593 increased the inward current in a different oocyte, and the effect was also blocked by 100 nM nor-BNI (Fig. 1B). Neither 10 ␮M salvinorin A nor U69593 increased the membrane conductance of oocytes expressing Kir3 without KOR (data not shown). Concentration-response curves of salvinorin-A and ␬-agonists U69593 and U50488 were compared (Fig. 4). Each point

Discussion The principal finding of this study is that salvinorin A is an extraordinarily potent full agonist at hKORs. Additionally, we report that salvinorinyl-2-propionate is a potent partial agonist at KORs and also demonstrate that the nature of the 2-substituent of the salvinorin scaffold is critically important for agonist efficacy and potency. We also have obtained data with KOR-knockout and wild-type mice that the actions of salvinorin A are mediated by KOR in vivo (J. Pintar, personal communication). Together, these results imply that the profound effects of salvinorin A on human consciousness are mediated by potent and highly efficacious activation of KORs. In prior reports, we have suggested that because salvinorin A is a potent hallucinogen that is apparently selective for KORs, and that targeting KORs might lead to novel medications for the treatment of diseases manifested by hallucinatory experiences (e.g., schizophrenia, affective disorders, and dementia) (Roth et al., 2002; Sheffler and Roth, 2003). In this regard, studies with nonselective opioid antagonists that possess KOR actions in schizophrenia have been mixed (Rapaport et al., 1993; Sernyak et al., 1998), although there are no studies in which selective KOR antagonists have been tested. Because of anecdotal reports that extracts of S. divinorum may possess antidepressant actions (Hanes, 2001), and published studies in rodents that KOR antagonists block stressinduced responses (McLaughlin et al., 2003), KOR antagonists could possess antianxiety/antidepressant actions as well. Indeed, a recent study (Mague et al., 2003) suggested that ␬-selective antagonists might have intrinsic antidepressant actions. Our current studies suggest that novel KORselective agents might be obtained by selective modification of the salvinorin scaffold. Whether such agents might possess antidepressant or antipsychotic activity is unknown. As shown in these studies, salvinorin A and salvinorinyl2-propionate are potent agonists at KORs with salvinorin A being a full agonist in most assay systems, whereas salvinorinyl-2-propionate is likely a partial agonist. Salvinorin B and all other tested salvinorin derivatives were devoid of

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Salvinorin A Propionate Heptanoate Privalate p-Bromobenzoate 2,2,2-Triethylcarbonate Ethylcarbonate Piperonylate 1-Napthoate Cyclopropanecarboxylate Salvinorin B

Ki ⫾ S.E.M. (nM)

represents the mean response measured in four to seven different oocytes. Data were collected from multiple batches of oocytes and merged by normalizing the responses to the average maximal response produced by salvinorin A on that recording day. Based on these results, salvinorin A was not significantly more potent (EC50 ⫽ 69 nM; confidence intervals 50 –94 nM) than U69593 (EC50 ⫽ 224 nM; confidence intervals 51–157 nM) or U50488 (EC50 150 nM; confidence intervals 50 –194 nM). Under these expression conditions, there was an apparent lack of spare ␬-receptors. Increasing the ␬-receptor cRNA from 0.5 ng/oocte to 1.0 ng increased the average U69593 response from 1.63 ⫾ 0.57 to 2.76 ⫾ 1.04 ␮A (n ⫽ 7 or 8). Based on the lack of spare receptors, we directly compared the maximal responses evoked by 10 ␮M each of the ␬-agonists (Fig. 5) with that of dynorphin A. In this assay, propionyl-salvinorin also acted as a partial agonist whose maximal activity was less than salvinorin A. The response to salvinorin A was significantly greater than that to U69593 and U50488 (p ⬍ 0.05), but not significantly greater to that of dynorphin A.

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Downloaded from jpet.aspetjournals.org at ASPET Journals on October 23, 2014 Fig. 2. Salvinorin A mobilizes intracellular Ca2⫹ when hKORs are cotransfected with the universal G protein G16 or the chimeric G protein. For these studies human embryonic kidney-293 cells were transfected with hKOR and either Gqi5 or G16 and the mobilization of intracellular calcium quantified as described previously (Rothman et al., 2004) using a 96-well FLEXSTATION. A and B, representative results with increasing doses of U69593 or salvinorin A (0, 10, and 100 nM), whereas hKORs were cotransfected with G16. C and D, results obtained when hKORs were cotransfected with Gq-i5. E, average for n ⫽ 3 separate experiments for dose-response studies to salvinorin A and U69593.

significant activity. One potential complication of the studies performed on recombinant, overexpressed receptors relates to the issue of receptor reserve. Thus, it is widely appreciated that overexpressing G proteins and/or receptors in heterologous expression systems leads to inaccurate estimates of agonist potencies and maximal responses (for review, see Kenakin, 1997). Accordingly, we also evaluated the agonist

actions of salvinorin A and other compounds at KORs expressed in Xenopus oocytes. KOR expressed in Xenopus oocytes activate intrinsic G proteins that then increase the conductance of coexpressed G protein-coupled inwardly rectifying potassium channels (GIRK and Kir3) (Henry et al., 1995). Injection of cRNAs coding for the mammalian receptor and channel has been

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TABLE 3 Salvinorin A and salvinorinyl-2-propionate are agonist at hKORstimulated intracellular Ca2⫹ mobilization: comparison with reference compounds Data represent mean ⫾ S.D. of quadruplicate determinations for EC50 and Emax for mobilization of intracellular calcium. Drug

EC50 in nM (pEC50 ⫾ SD)

Emax (Relative to U69593)

U69593 U50488 Salvinorin A Dynorphin A Salvinorinyl-2propionate Salvinorin B

13 (1.14 ⫾ 0.2) 24 (1.39 ⫾ 0.14) 7 (0.84 ⫾ 0.07) 83 (1.92 ⫾ 0.17) 17.3 (1.23 ⫾ 0.18)

100 102 ⫾ 4 104 ⫾ 7 107 ⫾ 8 102 ⫾ 8

No activity

No activity

Fig. 4. Concentration-response curve of salvinorin A, and ␬-agonists U69593 and U50488. Cumulatively higher concentrations of salvinorin A and the ␬-agonists were applied to the bath. The agonist response at each concentration was normalized as a percentage of the maximal salvinorin A response. Each point represents the mean response measured in four to seven different oocytes.

length must not be the only factor, because the short-chain ethylcarbonate derivative is absent of activity. The current results support the conclusion that just as morphine is a natural plant product able to activate the ␮-opioid receptor, salvinorin A is a natural plant product able to activate the KOR. The strongly psychotomimetic actions of salvinorin A suggest that the dynorphin/␬-opioid system may have a role in the regulation of cognition and perception and support earlier proposals that some forms of schizophrenic hallucinations may be caused by hyperactive endogenous opioid systems (Gunne et al., 1977). Recent data implicating the KOR-dynorphinergic system in modulating stress and anxiety responses in rodents suggest that targeting KORs might also lead to novel antidepressant and anxiolytic medications. Salvinorin A, by virtue of its potency, efficacy, and selectivity as a KOR agonist will be an important tool for discovering the role that the KOR-dynorphinergic system has in health and disease.

Fig. 3. Salvinorin A is a highly efficacious ␬-receptor agonist. Representative traces showing the change in current during a typical experiment. A large inward current was apparent as the K⫹ concentration was increased from 2 to 24 mM in normal oocyte saline buffer. Salvinorin A (1 ␮M) and U69593 (1 ␮M) in the buffer (24 mM K⫹) further increased Kir3 currents, and the response was reversed by nor-BNI (100 nM), a ␬-antagonist.

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demonstrated to faithfully reconstitute opioid signaling in oocytes equivalent to that observed in guinea pig substantia gelatinosa neurons (Grudt and Williams, 1993). In addition, by controlling the levels of receptor and channel expression, spare receptors can be avoided and the peak responses produced by different drugs can be a direct measure of agonist efficacy. The in vitro bioassay also eliminates pharmacokinetic barriers, and the electrophysiological recording of channel activation provides a rapid measure of receptor activation. In this study, we compared the relative activity of salvinorin A with three compounds having established ␬-opioid receptor agonist activity. Salvinorin A was found to be more potent and have higher efficacy than either U50488 and U69593. The agonist efficacy of salvinorin A was not significantly different from dynorphin A(1-17), an endogenous neurotransmitter of the ␬-opioid receptor (Chavkin et al., 1982). Structure-activity relationship studies show that the KOR agonistic activity of salvinorin derivatives depend largely on the size and character of the substituent on the 2-ester moiety. Generally, the studied derivatives have either lower affinity for KOR than salvinorin A or are completely devoid of activity. The two active derivatives, the propionate and the heptanoate, demonstrate that as the alkyl chain is lengthened, KOR affinity diminishes. Interestingly however, chain

Salvinorin A Activates ␬-Opioid Receptors

References Chavkin C, James IF, and Goldstein A (1982) Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science (Wash DC) 215:413– 415. Conklin BR, Farfel Z, Lustig KD, Julius D, and Bourne HR (1993) Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature (Lond) 363:274 –276. Giroud C, Felber F, Augsburger M, Horisberger B, Rivier L, and Mangin P (2000) Salvia divinorum: an hallucinogenic mint which might become a new recreational drug in Switzerland. Forensic Sci Int 112:143–150. Grudt TJ and Williams JT (1993) kappa-Opioid receptors also increase potassium conductance. Proc Natl Acad Sci USA 90:11429 –11432. Gunne LM, Lindstrom L, and Terenius L (1977) Naloxone-induced reversal of schizophrenic hallucinations. J Neural Transm 40:13–19. Hanes KR (2001) Antidepressant effects of the herb Salvia divinorum: a case report. J Clin Psychopharmacol 21:634 – 635. Henry DJ, Grandy DK, Lester HA, Davidson N, and Chavkin C (1995) Kappa opioid

receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes. Mol Pharmacol 47:551–557. Kenakin T (1997) Differences between natural and recombinant G protein-coupled receptor systems with varying receptor/G protein stoichiometry. Trends Pharmacol Sci 18:456 – 464. Kenakin T (2002) Efficacy at G-protein-coupled receptors. Nat Rev Drug Discov 1:103–110. Mague SD, Pliakas AM, Todtenkopf MS, Tomasiewicz HC, Zhang Y, Stevens WC Jr, Jones RM, Portoghese PS, and Carlezon WA Jr (2003) Antidepressant-like effects of kappa-opioid receptor antagonists in the forced swim test in rats. J Pharmacol Exp Ther 305:323–330. McLaughlin JP, Marton-Popovici M, and Chavkin C (2003) Kappa opioid receptor antagonism and prodynorphin gene disruption block stress-induced behavioral responses. J Neurosci 23:5674 –5683. Offermanns S and Simon MI (1995) G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J Biol Chem 270:15175–15180. Rapaport MH, Wolkowitz O, Kelsoe JR, Pato C, Konicki PE, and Pickar D (1993) Beneficial effects of nalmefene augmentation in neuroleptic-stabilized schizophrenic patients. Neuropsychopharmacology 9:111–115. Roth BL, Baner K, Westkaemper R, Siebert D, Rice KC, Steinberg S, Ernsberger P, and Rothman RB (2002) Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist. Proc Natl Acad Sci USA 99:11934 –11939. Rothman RB, Vu N, Partilla JS, Roth BL, Hufeisen SJ, Compton-Toth BA, Birkes J, Young R, and Glennon RA (2003) Related in vitro characterization of ephedrinerelated stereoisomers at biogenic amine transporters and the receptorome reveals selective actions as norepinephrine transporter substrates. J Pharmacol Exp Ther 307:138 –145. Sernyak MJ, Glazer WM, Heninger GR, Charney DS, Woods SW, Petrakis IL, Krystal JH, and Price LH (1998) Naltrexone augmentation of neuroleptics in schizophrenia. J Clin Psychopharmacol 18:248 –251. Sheffler DJ and Roth BL (2003) Salvinorin A: the “magic mint” hallucinogen finds a molecular target in the kappa opioid receptor. Trends Pharmacol Sci 24:107–109. Shi Q, Savage JE, Hufeisen SJ, Rauser L, Grajkowska E, Ernsberger P, Wroblewski JT, Nadeau JH, and Roth BL (2003) L-Homocysteine sulfinic acid and other acidic homocysteine derivatives are potent and selective metabotropic glutamate receptor agonists. J Pharmacol Exp Ther 305:131–142. Siebert DJ (1994) Salvia divinorum and salvinorin A: new pharmacologic findings. J Ethnopharmacol 43:53–56. Snutch TP (1988) The use of Xenopus oocytes to probe synaptic communication. Trends Neurosci 11:250 –256. Valdes LJ, Butler WM, Hatfield GM, Paul AG, and Koreeda M (1984) Divinorin A: a psychotropic terpenoid and divnorin B from the hallucinogenic Mexican mint Salvia divinorum. J Org Chem 49:4716 – 4720. Valdes LJ 3rd, Diaz JL, and Paul AG (1983) Ethnopharmacology of ska Maria Pastora (Salvia divinorum, Epling and Jativa-M.). J Ethnopharmacol 7:287–312. Wasson RG (1962) A new Mexican psychotropic drug from the mint family. Bot Mus Leaflets Harvard Univ 20:77– 84. Wasson RG (1963) Notes on the present status of ololuiqui and the other hallucinogens of Mexico. Bot Mus Leaflets Harvard Univ 20:163–193. Woolf PJ, Kenakin TP, and Linderman JJ (2001) Uncovering biases in high throughput screens of G-protein coupled receptors. J Theor Biol 208:403– 418.

Address correspondence to: Dr. Bryan Roth, Department of Biochemistry; Room RT500-9, Case Western Reserve University Medical School, 2109 Adelbert Rd., Cleveland, OH 44106. E-mail: [email protected]

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Fig. 5. Salvinorin A is more efficacious than U69593 and U50488 in ␬-receptor-mediated activation of Kir3 currents. At saturating concentration, salvinorin A (10 ␮M) evoked a large Kir3 currents, which were significantly higher than the response evoked by U69593 (10 ␮M) or U50488 (10 ␮M). Data are mean ⫾ S.E.M.; ⴱⴱ, p ⬍ 0.05. Dynorphin A (10 ␮M) produced a response that was not significantly different from salvinorin A.

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R. Bücheler1,2 C. H. Gleiter1 P. Schwoerer2 I. Gaertner3

Use of Nonprohibited Hallucinogenic Plants: Increasing Relevance for Public health? A Case Report and Literature Review on the Consumption of Salvia divinorum (Diviner's Sage)

ing or chewing, induces a short-lived inebriant state with intense, bizarre feelings of depersonalization. This article wants to be a signal for physicians or psychotherapists to take Salvia into consideration, when exploring young people for drug use. Methods: We report the individual perceptions of a young man consuming Salvia divinorum. We review the scarce scientific literature and consider relevant internet websites. Discussion: We define open issues for further investigations and try to discuss why Salvia divinorum may be of interest for teenagers and young adults in Europe.

Introduction

the state of Oaxaca, Mexico, in healing and divination ceremonies. In 1962, however, it was characterized botanically for the first time by Epling and Jµtiva [13]. For ritual purposes, five up to 80 pairs of fresh leaves are chewed or crushed and prepared as a bitter tasting, foamy infusion [43]. To date, six different ingredients (salvinorin A±F) have been isolated from its leaves [27]. In 1982 the main psychoactive compound, salvinorin A (Divinorin A), was identified by two independent research groups [29, 45]. Salvinorin A, which is not water-soluble, is only absorbed by the respiratory and, to a lesser extent, by the oral mucosa. Dried leaves of Salvia divinorum are smoked as a joint, consumed in water pipes or vaporized and inhaled. About 1.5 g of pure salvinorin A can be extracted from one kilogram of air-dried leaves, gained from about 8 kg of fresh leaves [37, 43]. According to biochemical reports, it is easier to obtain salvinorin A than to extract

In 2002 the number of offences, especially among children and adolescents involving the possession and purchase of cannabis, rose by more than 6 % to 139 082/year in Germany [3]. Similar observations are documented from abroad [7, 9]. These figures may reflect a changing attitude of young people towards drug use in general. Intoxication by nonprohibited drugs of herbal origin like Datura (Datura Stramonium) or Angel's Trumpet (Brugmansia suaveolens or sanguinea) plays an increasing role in emergency medicine [10,18, 28]. We want to point out a newcomer among drugs of herbal origin: A Mexican mint, a sage plant, called ªSalvia divinorumº. For many centuries, it has been used by shamans of the Mazatec Indians in

Affiliation Abteilung Klinische Pharmakologie, Universitätsklinikum Tübingen, Otfried-Müller Strasse 45, 72076 Tübingen, Germany 2 Medizinischer Dienst der Krankenversicherung (MDK) Baden-Württemberg, 77933 Lahr, Germany 3 Abteilung Allgemeine Psychiatrie und Psychotherapie mit Poliklinik, Universitätsklinikum Tübingen, Osianderstraûe 24, 72076 Tübingen, Germany 1

Correspondence Prof. Dr. med. Christoph H. Gleiter ´ Abteilung Klinische Pharmakologie ´ Universitätsklinikum Tübingen ´ Otfried-Müller-Strasse 45 ´ 72076 Tübingen ´ Germany ´ Fax: 07071 29 5035 ´ E-Mail: [email protected] Received 1.10.2003 ´ Revised 11.3.2004 ´ Accepted 17.3.2004 Bibliography Pharmacopsychiatry 2005; 38: 1±5 ´  Georg Thieme Verlag KG Stuttgart ´ New York DOI 10.1055/s-2005-837763 ISSN 0176-3679

Original Paper

Introduction: We want to call attention to a mint plant, called diviner's sage (Salvia divinorum), originally used in shamanic ceremonies of the Mazatec Indians of Mexico. On numerous websites of the internet, this ancient herbal drug and its extracts are offered as a legal means of widening individual awareness. Regarding its dose-response relationship, the active ingredient, salvinorin A, is one of the most potent naturally occurring hallucinogens. Laws on controlled substances, except for Finland, Denmark and Australia, do not prohibit cultivating, consuming or dealing with Salvia divinorum. Ingestion by smoking, vaporis-

1

lysergic acid diethylamide (LSD) or than to produce phencyclidine derivatives [44]. In minimum doses above 200 to 500 mg, purified salvinorin A has shown intense psychoactive effects [37].

Original Paper

As the content of salvinorin A in one gram of dried leaves may vary from 0.9 to 3.8 mg [19], only 0.1±0.5 g of these leaves are required for a hallucinogenic trip, when inhaled. Fortified plant-extracts however, can also be ordered via the internet. They contain up to 25 mg salvinorin A per gram. On the internet, esoteric websites or ethnobotanical shops openly offer Salvia as a means of improving the air in rooms or as a legal hemp alternative at an affordable price [e. g. 2,16,25,31]. Five grams of dried Salvia leaves cost between 5 to 12.50 E, not including shipping charges. The cultivation of Salvia divinorum has spread from South and North America to Canada and Europe. Recently, the plant was identified in the greenhouses of a Swiss horticulturist [17]. As clinical effects of Salvia divinorum in adolescents have not been described in medical literature, we would now like to recount the psychedelic experiences of a teenager. We have also reviewed available scientific articles as well as trip reports and accessible sites of the internet.

Case report

2

In February 2003, the mother of a 19-year old high school student preparing for his A-levels consulted the Department of Clinical Pharmacology of the University Hospital Tuebingen for information concerning the potential health risks of Salvia divinorum. She had accidentally noticed a dreadful offensive odour coming from her son's room. While smoking the dried leaves, the young man hardly reacted to her approach and seemed to suffer from a reduced awareness. His face had a strange, transcendental and mask-like expression. The young man was a good student. Apart from habitual smoking, no other drug use was reported by the parents who thought him to be ªnormalº regarding his social and academic skills. His IQ was tested as above 140. A standardized psychiatric interview revealed neither personal nor family histories of major psychiatric disorders. The young man reported that he had been chewing or inhaling dried leaves of Salvia divinorum twice a week for about six months, alone or in the company of friends. His most important motivation to consume Salvia is the unique sensation of being disconnected from his own body during the trip. This extracorporal existence in a new ªastral bodyº gives him a very ªgoodº feeling of recreation. He also describes vestibular hallucinations that provide an illusion of hovering above the floor, or penetrating the natural limits of his own room. In these moments, he believes to gain a more mature insight not only into his own personality but also into philosophical or ethical problems. Almost immediately ± he estimates in less than five minutes after inhaling ± the peak of psychotropic effects seems to be reached. During the trip, he experiences somatic sensations like prickling of the skin, fever-like hot flashes, muscular tremor, and a sort of ringing in the ears. All these effects, including the desired feeling of changing his personality and an increased status of self-consciousness, completeBücheler R et al. Use of Nonprohibited ¼ Pharmacopsychiatry 2005; 38: 1 ± 5

ly disappear within 30 minutes. For some hours afterwards, he reports shivering and exhaustion that render him unable to learn or memorize school assignments. The young student attributes this lack of concentration not to a prolonged drug effect but to the need of reflecting the overwhelming perceptions during the trip. He denies having optical or verbal hallucinations, ªbad tripsº nor fits of panic. During the last months, his trips seemed to follow similar patterns as described above. Nevertheless, he reports his impression that the amount of Salvia material, necessary for one trip, will have to be increased gradually in order to maintain the original effective strength. The young man says that he has a good feeling about the safety of Salvia divinorum. He is convinced to be well informed by numerous websites on the internet which do not describe severe short term nor long term health risks such as intoxication or an induction of psychoses.

Discussion Sources of information on Salvia divinorum Reliable, systematic observations on the psychotropic activities of Salvia divinorum or its ingredients in human beings are scarce: Searching databases like MEDLINE or BIOSIS, we found a publication on psychotropic effects following the use of fresh Salvia leaves in six human volunteers, as well as after the application of purified salvinorin A in 20 volunteers [37]. In another paper, two ethnobotanical researchers report their own observations after drinking a Salvia infusion in two different concentrations [43]. On the internet however, ªSalvia divinorumº is linked to numerous websites of ethnobotanical shops, consumers and ªexperienced specialistsº that provide details on botanical cultivation, on dosing and sometimes even publish ªguidelinesº for a safe and satisfiying use of ªthe magic mintº [e. g. 1,16,35,42]. In chatrooms such as [33], users communicate and discuss their experiences during the trips. Amateur researchers even publish the results of their private ªdouble-blindº and ªplacebo-controlledº tests in search of the optimal Salvia dose for meditation [40]. Clinical effects The young boy, who lives about 100 km away from our clinic told us about his trip experiences in a clinically drug-free condition. As Salvia and its ingredients cannot be detected by usual drugscreening methods, we did not perform blood or urine analysis. We excluded the concomitant use of other hallucinogens by interview. The student reported Salvia effects like depersonalization, widening of consciousness, the subjective illusion of rapid movements, flying or hovering, as being comfortable feelings. They seem to outreach negative side effects of Salvia use such as impaired vigilance and coordination. Although horror-trips appear to be rare, on the internet, some consumers delineate frightening attacks of panic, mostly due to the loss of self-control and to the profound experience of losing contact with consensual reality. As this is ªnothing for beginnersº, most of the websites recommend the presence of a sober ªtrip sitterº [14, 35]. He should also protect the Salvia consumer against injury due to somnambulistic activities or to coordination disturbances [38].

Onset and duration of the young man's trips correspond to the data reported on websites and in a scientific publication: After inhaling a bolus of the active ingredient, hallucinogenic feelings are intense but very short-lived. They occur rapidly after 30 seconds and disappear within one hour [38]. Hallucinogenic effects after oral ingestion of salvinorin A begin within 3±5 minutes. These perceptual distortions may return for up to 4 hours, sometimes experienced as ªflashbacksº [43]. Hysterical laughter is observed, but Salvia divinorum is said to have only a weak influence on the prevailing mood of the consumer and rarely changes it [37]. This is an important difference to LSD or hallucinogenic mushrooms.

Doses of salvinorin A needed for hallucinogenic effects, vary from one individual to the other. Different Salvia websites report that about 10 to 15 % of the consumers do not experience any psychotropic effects at all! In doses exceeding 1 mg salvinorin A, out-of body experiences, i. e. advanced ªtrip levelsº, are frequent [37]. On awakening after very high trip levels, the consumer may completely have lost his recollection of having taken any drug [35]. These psychotomimetic effects of Salvia divinorum closely resemble schizophrenia symptoms induced by other distinct classes of drugs: Serotonergic agonists (e. g. LSD) and especially antagonists of the NMDA (N-methyl-D-aspartate) glutamate receptor like phencyclidine (PCP, Angel dust) or ketamine [21, 22]. Indeed, web-reports describe similarities of Salvia associated perceptions with LSD or Ketamine [1, 34], but it is often emphasized, that the depersonalization caused by Salvia has a unique and specific character [37]. In 2002, the active ingedient, salvinorin A, has been shown to be a potent and strong agonist of cerebral kappa-opioid receptors (KOR) [6, 32, 36]. This interaction may cause the reported vegetative reactions to Salvia like sweating, chill and increased diuresis, which may be related to the interaction with KOR. The same effects were shown by synthetic agents stimulating KOR like spiradoline in humans [46]. Salvia induced illusions are intensified by rest and darkness [43]. The afterglow of former Salvia trips as well as the concomitant intake of other psychoactive agents like ethanol, cannabis, LSD or hallucinogenic mushrooms have been mentioned to determine the individual feeling [35]. Pharmacokinetics and pharmacodynamics of the active ingredient, Salvinorin A Salvinorin A, a neoclerodane diterpene, is the only known nonalkaloidal hallucinogen [6]. Beside the fact, that it is not easily absorbed by the gastrointestinal system [37], data on bioavailability, on metabolism or excretion and on interactions with food, drugs or narcotic agents are not published. In terms of its psychoactive effects doses of above 200 mg, salvinorin A rivals in potency with the synthetic hallucinogen LSD acting in doses of 50 ± 250 mg [44].

Due to their reduced affinity to KOR, therapeutically used opioidantagonists like naloxone or naltrexone are not regarded as a very potent antidote for salvinorin A [15, 23, 24]. Pharmacotherapeutic potential KOR mediated neurotropic effects are analgesia, sedation, dysphoria and perceptual distortions [12,15, 30]. Selective stimulation of KOR by salvinorin A may be a pharmacological model to study the promotion of schizophrenia, dementia or bipolar depression. KOR-antagonists like nor-binaltorphimine, have shown antidepressant effects by ameliorating psychomotoric functions in rats [26]. Paradoxically, a case-report of a 26-year old woman documents the complete resolution of a perennial depression since ingesting 0.5 ± 0.75 g of Salvia leaves three times per week sublingually [20]. This is surprising, since the dysphoric side effects of KOR-agonists normally form an obstacle to their use as analgesics, for instance, see [46]. Stimulation or blockade of cerebral KOR may also modulate cardiovascular functions. Experimental investigations in animals show an influence on blood pressure, on the ischemic tolerance of the myocardium and on the induction of cardiac arrhythmias [8, 47, 48]. User population and legal aspects To date, the vast majority of Salvia divinorum consumers are younger adults and adolescents. As ªDiviner's mintº is not a party-drug [35, 43], it appeals to individual experimentalists. On the internet, they define themselves as a kind of community, ingesting the plant or its extracts not to satisfy an addiction, but as a tool for meditative introspection [5, 42]. In international conferences, psychotherapists, artists, ethnobotanists, anthropologists, pharmacologists and consumers discussed, how the plant could serve modern people in daily life to perform meditation or healing rituals [40]. Salvinorin A fails to meet the criteria of chemical similarity to other hallucinogens. Therefore, in most of the countries the plant and its compounds are not banned by national laws for controlled substances. In Austalia however, the possession of Salvia divinorum is illicit [14]. This is officially justified by concerns about its unknown addictive potential and long-term effects. In Europe, only Finland and Denmark have added Salvia to the list of controlled plants. In Norway, Salvia divinorum is not controlled, but has the status of a psychoactive drug. The American Drug EnforceBücheler R et al. Use of Nonprohibited ¼ Pharmacopsychiatry 2005; 38: 1 ± 5

Original Paper

Salvia provides the experience of voyages leading the individual to places of the past, especially from childhood. It may cause vivid illusions of a self-metamorphosis into things like water or animals, culminating in the individual conviction of having definitely abandoned human existence [37].

Pharmacodynamic aspects of salvinorin A and its derivatives have been studied more exactly in the last decade [6, 32, 36]. In human and nonhuman cell cultures, salvinorin A has proven to be a selective, full and very efficacious agonist for KOR [6, 32]. It does not interact with 5-hydroxytryptamine 2A-receptors, like classical hallucinogens such as LSD, psilocybin or mescaline do, and shows no affinity to m- or d-opioid receptors nor did it interact with binding-sites for norepinephrine, dopamine, glutamine and GABA-transporters [32]. Psychotropic effects of salvinorin A appear to be the result of KOR stimulation. This hypothesis is supported by the recently published behavioral effects of salvinorin A in primates [4]. Salvinorin A is the first naturally occurring non-nitrogenous agent and stimulates KOR to the same extent like dynorphin, the endogenous KOR-agonist [6].

3

ment Agency (DEA) has placed Salvia divinorum on a list of drugs or chemicals ªof concernº, without legal implications at present. Consumers have meanwhile founded a ªSalvia Divinorum Defense Fundº in order to prevent more restrictions on Salvia use [5, 33].

Conclusion

Original Paper

Salvia divinorum might become increasingly attractive to adolescents and young adults for several reasons: ± It can be easily ordered at an affordable price. ± The use of Salvia divinorum promises philosophical insights or escapism for young people seeking their own personality. Furthermore, adherence to an international ªSalvia communityº may be socially attractive. ± Numerous internet sources offer a mixture of esoteric advice, practical warnings and instructions on the use of the plant. The consumer may take this subtle promotion of Salvia products as ªevidence-basedº in a scientific sense and underestimate known and unknown health risks.

Open issues

4

As a consequence, the following questions deserve more attention in research: 1. Unidentified, salvinorin-induced intoxications by an unintentional intake of more than 500±1000 mg salvinorin A may be more frequent than presumed, because salvinorin A in blood or urine is not examined by the drug screenings, available at the moment. 2. The influence of Salvia use on social behavior and on daily activities like driving a car or handling technical devices should be observed. 3. Psychotomimetic effects of Salvia divinorum, especially in teenagers and young adults should be documented systematically, e. g. by using a standardized questionnaire to assess altered states of consciousness [11]. 4. Long term effects of Salvinorin A especially in combination with conventional hallucinogens or psychoactive drugs must be watched carefully. They might promote the manifestation of endogenous psychoses in predisposed persons. 5. The addictive potential of Salvia divinorum is still a matter of debate. Stimulated cerebral KOR may develop mechanisms of tolerance that mediate withdrawal behavior [39, 41]. 6. Pharmacokinetics and molecular mechanisms of salvinorin A as well as interactions with ethanol or psychoactive drugs should be investigated. Finally the potential of this naturally occurring KOR-agonist for exploring and alleviating psychiatric conditions, has to be determined.

Acknowledgments Funding: C.H. Gleiter is supported by the BMBF (Deutsches Bundesministerium für Bildung und Forschung), grant 01 EC 0001. I. Gaertner is supported by the AKF-program in therapeutic approaches to opioid dependence (University of Tuebingen 2003). Bücheler R et al. Use of Nonprohibited ¼ Pharmacopsychiatry 2005; 38: 1 ± 5

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Original Paper

Munro TA, Rizzacasa MA. Salvinorins D-F new neoclerdane diterpenoids from Salvia divinorum and an improved method for the isolation of Salvinorin A. J Nat Prod 2003; 66: 703 ± 705 28 Niess C, Schnabel A, Kauert G. Angel trumpet: a poisonous garden plant as a new addictive drug?. Dtsch Med Wochenschr 1999; 124: 1444 ± 1447 29 Ortega A, Blount JF, Machand PS. Salvinorin a new trans-neoclerodane diterpene from Salvia divinorum (Labiatae). J Chem Soc Perkin Trans 1982; 1: 2505 ± 2508 30 Pfeiffer A, Brantl V, Herz A, Emrich HM. Psychotomimesis mediated by kappa opiate receptors. Science 1986; 233: 774 ± 776 31 Pflanzenpfade Online Shop 2004. http://www.pflanzenpfade.de/start2. html (01.03.2004). 32 Roth BL, Baner K, Westkaemper R, Siebert D, Rice KC, Steinberg Set al. Salvinorin A: A potent naturally occurring nonnitrogenous opioid selective agonist. PNAS 2002; 99: 11934 ± 11939 33 Salvia Divinorum Alliance. (2004) http://groups.yahoo.com/group/ SalviaD_Alliance/message/11626 (27.02.2004). 34 Schabner D. A new LSD? Mexican herb for sale online comes with divine claims warnings. ABCNews com 2002 April 1 http://www.maps. org/media/abcsalvia.html (01.03.2004). 35 Schizo 2004. http://www.goatrance.de/goacidia/salvia/de-salvia-faq. html (01.03.2004). 36 Sheffler DJ, Roth BL. Salvinorin A: the ªmagic mintº hallucinogen finds a molecular target in the kappa opioid receptor. Trends Pharmacol Sci 2003; 24: 107 ± 109 37 Siebert DJ. Salvia divinorum and Salvinorin A: new pharmacologic findings. J Ethnopharmacol 1994; 43: 53 ± 56

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Bücheler R et al. Use of Nonprohibited ¼ Pharmacopsychiatry 2005; 38: 1 ± 5

Biochemistry 2005, 44, 8643-8651

8643

Identification of the Molecular Mechanisms by Which the Diterpenoid Salvinorin A Binds to κ-Opioid Receptors† Feng Yan,‡,§ Philip D. Mosier,‡,| Richard B. Westkaemper,| Jeremy Stewart,⊥ Jordan K. Zjawiony,⊥ Timothy A. Vortherms,§ Douglas J. Sheffler,§ and Bryan L. Roth*,§,# Departments of Biochemistry, Psychiatry, and Neurosciences and ComprehensiVe Cancer Center, Case Western ReserVe UniVersity Medical School, CleVeland, Ohio 44106, Department of Medicinal Chemistry, Virginia Commonwealth UniVersity, Richmond, Virginia 23284, and Department of Pharmacognosy, UniVersity of Mississippi, UniVersity, Mississippi 38677 ReceiVed March 16, 2005; ReVised Manuscript ReceiVed May 2, 2005

ABSTRACT: Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant SalVia diVinorum that selectively and potently activates κ-opioid receptors (KORs). Salvinorin A is unique in that it is the only known lipid-like molecule that selectively and potently activates a G-protein coupled receptor (GPCR), which has as its endogenous agonist a peptide; salvinorin A is also the only known non-nitrogenous opioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high binding affinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilized in the binding pocket by interactions with tyrosine residues in helix 7 (Tyr313 and Tyr320) and helix 2 (Tyr119). Intriguingly, activation of KORs by salvinorin A required interactions with the helix 7 tyrosines Tyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KOR agonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differential requirements for these three residues for binding and activation. We also employed a novel approach, whereby we examined the effects of cysteine-substitution mutagenesis on the binding of salvinorin A and an analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to be in close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showed enhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that the diterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectively activate KORs.

Salvinorin A (Figure 4) is the major active ingredient of SalVia diVinorum, a hallucinogenic plant that has been used historically in the traditional shamanic practices of the Mazatec people of Oaxaca, Mexico (1-3). We recently discovered that salvinorin A, a neutral diterpenoid, activates κ-opioid receptors (KORs)1 (4) and is unique in that it represents the only known lipid-like small molecule that selectively and potently activates a peptidergic G-protein coupled receptor (GPCR) (5, 6). Salvinorin A is highly selective for KOR and has no significant activity at µ, δ, or ORL1-opioid receptors (4, 7) nor other tested GPCRs, neurotransmitter transporters, or ion channels (4). Because of its unique structure and selectivity for a single GPCR, † This research was supported in part by RO1DA017204, KO2MH01366, and the NIMH Psychoactive Drug Screening Program to B.L.R. * To whom correspondence should be addressed: Department of Biochemistry, RM W441, School of Medicine, Case Western Reserve University, 2109 Adelbert Road, Cleveland, OH 44106. Telephone: 216-368-2730. Fax: 216-368-3419. E-mail: [email protected]. ‡ These authors contributed equally to this work. § Department of Biochemistry, Case Western Reserve University Medical School. | Virginia Commonwealth University. ⊥ University of Mississippi. # Departments of Psychiatry and Neurosciences and Comprehensive Cancer Center, Case Western Reserve University Medical School. 1 Abbreviations: KOR, κ-opioid receptor; GPCR, G-protein coupled receptor.

FIGURE 1: Comparison of the e2 loop structures. Comparison of the e2 loop structures from the initial minimized KOR (orange), the MD-averaged and minimized structure (green), and the minimized structure of the MD snapshot taken at 2225 fs (magenta). The position of Tyr313 in TM7 is illustrated.

the salvinorin A-KOR receptor-ligand complex provides a model system for exploring the molecular and atomic features responsible for small-molecule selectivity among highly homologous receptors. Salvinorin A also provides a tool for further study of the activation mechanisms of GPCRs.

10.1021/bi050490d CCC: $30.25 © 2005 American Chemical Society Published on Web 05/26/2005

8644 Biochemistry, Vol. 44, No. 24, 2005

FIGURE 2: Effects of various mutations on salvinorin A binding to KORs reveals the importance of Y313. Shown are representative competition binding isotherms for inhibition of 3H-diprenorphine binding to cloned KORs transiently expressed in HEK-293-T cells. Curves represent the theoretical fits for a single binding site model. Ki values are found in Table 2. For the sake of clarity, error bars are omitted but were typically > 1a (EC50 = 2.2 nM) > 18 (EC50 = 73.6 nM). Interestingly, 1a was found to be 40-fold less potent in promoting internalization of the hKOR compared to U50,488 and showed little anti-scratching activity and no antinociception in mice (Wang et al., 2005). It has been speculated that the divergence between the in vivo and in vitro effects of 1a may be due to in vivo metabolism of 1a to metabolites that are inactive at the n opioid receptor (Wang et al., 2005). Another possibility for the discrepancies is that 1a may be interacting with additional receptors, ion channels, and/ or transporters. Recently, systemic administration of 1a has been found to elevate intracranial self-stimulation levels (ICSS) in rats (Todtenkopf et al., 2004). This depressive-like effect was found to be qualitatively similar to the systemic administration of U69,593. Pretreatment with the selective n opioid antagonist ANTI (5V-acetylamidinoethylnaltrindole) dose dependently blocked elevations in ICSS threshold effects. This finding then suggests that stimulation of n receptors in rats triggers depressive-like signs in a behavioral model. Toxicology The potenital toxicity and metabolism of 1a has not been fully investigated in laboratory animals or humans. An initial study examined the potential toxicity of 1a in rodents (Mowry et al., 2003). This study showed that little to no toxicity associated with high doses of 1a in mice. However, the study was carried out for only two weeks. No significant histologic differences between the control mice and the ones treated with doses of 1a were found. However, this does not mean that potential toxicities do not exist. Presently, the identity of the metabolites of 1a are not definitely known. It was suggested that 1b is a metabolite of 1a (Roth et al., 2004; Va´ldes et al., 2001). However, there are few analytical methods to study the routes of metabolism of 1a in vitro or in vivo. One method for determining the concentration of 1a in human and rhesus monkey plasma, rhesus monkey cerebrospinal fluid, and human urine by negative ion LC-MS/ APCI has recently been described (Schmidt et al., 2005a). The fully validated method had a lower limit of detection using FDA guidelines of 2 ng/mL for 0.5 mL plasma samples; the linear range was from 2– 1000 ng/mL. Several derivatives in the salvinorin family can also be analyzed by this method. The method has been used to establish that 1b is the principal metabolite of 1a ex vivo. However, 1b was not found in significant amounts in plasma of nonhuman primates. A

preliminary study indicated that the elimination half-life of 1a in nonhuman primates was found to be 56.6 T 24.8 min for all subjects tested (Schmidt et al., 2005b). Conclusion S. divinorum is an unregulated hallucinogenic plant whose use is increasing. The active component of S. divinorum is the neoclerodane diterpene salvinorin A (1a). In vitro pharmacological studies have found 1a to be a potent and selective n agonist in vitro. In vivo studies indicate that 1a produces discriminative stimulus effects similar to those of a high efficacy n agonist. However, there are discrepancies between the in vitro and in vivo effects of 1a. Preliminary structure activity relationship data has suggested that the 2-position is critical for n opioid receptor binding and activation. Toxicological studies have not identified significant differences in histology between the control mice and the ones treated with doses of 1a. However, this does not rule out the possibility that toxicities do exist. Currently, the metabolites of 1a are not definitively known, however, the half-life of 1a in nonhuman primates is 56.6 T 24.8 min. The body of knowledge of S. divinorum continues to grow and has the potential to identify novel opioid receptor modulators and give greater insight into opioid receptor mediated phenomena. Acknowledgements The author wishes to thank Leander J. Valde´s III for a critical reading of the manuscript and the Biological Sciences Funding Program of the University of Iowa and the National Institute on Drug Abuse for financial support of this work. References Beguin, C., Richards, M.R., Wang, Y., Chen, Y., Liu-Chen, L.Y., Ma, Z., Lee, D.Y., Carlezon Jr., W.A., Cohen, B.M., 2005. Synthesis and in vitro pharmacological evaluation of salvinorin A analogues modified at C(2). Bioorganic and Medicinal Chemistry Letters 15 (11), 2761 – 2765. Bigham, A.K., Munro, T.A., Rizzacasa, M.A., Robins-Browne, R.M., 2003. Divinatorins A – C, new neoclerodane diterpenoids from the controlled sage Salviab divinorum. Journal of Natural Products 66 (9), 1242 – 1244. Butelman, E.R., Harris, T.J., Kreek, M.J., 2004. The plant-derived hallucinogen, salvinorin A, produces kappa-opioid agonist-like discriminative effects in rhesus monkeys. Psychopharmacology 172 (2), 220 – 224. Chavkin, C., Sud, S., Jin, W., Stewart, J., Zjawiony, J.K., Siebert, D.J., Toth, B.A., Hufeisen, S.J., Roth, B.L., 2004. Salvinorin A, an active component of the hallucinogenic sage Salvia divinorum is a highly efficacious n-opioid receptor agonist: structural and functional considerations. Journal of Pharmacology and Experimental Therapeutics 308 (3), 1197 – 1203. Egan, C.T., Herrick-Davis, K., Miller, K., Glennon, R.A., Teitler, M., 1998. Agonist activity of LSD and lisuride at cloned 5HT2A and 5HT2C receptors. Psychopharmacology 136 (4), 409 – 414. Glennon, R.A., Titeler, M., McKenney, J.D., 1984. Evidence for 5-HT2 involvement in the mechanism of action of hallucinogenic agents. Life Sciences 35 (25), 2505 – 2511. Harding, W.W., Tidgewell, K., Byrd, N., Cobb, H., Dersch, C.M., Butelman, E.R., Rothman, R.B., Prisinzano, T.E., 2005. Neoclerodane diterpenes as a novel scaffold for A opioid receptor ligands. Journal of Medicinal Chemistry 48 (15), 4765 – 4771.

T.E. Prisinzano / Life Sciences 78 (2005) 527 – 531 Harding, W.W., Tidgewell, K., Shah, K., Dersch, C.M., Synder, J., Parrish, D., Deschamps, J.R., Rothman, R.B., Prisinzano, T.E., in press. Salvinicin A and B, new neoclerodane diterpenes from Salvia divinorum. Organic Letters. Hazelden Foundation, 2004. Drug Abuse Trends. June 2004. www.research. hazelden.org. Hofmann, A., 1980. LSD, My Problem Child. McGraw-Hill, New York. Mowry, M., Mosher, M., Briner, W., 2003. Acute physiologic and chronic histologic changes in rats and mice exposed to the unique hallucinogen salvinorin A. Journal of Psychoactive Drugs 35 (3), 379 – 382. Munro, T.A., Rizzacasa, M.A., 2003. Salvinorins D – F, new neoclerodane diterpenoids from Salvia divinorum, and an improved method for the isolation of salvinorin A. Journal of Natural Products 66 (5), 703 – 705. Munro, T.A., Rizzacasa, M.A., Roth, B.L., Toth, B.A., Yan, F., 2005. Studies toward the pharmacophore of salvinorin A, a potent kappa opioid receptor agonist. Journal of Medicinal Chemistry 48 (2), 345 – 348. National D.I.C., 2003. Salvia divinorum. Information Bulletin. U.S. Department of Justice, Johnstown, PA. Nichols, D.E., 2004. Hallucinogens. Pharmacology and Therapeutics 101 (2), 131 – 181. Ortega, A., Blount, J.F., Manchand, P.S., 1982. Salvinorin, a new transneoclerodane diterpene from Salvia-divinorum (Labiatae). Journal of the Chemical Society. Perkin Transactions 1 (10), 2505 – 2508. Reisfield, A.S., 1993. The botany of Salvia divinorum (Labiatae). SIDA 15 (3), 349 – 366. Roth, B.L., Baner, K., Westkaemper, R., Siebert, D., Rice, K.C., Steinberg, S., Ernsberger, P., Rothman, R.B., 2002. Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist. Proceedings of the National Academy of Sciences of the United States of America 99 (18), 11934 – 11939. Roth, B.L., Lopez, E., Beischel, S., Westkaemper, R.B., Evans, J.M., 2004. Screening the receptorome to discover the molecular targets for plantderived psychoactive compounds: a novel approach for CNS drug discovery. Pharmacology and Therapeutics 102 (2), 99 – 110. Schmidt, M.D., Schmidt, M.S., Butelman, E.R., Harding, W.W., Tidgewell, K., Murry, D.J., Kreek, M.J., Prisinzano, T.E., 2005a. Pharmacokinetics of the plant-derived n-opioid hallucinogen salvinorin A in nonhuman primates. Synapse 58 (3), 208 – 210. Schmidt, M.S., Prisinzano, T.E., Tidgewell, K., Harding, W.W., Butelman, E.R., Kreek, M.J., Murry, D.J., 2005b. Determination of salvinorin A in body fluids by high proformance liquid chromatography — atmospheric

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pressure chemical ionization. Journal of Chromatography. B 818 (2), 221 – 225. Sheffler, D.J., Roth, B.L., 2003. Salvinorin A: the ’Magic mint’ hallucinogen finds a molecular target in the kappa opioid receptor. Trends in Pharmacological Sciences 24 (3), 107 – 109. Siebert, D.J., 1994. Salvia-divinorum and salvinorin-a — new pharmacological findings. Journal of Ethnopharmacology 43 (1), 53 – 56. Siebert, D.J., 2004. Localization of Salvinorin A and related compounds in glandular trichomes of the psychoactive sage, Salvia divinorum. Annals of Botany 93 (6), 763 – 771. Tidgewell, K., Harding, W.W., Schmidt, M., Holden, K.G., Murry, D.J., Prisinzano, T.E., 2004. A facile method for the preparation of deuterium labeled salvinorin A: synthesis of [2,2,2-2H3]-salvinorin A. Bioorganic and Medicinal Chemistry Letters 14 (20), 5099 – 5102. Todtenkopf, M.S., DiNieri, J.A., Beguin, C., Portoghese, P.S., Lee, D.Y., Cohen, B.M., Carlezon, W.A. Jr., 2004. Regulation of mood in rats by kappa opioid receptor ligands. Neuropsychopharmacology 29 (Supplement 1), S212. Va´ldes III, L.J., 1983. The Pharmacognosy of Salvia divinorum (Epling and Jativa-M): an Investigation of Ska Maria Pastora. University of Michigan, Ann Arbor, pp. 1 – 237. Va´ldes III, L.J., 1994. Salvia divinorum and the unique diterpene hallucinogen, Salvinorin (Divinorin) A. Journal of Psychoactive Drugs 26 (3), 277 – 283. Va´ldes III, L.J., Diaz, J.L., Paul, A.G., 1983. Ethnopharmacology of Ska-MariaPastora (Salvia, Divinorum, Epling and Jativa-M). Journal of Ethnopharmacology 7 (3), 287 – 312. Va´ldes III, L.J., Butler, W.M., Hatfield, G.M., Paul, A.G., Koreeda, M., 1984. Divinorin A, a psychotropic terpenoid, and Divinorin B from the hallucinogenic Mexican mint Salvia divinorum. Journal of Organic Chemistry 49, 4716 – 4720. Va´ldes III, L.J., Chang, H.M., Visger, D.C., Koreeda, M., 2001. Salvinorin C, a new neoclerodane diterpene from a bioactive fraction of the hallucinogenic Mexican mint Salvia divinorum. Organic Letters 3 (24), 3935 – 3937. Wang, Y., Tang, K., Inan, S., Siebert, D., Holzgrabe, U., Lee, D.Y., Huang, P., Li, J.G., Cowan, A., Liu-Chen, L.Y., 2005. Comparison of pharmacological activities of three distinct k ligands (Salvinorin A, TRK-820 and 3FLB) on n opioid receptors in vitro and their antipruritic and antinociceptive activities in vivo. Journal of Pharmacology and Experimental Therapeutics 312 (1), 220 – 230.

0022-3565/06/3182-641–648 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS U.S. Government work not protected by U.S. copyright JPET 318:641–648, 2006

Vol. 318, No. 2 101998/3125665 Printed in U.S.A.

Antinociceptive and Hypothermic Effects of Salvinorin A Are Abolished in a Novel Strain of ␬-Opioid Receptor-1 Knockout Mice Michael A. Ansonoff, Jiwen Zhang, Traci Czyzyk, Richard B. Rothman, Jeremy Stewart, Heng Xu, Jordan Zjwiony, Daniel J. Siebert, Feng Yang, Bryan L. Roth, and John E. Pintar Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School (UMDNJ-RWJMS), Piscataway, New Jersey (M.A.A., J.Z., T.C., J.E.P.); Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio (J.S., J.Z., F.Y., B.L.R.), Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland (R.B.R., H.X.); and Malibu, California (D.J.S.) Received January 27, 2006; accepted May 2, 2006

ABSTRACT Salvia divinorum is a natural occurring hallucinogen that is traditionally used by the Mazatec Indians of central Mexico. The diterpene salvinorin A was identified as an active component of S. divinorum over 20 years ago, but only recently has biochemical screening indicated that a molecular target of salvinorin A in vitro is the ␬-opioid receptor. We have examined whether salvinorin A, the C2-substituted derivative salvinorinyl-2-propionate, and salvinorin B can act as ␬-opioid receptor agonists in vivo. We found that following intracerebroventricular injection over a dose range of 1 to 30 ␮g of both salvinorin A and salvinorinyl-2-propionate produces antinociception in wild-type mice but not in a novel strain of ␬-opioid receptor knockout

Salvia divinorum is a natural occurring hallucinogen that has been used traditionally for divination and other spiritual practices by the Mazatec people of Oaxaca, Mexico (Valdes et al., 1983) and more recently as a legal hallucinogen (Giroud et al., 2000; Sheffler and Roth, 2003). Studies in the 1980s identified an active component of S. divinorum to be salvinorin A (Ortega et al., 1982; Valdes et al., 1983), which was

This work was supported by National Institutes of Health (NIH) Grants DA-009040 and DA-015237 (to J.E.P.), the Intramural Research Program of the NIH, and National Institute of Drug Abuse (NIDA) (to R.B.R.), NIH, Grants DA-017204 (to B.L.R.) and MH/AG 19957 and F32 DA-14755 (to M.A.A.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.101998.

mice. Moreover, both salvinorin A and salvinorinyl-2-propionate reduce rectal body temperature, similar to conventional ␬-opioid receptor agonists, in a genotype-dependent manner. In addition, we determined that salvinorin A has high affinity for ␬1- but not ␬2-opioid receptors, demonstrating selectivity for this receptor subclass. Finally, treatment over the same dose range with salvinorin B, which is inactive in vitro, produced neither antinociceptive nor hypothermic effects in wild-type mice. These data demonstrate that salvinorin A is the active component of S. divinorum, selective for ␬1-opioid receptors, and that salvinorin A and specific structurally related analogs produce behavioral effects that require the ␬-opioid receptor.

later determined to be the primary psychoactive molecule in the plant (Siebert, 1994). Salvinorin A is a neoclerodane diterpene whose absolute configuration and structure have been determined by NMR and single-crystal X-ray analysis (Ortega et al., 1982; Koreeda et al., 1990; Valdes, 1994), and it is quite distinct from other natural hallucinogens. The putative molecular target of salvinorin A remained elusive until recently when radioligand guanosine 5⬘-3-O-(thio)triphosphate and adenylyl cyclase assays all identified salvinorin A to be an agonist at ␬-opioid receptors (KORs) in vitro (Roth et al., 2002; Chavkin et al., 2004; Nichols, 2004; Yan and Roth, 2004). It is noteworthy that there is no in vitro agonist activity at the 5-hydroxytryptamine 2A serotonin receptors that mediate actions of most other known halluci-

ABBREVIATIONS: KORs, ␬-opioid receptors; i.c.v., intracerebroventricularly; KO, knockout; kb, kilobase; neo, neomycin; U69,593, (⫹)-(5a,7a,8b)N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspirodec-8-yl]-benzeneacetamide; HRMS, high-resolution mass spectrometry; BIT, 2-(p-ethoxybenzyl)-1DEAE-5-isothiocyanatobenzimidazole-HCl; FIT, N-phenyl-N-[1-(2-(p-isothiocyanato)phenylethyl)-4-piperidinyl]propanamide-HCl; DMSO, dimethyl sulfoxide; %MPE, percentage maximal possible effect; U50,488H, (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide; ANOVA, analysis of variance; PLSD, protected least significant difference; ES, embryonic stem. 641

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nogens (Roth et al., 2002; Chavkin et al., 2004; Nichols, 2004). These earlier studies established that salvinorin A was a potent and selective KOR agonist. However, it is still unclear whether salvinorin A is selective for ␬1- or ␬2-opioid receptors. The ␬2-opioid receptor was initially defined on the basis of ligand binding and physiological studies (Iyengar et al., 1986; Zukin et al., 1988; Rothman et al., 1990). Although there is no evidence for a gene coding for the ␬2-opioid receptor, recent data suggest that the ␬2-opioid receptor might result from ␦-␬ heterodimers (Jordan and Devi, 1999). We have used ligand binding to determine whether salvinorin A shows selectivity for ␬1- or ␬2-opioid receptors. Consistent with in vitro data, the reported physiologic effects of salvinorin A are consistent with prospective action through the KOR. For example, documented in vivo effects of S. divinorum and purified salvinorin A include sedation, antinociception, and production of hallucinations (Siebert, 1994; Valdes, 1994; Giroud et al., 2000; Hanes, 2001; Wang et al., 2005; McCurdy et al., 2006). Sedation and antinociception have long been identified as major effects of KOR agonists (Martin et al., 1976; Vonvoigtlander et al., 1983; Leighton et al., 1988), whereas activation of KORs has also been noted to be dysphoric and cause perception alterations (Pfeiffer et al., 1986). Consistent with the dysphoric effects in humans, administration of salvinorin A to mice produces a long-lasting decrease in extracellular dopamine (Zhang et al., 2005) that is consistent with KOR activation (Chefer et al., 2005). Based on the overall similarities between the behavioral effects of salvinorin A and ␬ agonists, we hypothesized that KOR could be the target for many if not all salvinorin A actions in vivo and thus could potentially mediate additional behavioral responses, such as hypothermia (Baker and Meert, 2002), that result from KOR activation. The most direct way to test the in vivo requirement of KOR is in a knockout (KO) model, so we have compared the responses of several salvinorin-related compounds in both wild-type mice and in a novel mouse strain containing a null mutation of the KOR-1 gene.

Materials and Methods All experiments were conducted in accordance with the guidelines of the Institutional Care and Use Committees of UMDNJ-RWJMS and the National Institute on Drug Abuse, National Institutes of Health in facilities accredited by the American Association for the Accreditation of Laboratory Animal Care. Adult male mice were used for all initial characterization of the KOR-1 KO mice and were derived from mating of either KOR-1 heterozygous mutant mice maintained on a mixed C57BL6/J⫻129S6 background or wild-type and KOR-1 mutant mice maintained on an inbred 129S6 background. Subsequent behavioral experiments used wild-type and KOR-1 KO mice of both sexes maintained on the 129S6 background. Production of KOR-1-Deficient Mice. A digoxigenin-labeled cDNA probe containing murine KOR-1 exons 2 and 3 (Yasuda et al., 1993) was used to screen a genomic library constructed from the 129/SwRe strain. One genomic clone containing exon 3 of KOR-1 was isolated, confirmed by sequencing, and used to construct the targeting vector. A 6-kb NotI/SpeI fragment 5⬘ of exon 3 and a 1.4-kb EcoRI fragment downstream of exon 3 were subcloned into pBS-KO vector (obtained from Dr. Steven Potter, University of Cincinnati, Cincinnati, OH) containing neomycin (neo) and thymidine kinase selection markers. Replacement of exon 3 with a neo cassette would then occur following successful homologous recombination (Fig. 1A).

Fig. 1. Targeting of KOR-1. A, KOR-1 genomic clone 3 was restrictionmapped, and exon 3 was positioned ⬃8 kb from the 5⬘ end and ⬃4 kb from the 3⬘ end of the clone. The KOR-1-targeting vector was constructed by subcloning ⬃6 kb 5⬘ of exon 3 and 1.4 kb 3⬘ of exon 3 into the KO vector, such that the neo gene replaced a 2-kb KOR-1 sequence containing exon 3. In the predicted targeted locus, the neo gene would introduce an extra BamHI site. Using a screening probe outside the sequence used for targeting, Southern blot analysis detected an 8-kb wild-type allele and a 5-kb targeted allele after homologous recombination. This screening strategy was used to identify a positively targeted ES cell line after electroporation (B) and to demonstrate mice of all genotypes from offspring of heterozygous mating (C). D, [3H]U69,593 was used to assess ␬ receptor binding in adult brain homogenates from wild-type, heterozygous, and homozygous KOR-1 littermates. Binding was undetectable in KOR-1 mice and reduced to ⬃50% in heterozygous mice. (⫹/⫹, Bmax ⫽ 8.2 fmol/mg protein; Kd ⫽ 0.64 nmol; ⫹/⫺, Bmax ⫽ 3.24 fmol/mg protein; Kd ⫽ 0.59; ⫺/⫺, Bmax ⫽ N/A; Kd ⫽ N/A (n ⫽ 3).

The targeting vector was linearized and electroporated into 129SvEv-derived CCE ES cells (provided by Elizabeth Robertson, Harvard University, Cambridge, MA) as described previously (Schuller et al., 1999). Individual ES colonies that survived the G418 and ganciclovir double selection were screened, and targeted ES cell lines were identified following hybridization with a 0.6-kb screening probe from a genomic region 3⬘ of the region incorporated into the targeting vector. This probe hybridized to a 8-kb wild-type fragment, and a diagnostic 5-kb band was derived from the targeted allele following BamHI digestion (Fig. 1B). Targeted ES cells were injected into blastocysts, and chimeras were mated with C57BL6/J and 129S6 female mice. Heterozygous mice were identified using the screening strategy described above and used to establish and maintain the mixed C57BL/6⫻129S6 strain and produce an inbred 129S6 line. Homogenate Binding Assays. Adult brains from wild-type and homozygous KOR-1 mutant mice were isolated and homogenized in 30 volumes of 50 mM Tris-HCl, pH 7.4, at 4°C and then centrifuged twice at 30,000g for 15 min at 4°C with the supernatant discarded. Brain membranes were suspended in 30 volumes of buffer and incubated for 30 min at 37°C to dissociate bound endogenous ligand before recentrifugation. Resuspended brain aliquots [400 mg wet weight in 30 ml of Tris buffer, 400-␮l aliquots of membrane suspension (⬃0.25 mg of protein)] were incubated with eight concentrations of [3H]U69,593 DuPont, Wilmington, DE and PerkinElmer Life and Analytical Sciences, Boston, MA) in 50 mM Tris-HCl, pH 7.4, for 90 min. Nonspecific binding was assessed using 10 nM naloxone. The homog-

Salvinorin A Is a ␬-Opioid Agonist in Vivo enates were filtered under reduced pressure through Whatman GF/B filters (Whatman, Inc., Florham Park, NJ) using a Brandel cell harvester (Brandel, Inc., Gaithersburg, MD). Filters were washed three times with 50 mM Tris-HCl, pH 7.4, at 4°C to remove free radioligands and then assayed by liquid scintillation spectrometry. Protein concentrations were determined by the Lowry procedure (Sigma, St. Louis, MO). Binding affinities and capacities were determined by Scatchard analysis (Munson, 1983). Chemistry. Salvinorin A was isolated from dried leaves of S. divinorum by the method reported by Valdes et al. (1994). Salvinorin B and salvinorinyl-2-propionate were prepared as detailed previously (Chavkin et al., 2004). Salvinorin B was characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS) and found to be authentic by comparison with literature values (Valdes, 1994). The reported esters were characterized by high-performance liquid chromatography and HRMS. NMR (1H and 13C) spectra were recorded on a Bruker AMX-NMR spectrometer in CDCl3. The HRMS spectra were measured using a Bioapex FT mass spectrometer (Bruker Daltonics, Billerica, MA) with electrospray ionization. High-performance liquid chromatography was conducted on a Waters Deltaprep 4000 system (Waters, Milford, MA) using a Waters Xterra RP18, 5 ␮m, 4.6 ⫻ 150-mm column, with mobile phase H2O/acetonitrile (1:1). Thin layer chromatography analyses were carried out on precoated Si gel G254, 250-␮m plates, with the developing system hexane/EtOAc (2:1) and visualized with vanillin/H2SO4 in EtOH. Radioligand Binding Assays. Assays of rat brain ␬2-opioid receptor binding sites followed published procedures (Rothman et al., 1992). In brief, membranes prepared from whole-rat brain, pretreated with 1 ␮M 2-(p-ethoxybenzyl)-1-DEAE-5-isothiocyanatobenzimidazole-HCl (BIT) and 1 ␮M N-phenyl-N-[1-(2-(p-isothiocyanato)phenylethyl)-4-piperidinyl]propanamide-HCl (FIT) to deplete ␮- and ␦-opioid receptor binding sites, washed by repeated centrifugation and resuspension, and then assayed with [3H]bremazocine (2.5 nM) in 50 ␮M potassium phosphate buffer, pH 7.4, for 4 to 6 h at 0°C. The incubations were terminated by rapid filtration over Whatman GF/C filters. Further details of this assay have been published (Rothman et al., 1990). Drug Administration. All experimental drugs were dissolved in 50, 60, or 100% DMSO. Controls contained equal concentrations of DMSO mixed with saline. Intracerebroventricular (i.c.v.) injections were performed as described elsewhere (Haley, 1957). In brief, mice were exposed to an isoflurane and oxygen combination for approximately 2 to 3 min until full anesthesia was observed. A midline incision was made along the sagittal suture, and a microinjection of 3 ␮l was administered in the lateral ventricle at 2 mm anterior to the lambda suture and 3 to 3.5 mm lateral to the midline suture that extended 2 mm below the surface of the skull. Nearly complete locomotor/nociceptive recovery from anesthesia was observed within 15 min. Analgesic Testing. Analysis of salvinorin A, salvinorin B, and salvinorinyl-2-propionate analgesia was performed on wild-type and KOR-1 KO mice of both sexes, maintained on a 129S6 inbred back-

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ground using the radiant heat tail-flick assay of nociception. Intensity of the beam was adjusted to yield baseline tail-flick latencies between 2 and 3 s, and cutoff of 10 s was employed to reduce tissue damage. Percentage maximal possible effect (%MPE) was determined according to the following formula: [(postinjection latency ⫺ preinjection latency)/(10 ⫺ preinjection latency)] ⫻ 100. Nociceptive thresholds were determined prior to drug administration. Mice were then injected i.c.v. with drug and tested for analgesia 15 min afterward. For time courses, mice were injected i.c.v. with drug and tested for analgesia every 15 min for 1 h. For cumulative dose-response curves, mice were injected i.c.v. with drug and tested for analgesia 15 min afterward. Immediately after testing, mice were injected with the next highest dose. This procedure was repeated until all doses had been administered. Initial experiments were analyzed using one-way ANOVA. Differences were isolated using Fisher’s PLSD. Time dependence was evaluated using repeated measures ANOVA with time as the dependent variable and treatment as an independent variable. Individual differences were isolated using one-way ANOVA at individual time points and Fisher’s PLSD for post hoc tests. Dose-dependent analgesia was evaluated using repeated measures ANOVA with dose as the dependent variable (p ⬍ 0.05). Both salvinorin A and salvinorinyl-2-propionate show a significant effect of dose in wild-type mice. Salvinorin B in wild-type mice and salvinorin A and salvinorinyl-2-propionate in KOR-1 KO mice do not show a significant effect of dose. Individual doses were analyzed using Fisher’s PLSD to determine significance between wild-type and KOR-1 KO responses. ED50 values were determined using a nonlinear curve-fitting program for data expressed as both %MPE and percentage mice that became analgesic (Prism; GraphPad Software, Inc., San Diego, CA). Mice were considered analgesic when postinjection latency was greater than twice baseline line tail-flick latency. No difference in drug response was observed between sexes. Rectal Temperature Measurement. Effects of salvinorin A and salvinorinyl-2-propionate on rectal body temperature was performed on wild-type and KOR-1 KO mice of both sexes maintained on a 129S6 inbred background. All measurements were made with a 2.5-cm rectal temperature probe (Model BAT-12; Physitemp, Clifton, NJ). The probe was inserted 2.5 cm and allowed to reach temperature for 5 to 6 s, and the measurement was then recorded. Rectal temperature was first determined before drug administration. Mice were then injected i.c.v. with drug and then measured for rectal body temperature every 15 min for 2 h. Differences were isolated using one-way ANOVA at individual time points with treatment as the independent factor and Fisher’s PLSD for post hoc tests. No difference in drug response was observed between sexes.

Results Pharmacological Characterization of Salvinorin A, Salvinorin B, and Substituted Salvinorin A Derivatives. Both salvinorin A and salvinorinyl-2-propionate sig-

TABLE 1 Effect of Salvinorin A derivatives on KOR subtype binding and KOR-1-stimulated Ca2⫹ mobilization KOR-2 binding experiments were done as described under Materials and Methods. The salvinorin A derivatives were screened using a large panel of cloned human receptors, ion channels, and transporters (see Roth et al., 2002 for list) and were inactive (data not shown) Drug

Ki at KOR-1

Ki at KOR-2A

U69,593 U50,488H Salvinorin A Salvinorinyl-2-propionate Salvinorin B

0.7 ⫾ 0.05b 0.2 ⫾ 0.05b 18.7 ⫾ 3.4a 32.6 ⫾ 15.7a ⬎10,000a

121 ⫾ 10c 55.6 ⫾ 7.4c ⬎10,000 ND ⬎10,000

Ki at KOR-2B

pEC50a

⬎10,000c 20,400 ⫾ 5160c ⬎10,000 ND ⬎10,000

1.14 ⫾ 0.02 1.39 ⫾ 0.14 0.84 ⫾ 0.07 1.23 ⫾ 0.18 NA

nM ⫾ S.E.M.

Emaxa

nM ⫾ S.D.

NA, no activity; ND, not done. a The results for KOR-1 binding and KOR-1-stimulated Ca2⫹ mobilization are taken from Chavkin et al. (2004). b Data from Toll et al. (1998). c Data from Rothman et al. (1990).

100 102 ⫾ 4 104 ⫾ 7 102 ⫾ 8 NA

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Fig. 2. Salvinorin A and salvinorinyl-2-propionate elicit antinociception. A, wild-type mice were injected i.c.v. with 7.5 ␮g of salvinorin A (black bars), salvinorinyl-2-propionate (dotted bars), and salvinorin B (diagonal striped bars) dissolved in 50% DMSO or 50% DMSO (Control) (white bars); n ⫽ 6 –18. ⴱ, p ⬍ 0.05 versus Control. B, wild-type mice were injected i.c.v. with 13 ␮g of salvinorinyl-2-propionate (dotted bars), salvinorin B (diagonal striped bars) dissolved in 100% DMSO, or 100% DMSO (Control) (white bars); n ⫽ 4 –9. ⴱ, p ⬍ 0.05 versus Control.

nificantly inhibit ␬1-opioid receptor binding, whereas neither salvinorin A nor salvinorinyl-2-propionate significantly inhibited ␬2-opioid receptor binding (Table 1). In contrast to the weak interaction of salvinorin A with ␬2-opioid receptors in rat brain, other agents that activate pharmacologically defined ␬2-opioid receptors, such as (⫺)-ethylketocyclazocine (Sheffler and Roth, 2003), have high affinity for the ␬2-opioid receptor site (Iyengar et al., 1986). Thus, based on receptor binding affinity, salvinorin A and its derivatives are ␬1-opioid receptor-selective. Production and Pharmacologic Characterization of KOR-1-Deficient Mice. To evaluate the actions of salvinorin-related compounds in vivo, a novel line of KOR-1 KO mice was used. ES cells from one of two KOR-1-targeted ES clones were injected into blastocysts, and germline-transmitting chimeras were identified. Mice heterozygous for the mutant KOR-1 allele were mated, and homozygous mutant mice were identified that were viable and fertile with no obvious morphological abnormalities (Fig. 1C). Genotypes of

offspring from heterozygous matings arose in the predicted Mendelian frequency (wild type, 28.2%; heterozygous, 48.4%; and homozygous, 23.4%; n ⫽ 273). The effect of the exon 3 KOR-1 gene deletion on binding of the selective ␬ agonist [3H]U69,593 was determined by homogenate binding assays on adult brain membrane fractions from ⫹/⫹, ⫹/⫺, and ⫺/⫺ genotypes. U69,593 binding was absent from KOR-1 KO mice, whereas binding in heterozygous mice was ⬃50% that of wild-type levels (Fig. 1D), similar to another line of KOR-1 KO (Simonin et al., 1998) mice in which binding CI-977 binding was measured. Antinociceptive Effects of Salvinorin A, Salvinorin B, and Salvinorinyl-2-propionate. To determine whether salvinorin A has analgesic potency, we initially injected salvinorin A at a dose of 5 mg/kg i.p. and observed no effect 30 min later (data not shown; also see Wang et al., 2005). We next determined whether salvinorin A has potency when injected supraspinally. Salvinorin A (7.5 ␮g) injected i.c.v. showed antinociceptive potency when nociception was measured 15 min later (Fig. 2A). We next tested salvinorinyl-2-propionate and salvinorin B supraspinally at the same dose (Fig. 2A). Consistent with the slight reduction in affinity compared with salvinorin A observed in previous studies (Chavkin et al., 2004), salvinorinyl-2-propionate produced a more limited antinociception at 7.5 ␮g than salvinorin A. In contrast, salvinorin B demonstrated no analgesic potency. We then increased the dose to 13 ␮g for savinorinyl-2-propionate and salvinorin B (Fig. 2B) and measured nociception 15 min later. At this dose, salvinorinyl-2-propionate produced a significant antinociceptive response, whereas salvinorin B remained inactive. Thus, in vivo analgesic activity could be elicited by only salvinorin A-like compounds active in vitro. To further characterize salvinorin A-elicited analgesia, time-course studies for the analgesic actions of salvinorin A and salvinorinyl-2-propionate were performed. Intracerebroventricular injection of either 7.5 ␮g of salvinorin A or 10 ␮g of salvinorinyl-2-propionate produced significant analgesia as early as 15 min (Fig. 3). By 30 min, the tail-flick latencies for both the salvinorin A and salvinorinyl-2-propionate-injected groups were still elevated but not significantly different from controls. By 45 min after injection, the tail-flick latencies for all treated mice had returned to baseline values. To better compare drug time courses between behavioral

Fig. 3. Salvinorin A and salvinorinyl-2-propionate antinociception is short-acting. Wild-type mice were injected i.c.v. with 3 ␮l of 100% DMSO (Control) (open triangles), 7.5 ␮g of salvinorin A (closed squares), 50 ␮g of salvinorin A (open squares), or 10 ␮g of salvinorinyl-2-propionate (closed circles) dissolved in 100% DMSO. Data are graphed as mean ⫾ S.E.M; n ⫽ 6 – 8. ⴱ, p ⬍ 0.05 (both 7.5 and 50 ␮g) salvinorin A versus Control; salvinorinyl-2-propionate versus Control.

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Fig. 4. Salvinorin A and salvinorinyl-2-propionate produced antinociception through activation of the KOR. A, wild-type (closed squares) or KOR-1 KO (open squares) mice were injected i.c.v. with an escalating dose of salvinorin A (1.5–15 ␮g) dissolved in 100% DMSO. Data are graphed as mean ⫾ S.E.M. n ⫽ 9 for wild-type; n ⫽ 8 for KOR-1 KO. ⴱ, p ⬍ 0.05 wild type versus KOR-1 KO. B, wild-type (closed squares) or KOR-1 KO (open squares) were injected i.c.v. with an escalating dose of salvinorinyl-2-propionate (1–30 ␮g) dissolved in 100% DMSO. Data are graphed as mean ⫾ S.E.M. n ⫽ 8 for wild type; n ⫽ 6 for KOR-1 KO. ⴱ, p ⬍ 0.05 wild-type versus KOR-1 KO. C, wild-type mice (closed squares) were injected i.c.v. with an escalating dose of salvinorin B (1–30 ␮g) dissolved in 100% DMSO. Data are graphed as mean ⫾ S.E.M.; n ⫽ 9. D, wild-type mice were repeatedly injected i.c.v. with either saline (closed circles) or 100% DMSO (open triangles). Data are graphed as mean ⫾ S.E.M.; n ⫽ 5 for saline; n ⫽ 7 for DMSO.

Fig. 5. Repeated i.c.v. injection of DMSO slightly elevates % MPE. Mice were repeatedly injected four times i.c.v. with salvinorin A [n ⫽ 9 for wild-type (open bars); n ⫽ 8 for KOR-1 KO (closed bars)]; salvinorinyl-2propionate (n ⫽ 8 for wild type; n ⫽ 6 for KOR-1 KO) or salvinorin B (n ⫽ 9) dissolved in 100% DMSO, 100% DMSO (n ⫽ 9), or saline (n ⫽ 5). Data presented is the final drug and/or vehicle injection value from Fig. 4, A to D. Data are graphed as mean ⫾ S.E.M. ⴱ, p ⬍ 0.05 versus wild-type 100% DMSO injected.

paradigms (e.g., hypothermic effects; see Fig. 6), antinociceptive effects were also evaluated after i.c.v. injection of 50 ␮g of salvinorin A. As seen in Fig. 3, injection of this supermaximal dose of salvinorin A increased the time course for significant drug action. Salvinorin A still acted rapidly and reached 100% MPE within 15 min, but a significant drug effect was maintained for at least 45 min. Dose-response curves examining the potency of salvinorin A, salvinorinyl-2-propionate, and salvinorin B analgesia were then produced with nociception measured 15 min postinjection. Both salvinorin A and salvinorinyl-2-propionate induced dose-dependent antinociception in wild-type mice with an ED50 of 1.5 and 2.0 ␮g (for data expressed as %MPE) or 1.4 and 1.7 ␮g (for data expressed as percentage

mice analgesic), respectively (Fig. 4, A and B), whereas salvinorin B was inactive in wild-type mice (Fig. 4C). As further demonstration that salvinorin A and salvinorinyl-2-propionate act specifically on KORs, neither salvinorin A nor salvinorinyl-2-propionate induced analgesia in KOR-1 KO mice (Fig. 4, A and B). Because i.c.v. injection of salvinorin A and salvinorinyl-2propionate in KOR-1 KO mice and salvinorin B in wild-type mice increased % MPE slightly at the highest doses of the dose-response curve, we wanted to distinguish whether this effect was an antinociceptive response at high doses of the compound or a nonspecific antinociceptive effect of the vehicle (100% DMSO) in which test compounds were dissolved. Intracerebroventricular injection of 100% DMSO alone consistently stimulated a greater antinociceptive response (⬃20%; Fig. 2) than previously seen following i.c.v. injection of saline alone (⬃10%; data not shown). We then assessed whether repeated injections of 100% DMSO i.c.v. produced significant antinociception above this baseline value. Figure 4D shows that repeated DMSO injection does induce slight antinociception in wild-type mice after repeated dosing compared with saline injection. However, the level of analgesia observed is significantly lower than that observed following i.c.v. injection of salvinorin A or salvinorinyl-2-propionate in wild-type mice and never reached statistical significance (Fig. 5). Thus, the level of nociception in dose-response curves for salvinorin B in wild-type mice or salvinorin A or salvinorinyl-2-propionate in KOR-1 KO mice after the final i.c.v. injection is comparable with the level of nociception observed after repeated i.c.v. injection of vehicle (100% DMSO) (Fig. 5). Effects on Rectal Body Temperature. Because KOR agonists are known to cause depression of rectal body temperature, we attempted to ascertain whether salvinorin A and salvinorinyl-2-propionate also affect rectal body temperature. Rectal body temperature is immediately suppressed in

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Fig. 6. Salvinorin A and salvinorinyl-2propionate reduce rectal body temperature via the ␬-opioid receptor. A, wildtype mice injected i.c.v. with 100% DMSO (Control) (closed circles), 7.5 ␮g of salvinorin A (closed squares), or 50 ␮g of salvinorin A (open squares) dissolved in 100% DMSO. Data are graphed as mean ⫾ S.E.M.; n ⫽ 4 –7. ⴱ, p ⬍ 0.05 salvinorin A versus Control. B, wildtype mice injected i.c.v. with 100% DMSO (Control) (closed circles) or 50 ␮g of salvinorinyl-2-propionate (closed squares) dissolved in 100% DMSO. Data are graphed as mean ⫾ S.E.M.; n ⫽ 7. ⴱ, p ⬍ 0.05 salvinorinyl-2-propionate versus Control. C, wild-type mice injected i.c.v. with 100% DMSO (Control) (closed circles) or 50 ␮g of salvinorin B (closed squares) dissolved in 100% DMSO; n ⫽ 5. D, KOR-1 KO mice injected i.c.v. with 100% DMSO (Control) (closed circles) or 50 ␮g of salvinorin A (closed squares) dissolved in 100% DMSO. Data are graphed as mean ⫾ S.E.M.; n ⫽ 6.

both control and drug-treated wild-type mice after i.c.v. injection because of anesthesia used during the procedure (Kushikata et al., 2005). However, rectal body temperature of vehicle-treated mice returns to baseline within 45 to 60 min of injection, whereas rectal body temperature of mice treated with 50 ␮g of salvinorin A or salvinorinyl-2-propionate shows prolonged suppression at least 120 min post-i.c.v. injection (Fig. 6, A and B). Although a trend for hypothermia after i.c.v. injection of 7.5 ␮g of salvinorin A is seen at 45 min postinjection, no significant effect on rectal body temperature was observed in wild-type mice (Fig. 6A). Intracerebroventricular injection of salvinorin B does not produce additional suppression of rectal body temperature compared with vehicle injection (Fig. 6C). Finally, to confirm that the reduction in rectal body temperature induced by i.c.v. injection of salvinorin A requires the KOR-1 gene, we injected salvinorin A i.c.v. in KOR-1 KO mice. Similar to the i.c.v. injection of salvinorin B, no difference was seen in rectal body temperature between vehicle and salvinorin A-injected KOR-1 KO mice (Fig. 6D).

Discussion The major finding of the present study is the genetic determination that the in vivo actions of salvinorin A and its propionate-derivative are KOR-1-dependent. Both salvinorin A and its derivative salvinorinyl-2-propionate have antinociceptive and hypothermic effects when injected i.c.v. into wildtype mice (Figs. 2, A and B, and 6, A and B), whereas both drugs were inactive in KOR-1 KO mice (Figs. 4, A and B, and 6D), demonstrating that a principal molecular target of these compounds in vivo is the KOR. The loss of antinociceptive and hypothermic effects of salvinorin A in the KOR-1 KO mouse does not rule out the possibility that salvinorin A has other sites of action for other behavioral responses still to be investigated, but what these other sites of action might be is unclear since prior studies have failed to find any other

molecular target for the actions of salvinorin A, despite screening a large number of neurotransmitter receptors, ion channels, and transporters (Roth et al., 2002). Taken together, the most parsimonious explanation is that the effects of salvinorin A in vivo are mediated principally, if not exclusively, by KORs. Indeed, recent reports have suggested that the psychological effects of salvinorin A in humans (Sheffler and Roth, 2003) and discriminative stimulus effects of salvinorin A in monkeys (Butelman et al., 2004) are mediated by KORs. Consistent with recent studies (Wang et al., 2005; McCurdy et al., 2006) showing that salvinorin A has low potency and a very short half-life in vivo, we demonstrate antinociceptive efficacy only for a transient time following an i.c.v. injection of an ED80 dose (7.5 ␮g) of salvinorin A (maximal response at 15 min and no effect by 45 min; Fig. 3). Hypothermic effects of salvinorin A are absent after i.c.v. injection of the same 7.5-␮g dose of salvinorin A (Fig. 6A). It is possible that salvinorin A may be efficacious at 7.5 ␮g i.c.v., but the hypothermic effects of the anesthesia required for the i.c.v. surgery may mask these effects at early time points. Intracerebroventricular injection of 25 ␮g of salvinorin A was also inactive in producing hypothermia (data not shown). We obtained significant hypothermic effects only after i.c.v. injection of 50 ␮g of salvinorin A (Fig. 6A). The hypothermic efficacy of salvinorin A at this higher dose appears to be relatively longer lasting (maximal effect after 75 min and significant effects out to 120 min) than significant antinociceptive effects. The remaining difference in time course may be due to physiological differences between the two behaviors. Although not well documented, differences in maximal time for ␬-opioid agonist action on nociception and rectal temperature have previously been observed. For example, Spencer et al. (1988) demonstrated that, although U50,488H administered i.c.v. to rats shows maximal antinociceptive efficacy after 10 min and all analgesic action is lost after 40 min, the same treatment has maximal efficacy on rectal

Salvinorin A Is a ␬-Opioid Agonist in Vivo

temperature after 20 min with significant effects still present 40 min later. Similar differences in time course are seen when comparing maximal efficacy for dynorphin A-(1–13) administered i.c.v. Nakazawa et al. (1985) showed a maximal efficacy for i.c.v. administered dynorphin A at 15 min postinjection, whereas dynorphin A-(1–13) effects on rectal body temperature reach maximal effect 60 min postinjection and are still present 2 h after the initial injection (Chen et al., 2005). Exactly why such a difference exists between the time course of effects of ␬ agonists on nociception and rectal temperature is not known. However, the difference may be due to how activation of ␬-opioid receptors influences these behaviors as well as the time required for physiological changes in these behaviors to be manifested. ␬-Opioid activation may rapidly affect nociception by directly inhibiting the nociceptive neural circuitry. After degradation of the drug, the effects on nociception disappear as rapidly as they appear. Changes in rectal body temperature may take longer to appear after ␬-opioid activation due to the requirement for changes in physiology to lower body temperature and may last longer due to the same requirement for physiological alterations to restore the body temperature following drug clearance. Finally, although the vehicle DMSO used in these studies has slight behavioral activity, it does not confound interpretation of the data. High concentrations of DMSO (50% or greater) were used to dissolve salvinorin A because of its relative insolubility in common vehicles used for i.c.v. injection. After a single i.c.v. injection of DMSO, we saw ⬃20% MPE. Subsequent i.c.v. injections revealed that this effect increased to a maximum of ⬃45% following a fourth injection (Fig. 4D). These results are consistent with literature showing that DMSO can have antinociceptive actions (Haigler and Spring, 1981, 1983). Testing salvinorin A in both wild-type and KOR-1 KO mice eliminates any ambiguity in our interpretation of these data. The decrease in the antinociceptive effect of salvinorin A seen in the KOR-1 KO could only be due to the loss of salvinorin A action on the ␬-opioid receptor. The remaining antinociceptive effect could then be attributed to DMSO. Therefore, by testing salvinorin A in wild-type and KOR-1 KO mice, we can definitively state that some if not all of salvinorin A antinociceptive effects are due to action at the ␬-opioid receptor. In addition, the slight elevation of % MPE in the dose-response curve from wild-type mice injected with salvinorin B is most likely not due to antinociceptive activity of the compound, but rather the action of DMSO vehicle (Fig. 5). In conclusion, these studies have accomplished several objectives. First, we have confirmed two behavioral responses for salvinorin A and one of its derivatives. These compounds have analgesic efficacy and depress rectal body temperature consistent with their identification as KOR agonists. Second, we show that salvinorin B is inactive at comparable doses, indicating that salvinorin A is, most likely, the main active component of S. divinorum in vivo as it is in vitro. In addition, we provide the first in vivo structure-function studies of salvinorin A at the KOR and have identified salvinorinyl-2propionate as a novel salvinorin A derivative with appreciable KOR actions in vivo. We also demonstrate that salvinorin A interacts with the ␬1-opioid receptor subtype but not the ␬2-opioid receptor subtype. Finally, by performing behavioral assays in wild-type and a novel strain of KOR-1 KO mice, we

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Address correspondence to: Dr. John E. Pintar, Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. E-mail: [email protected]

0022-3565/06/3161-440 –447 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS U.S. Government work not protected by U.S. copyright JPET 316:440–447, 2006

Vol. 316, No. 1 92304/3071860 Printed in U.S.A.

Depressive-Like Effects of the ␬-Opioid Receptor Agonist Salvinorin A on Behavior and Neurochemistry in Rats William A. Carlezon, Jr., Ce´cile Be´guin, Jennifer A. DiNieri, Michael H. Baumann, Michele R. Richards, Mark S. Todtenkopf, Richard B. Rothman, Zhongze Ma, David Y.-W. Lee, and Bruce M. Cohen Behavioral Genetics Laboratory (W.A.C., J.A.D., M.S.T.), Molecular Pharmacology Laboratory (C.B., M.R.R., B.M.C.), and Bioorganic and Natural Products Laboratory (Z.M., D.Y.-W.L.), Department of Psychiatry, Harvard Medical School, McLean Hospital, Belmont, Massachusetts; and Clinical Psychopharmacology Section (M.H.B., R.B.R.), Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland Received July 8, 2005; accepted October 12, 2005

ABSTRACT Endogenous opioids seem to play a critical role in the regulation of mood states. For example, there is accumulating evidence that stimulation of ␬-opioid receptors, upon which the endogenous opioid dynorphin acts, can produce depressivelike behaviors in laboratory animals. Here we examined whether systemic administration of salvinorin A (SalvA), a potent and highly selective ␬-opioid agonist, would produce depressivelike effects in the forced swim test (FST) and intracranial selfstimulation (ICSS) test, which are behavioral models often used to study depression in rats. We extracted, isolated, and purified SalvA from Salvia divinorum plant leaves and examined its effects on behavior in the FST and ICSS test across a range of doses (0.125–2.0 mg/kg) after systemic (intraperitoneal) administration. SalvA dose dependently increased immobility in the FST, an effect opposite to that of standard antidepressant

Although much research on depression has focused on brain norepinephrine and serotonin (5-HT) systems, there is substantial evidence that other systems have important roles in the neurobiology of mood and affective disorders. For example, the mesolimbic dopamine (DA) system— which projects from the ventral tegmental area to the nucleus accumbens (NAc)— contributes importantly to the hedonic (rewarding) effects of food, sexual behavior, and This work was supported by Grant MH63266 from the National Institute of Mental Health (to W.A.C.) and the Stanley Medical Research Institute (to B.M.C.). This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program from the National Center for Research Resources, National Institutes of Health (RR11213). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.092304.

drugs. Doses of SalvA that produced these effects in the FST did not affect locomotor activity in an open field. Furthermore, SalvA dose dependently elevated ICSS thresholds, an effect similar to that produced by treatments that cause depressive symptoms in humans. At a dose that caused the depressivelike effects in both the FST and ICSS assays, SalvA decreased extracellular concentrations of dopamine (DA) within the nucleus accumbens (NAc), a critical component of brain reward circuitry, without affecting extracellular concentrations of serotonin (5-HT). These data provide additional support for the hypothesis that stimulation of brain ␬-opioid receptors triggers depressive-like signs in rats and raise the possibility that decreases in extracellular concentrations of DA within the NAc contribute to these effects.

addictive drugs (see Wise, 1998; Nestler and Carlezon, 2005). It has been proposed that disruption of DA function within the NAc causes anhedonia (reduced ability to experience reward) (Wise, 1982), a hallmark sign of clinical depression. The mesolimbic DA system is modulated by noradrenergic and serotonergic inputs (Pasquier et al., 1977), as well as endogenous opioid peptides (Devine et al., 1993; Shippenberg and Rea, 1997; Svingos et al., 1999). Agents that selectively affect the function of ␬-opioid receptors cause profound alterations in mood in humans (Pfeiffer et al., 1986; Roth et al., 2002) and motivated behaviors in laboratory animals (Shippenberg and Herz, 1987; Todtenkopf et al., 2004), suggesting that manipulations targeting brain ␬-opioid systems might be useful in the study and treatment of depressive disorders.

ABBREVIATIONS: 5-HT, serotonin; NAc, nucleus accumbens; CREB, cAMP-response element-binding protein; E-2078, N-CH3-Tyr-Gly-GlyPhe-Leu-Arg-N-CH3-Arg-D-Leu-NHC2H5; HPLC, high-performance liquid chromatography; ICSS, intracranial self-stimulation; DMSO, dimethyl sulfoxide; FST, forced swim test; U-50488H, trans-(⫾)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methane sulfonate salt; U-69593; (5␣,7␣,8␤)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec8-yl)-benzenacetamide; SalvA, salvinorin A; SSRI, selective serotonin reuptake inhibitor; ANOVA, analysis of variance. 440

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Molecular studies in rodents suggest that complex experience-dependent alterations in the function of ␬-opioid systems within the NAc contribute to the development and expression of depressive behaviors. For example, stress elevates the activity of the transcription factor CREB within the NAc (Pliakas et al., 2001). Selective elevation of CREB function within the NAc increases immobility behavior in the forced swim test (FST) (Pliakas et al., 2001), a model often used to study depression. This effect is opposite to that caused by standard antidepressants and thus is a potential sign of depressive-like states (Porsolt et al., 1977; Cryan et al., 2002). Elevated expression of CREB in the NAc also reduces the rewarding effects of cocaine, a putative sign of anhedonia (Carlezon et al., 1998; Pliakas et al., 2001). The depressive-like behavioral effects that accompany elevated CREB function within the NAc seem related, at least in part, to CREB-regulated transcription of dynorphin, an endogenous ␬-opioid receptor ligand (Carlezon et al., 1998). The ␬-antagonist norbinaltorphimine attenuates the behavioral effects of elevated CREB expression within the NAc (Carlezon et al., 1998; Pliakas et al., 2001), suggesting a role for this receptor subtype in the expression of depressive-like behaviors. ␬-Antagonists also have antidepressant-like effects in normal rats (Pliakas et al., 2001; Mague et al., 2003; McLaughlin et al., 2003), even when microinjected directly into the NAc (Newton et al., 2002). In contrast, stimulation of ␬-receptors in rats produces complex behaviors that might reflect depressive- or dysphoric-like states. The ␬-agonist U-69593 establishes conditioned place aversions (Shippenberg and Herz, 1987), increases immobility in the FST (Mague et al., 2003), and elevates ICSS thresholds (Todtenkopf et al., 2004). These findings are consistent with the observation that ␬-agonists produce depressive or dysphoric states in humans (Pfeiffer et al., 1986). The mechanism of these effects likely involves reduced DA function. ␬-Agonists decrease extracellular concentrations of DA within the NAc (DiChiara and Imperato, 1988; Spanagel et al., 1992; Devine et al., 1993) through stimulation of ␬-receptors that regulate DA release from mesolimbic neurons (Donzanti et al., 1992; Shippenberg and Rea, 1997; Svingos et al., 1999). Considered together, these findings raise the possibility that elevated CREB-mediated transcription of dynorphin within the NAc leads to increased ␬-receptor activity, which decreases local DA function and triggers certain signs of depression. Salvinorin A (SalvA) is a psychoactive compound found in the leaves of Salvia divinorum, a plant from the mint family (Roth et al., 2002). Binding and function studies indicate that SalvA is a potent and highly selective ␬-agonist, with greater efficacy than that of the synthetic ␬-agonists U-50488H and U-69593 (Roth et al., 2002; Chavkin et al., 2004; Munro et al., 2005). SalvA is non-nitrogenous and has no structural similarity with other opioid agonists, making it an ideal agent with which to further examine relationships among dynorphin, ␬-opioid receptors, and depressive behavior. The present studies were designed to examine the effects of SalvA in tests (FST, ICSS) that have previously identified the depressive-like effects of U-69593 (Mague et al., 2003; Todtenkopf et al., 2004) and other treatment regimens (e.g., drug withdrawal) that cause depression in humans (Cryan et al., 2002). We also examined the effects of SalvA on locomotor activity to determine whether it causes nonspecific behavioral suppression that might complicate data interpretation.

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Finally, we examined the effects of SalvA on extracellular concentrations of DA and serotonin within the NAc using in vivo microdialysis in freely moving rats.

Materials and Methods Rats. A total of 129 male Sprague-Dawley rats (Charles River Laboratories, Boston MA) were used in these studies. Rats used for forced swim testing or locomotor activity testing were housed in groups of four and weighed 325 to 375 g at the time of testing, whereas those used for ICSS testing or brain microdialysis were housed singly and weighed 350 to 400 g at the time of stereotaxic surgery. All rats were maintained on a 12-h light (7:00 AM to 7:00 PM)/12-h dark cycle with free access to food and water, except during testing. Experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication 85-23, 1996) as well as McLean Hospital and National Institute on Drug AbuseIntramural Research Program policies. Salvinorin A. Dried S. divinorum leaves were purchased from Salvia Space (Lawrence, KS). SalvA (Fig. 1) was extracted, isolated, and purified using published methods with minor modifications (see Lee et al., 2005 and references therein). In brief, the leaves were treated sequentially with hexane and acetone. The acetone extract was purified by chromatography on activated carbon, and SalvA was crystallized from acetone and methanol to yield a white crystalline solid. Spectroscopic analyses confirmed that the SalvA obtained with these methods is chemically identical to that described in other reports (Roth et al., 2002). The samples used for testing were determined by HPLC to be ⬎99% pure. SalvA was dissolved in a vehicle of 75% dimethyl sulfoxide (DMSO), 25% distilled water and was administered by i.p. injection in a volume of 1 ml/kg. Forced Swim Test. Forty rats were used to study the effects of SalvA in the FST. The FST is a two-day procedure in which rats swim under conditions where escape is not possible. On the first day, the rats are forced to swim for 15 min. The rats initially struggle to escape from the water, but eventually they adopt a posture of immobility in which they make only the movements necessary to keep their heads above water. When the rats are retested 24 h later, immobility is increased. Treatment with standard antidepressant drugs within the 24-h period between the first exposure to forced swimming and retesting can block facilitated immobility, an effect correlated with antidepressant efficacy in humans (Porsolt et al., 1977; Detke et al., 1995). On the first day of the FST, rats were placed in clear 65-cm tall, 25-cm diameter Plexiglas cylinders filled to 48 cm with 25°C water.

Fig. 1. Chemical structure of salvinorin A, a potent, efficacious, and highly selective non-nitrogenous ␬-opioid receptor agonist.

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After 15 min of forced swimming, the rats were removed from the water, dried with towels, and placed in a warmed enclosure for 30 min. The cylinders were emptied and cleaned between rats. Rats (6 – 8 per treatment condition) received three separate injections of SalvA (0.125–2.0 mg/kg, i.p.) at 1, 19, and 23 h after the first exposure to forced swimming. This is a commonly used treatment regimen (Porsolt et al., 1977; Detke et al., 1995) that we followed to enable qualitative comparisons with previous studies of agents with antidepressant-like and prodepressant-like effects (Mague et al., 2003). At 24 h after the forced swim, rats were retested for 5 min (300 s) under identical swim conditions. Retest sessions were videotaped from the side of the cylinders and scored using a behavioral sampling method (Detke et al., 1995; Mague et al., 2003) by raters unaware of the treatment condition. Rats were rated at 5-s intervals throughout the duration of the retest session; at each 5-s interval, the predominant behavior was assigned to one of four categories: immobility, swimming, climbing, or diving. A rat was judged to be immobile if it was making only movements necessary to keep its head above water, climbing if it was making forceful thrashing movements with its forelimbs directed against the walls of the cylinder, swimming if it was actively making swimming movements that caused it to move within the center of the cylinder, and diving if it swam below the water toward the bottom of the cylinder. (Data quantifying diving behavior is not shown in the present report because it rarely occurred, and it was not affected by any of the treatments tested.) This behavioral sampling method differentiates certain classes of antidepressant drugs. For example, tricyclic antidepressants decrease immobility and increase climbing without affecting swimming, whereas selective serotonin reuptake inhibitors (SSRIs) decrease immobility and increase swimming without affecting climbing (Detke et al., 1995). It is also sensitive to treatments that cause depressive effects in humans, including antimanic agents and drug withdrawal (Carlezon et al., 2002; Cryan et al., 2002). The number of occurrences (to a maximum of 60) of each category of behavior was analyzed using separate one-way (treatment) analyses of variance (ANOVAs). Significant effects were analyzed using post hoc Newman-Keuls tests. Locomotor Activity. Fifty-eight rats were used to determine whether the doses of SalvA examined in the FST studies alter locomotor activity. These studies were conducted exactly as the FST studies had been conducted until the point of re-testing; i.e., all rats (8 –11/group) underwent the first day of the FST and were treated with SalvA at the normal pretreatment times (1, 19, and 23 h after swimming). At 24 h after the first exposure to forced swimming, the rats were placed for 1 h in automated 43.2 ⫻ 43.2 ⫻ 30.5 cm (L ⫻ W ⫻ H) activity chambers (MED Associates, St. Albans, VT) instead of being retested in the FST. The total number of activity counts (photocell beam breaks) during the 30-min test session was quantified in 5-min bins, and differences among the treatment groups were analyzed using a one-way ANOVA (for total counts) and a two-way (treatment ⫻ time) ANOVA with repeated measures. ICSS. Each of seven rats was anesthetized with a mixture of ketamine plus xylazine (80 mg/kg plus 12 mg/kg i.p.; Sigma-Aldrich, St. Louis, MO), and given subcutaneous atropine sulfate (0.25 mg/kg) to reduce bronchial secretions. Each rat was then implanted with a monopolar stainless steel electrode (0.250-mm diameter; Plastics One, Roanoke, VA) aimed at the left medial forebrain bundle at the level of the lateral hypothalamus (2.8 mm posterior to bregma, 1.7 mm lateral from the midsaggital suture, and 7.8 mm below dura; Paxinos and Watson, 1986). The electrodes were coated with polyamide insulation, except at the flattened tip. Skull screws (one of which served as the ground) and the electrode were secured to the skull with dental acrylic. After at least one week of recovery, the rats were trained on a continuous reinforcement schedule (FR1) to respond for brain stimulation using procedures described previously (Todtenkopf et al., 2004). Each lever-press earned a 0.5-s train of square-wave cathodal pulses (0.1-ms pulse duration) at a set frequency of 141 Hz. The

delivery of the stimulation was accompanied by the illumination of a 2-watt house light. Responses during the 0.5-s stimulation period did not earn additional stimulation. The stimulation current (100 –300 ␮A) was adjusted gradually to the lowest value that would sustain a reliable rate of responding (at least 40 rewards/min). Once the minimal effective current was found for each rat, it was held constant for all subsequent phases of training and testing. Each rat was then adapted to brief tests at its minimal effective current with each of the descending series of 15 stimulation frequencies. Each series comprised 1-min test trials at each frequency. For each frequency tested, there was an initial 5-s “priming” phase during which noncontingent stimulation was given followed by a 50-s test phase during which the number of responses was counted. Following the test phase, there was a 5-s time-out period during which no stimulation was available. The stimulation frequency was then lowered by approximately 10% (0.05 log10 units), and another trial was started. After responding had been evaluated at each of the 15 frequencies, the procedure was repeated such that each rat was given six such series per day (90 min of training). During the training procedure, the range of frequencies was adjusted for each rat so that only the highest seven to eight frequencies would sustain responding. To characterize the functions relating response strength to reward magnitude, a least-squares line of best fit was plotted across the frequencies that sustained responding at 20, 30, 40, 50 and 60% of the maximum rate using customized analysis software. ICSS threshold was defined as the frequency at which the line intersected the x-axis (␪-0; Miliaressis et al., 1986). Drug testing started when mean ICSS thresholds varied by less than 10% over three consecutive sessions. For drug testing, three rate-frequency functions (“curves”) were determined for each rat immediately before drug treatment. The first curve served as a warm-up period and was discarded because it tended to be unreliable. The second and third curves were averaged to obtain the baseline (threshold and maximal response rates) parameters. Each rat then received an i.p. injection of drug or vehicle, and four more 15-min rate-frequency curves were obtained (1 h of testing). All rats received the same daily treatments in a standardized order: saline, vehicle (75% DMSO), and SalvA at 0.125, 0.25, 0.5, 1.0, and 2.0 mg/kg i.p. The doses were given in ascending and then descending order, such that each rat received vehicle and each dose of the drug twice. In addition, on alternate days, rats were tested after injections of saline to ensure that they had recovered from prior treatment and to minimize the possibility of conditioned drug effects. To determine whether there were differences between the first and second tests with each treatment, the effects of saline, vehicle (75% DMSO), and SalvA on ICSS thresholds and maximal response rates over the test period were evaluated in separate two-way analyses of variance (ANOVAs) (drug dose ⫻ test number) with repeated measures. Data from the first and last tests with saline were used. The first and second tests at each dose were then combined into single means, and the drug effects on thresholds and maximum rates were evaluated with separate one-way ANOVAs. Significant effects were analyzed further using post hoc Newman-Keuls tests. In Vivo Microdialysis. Each of the 24 rats was anesthetized as described above, and an indwelling jugular catheter was implanted (Baumann et al., 2001) to enable administration of anesthetic during insertion of the microdialysis probes (see below). The rat was then placed in a stereotaxic instrument. A plastic intracerebral guide cannula (CMA 12; CMA/Microdialysis, Solna, Sweden) was implanted above the NAc (1.6 mm anterior to bregma, 1.6 mm lateral from the midsaggital suture, and 6.2 mm below dura; Paxinos and Watson, 1986) according to published methods (Baumann et al., 2001). The guide cannula was fixed to the skull using stainless steel screws and dental acrylic. Animals were singly housed postoperatively and were allowed 7 to 10 days to recover. On the evening before an experiment, rats were moved to the testing room and lightly anesthetized with an intravenous injection of 10 mg/kg methohexital, an ultra-rapid short-acting anesthesia. A microdialysis probe

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with a 2 ⫻ 0.5-mm exchange surface (CMA/12) was lowered into the guide cannula, and an extension tube (PE-50; Becton Dickinson, Sparks, MD) was attached to the jugular catheter. Each rat was placed into its own plastic container and was connected to a tethering system that allowed unrestricted movement within the container. The microdialysis inflow and outflow tubing, as well as the catheter extension tubing, were connected to a fluid swivel (Instech Laboratories, Inc., Plymouth Meeting, PA). Artificial Ringers’ solution containing 147.0 mM NaCl, 4.0 mM KCl, and 1.8 mM CaCl2 was pumped through the probe overnight at 0.5 ␮l/min. On the next morning, 10-␮l dialysate samples were collected at 20-min intervals. Samples were immediately assayed for DA and 5-HT by HPLC with electrochemical detection as described below. When three stable baseline samples were obtained, drug treatments were administered; rats received an i.p. injection of 1.0 mg/kg SalvA, 0.125 mg/kg SalvA, or vehicle (75% DMSO). Sampling continued for 2 h after treatment. Aliquots of the dialysate were injected into a microbore HPLC column (5 ␮m, C18, 100 ⫻ 1 mm, Unijet; BAS Bioanalytical Systems, West Lafayette, IN) that was coupled to an amperometric detector (Model LC-4C; BAS Bioanalytical Systems). A glassy carbon electrode was set at a potential of ⫹650 mV relative to an Ag/AgCl reference. Mobile phase consisting of 150 mM monochloroacetic acid, 150 mM NaOH, 2.5 mM sodium octanesulfonic acid, and 250 ␮M disodium EDTA with 1 ml of triethylamine, 6% MeOH, and 6% CH3CN per liter of water (final pH ⫽ 5) was pumped (model 260D; ISCO, Lincoln, NE) at a rate of 60 ␮l/min. Chromatographic data were acquired on-line and exported to a Millennium software system (Waters, Milford, MA) for peak amplification, integration, and analysis. Standards of DA and 5-HT were run daily before dialysate samples, and standard curves were linear over a wide range of concentrations (0.1–100 pg). A monoamine standard mixture containing DA, 5-HT, and their respective acid metabolites was injected before and after the experiment to ensure validity of the constituent retention times. Peak heights of unknowns were compared with peak heights of standards and the lower limit of assay sensitivity (3⫻ baseline noise) was 50 fg/5-␮l sample. Extracellular concentrations of DA and 5-HT were expressed as percentage of baseline, and the data for each neurotransmitter were analyzed separately. Differences among the treatment groups were analyzed using two-way (treatment ⫻ time) ANOVA with repeated measures on the time factor. Significant effects were analyzed further using post hoc Newman-Keuls tests. Histology. Rats that had undergone stereotaxic surgery to implant ICSS electrodes or microdialysis probes were overdosed with pentobarbital (130 mg/kg i.p.) and perfused with 4% paraformaldehyde. The fixed brains were sliced in 40-␮m sections for cresyl violet staining to confirm placements.

Results In the FST, SalvA produced effects on behavior (Fig. 2a) that were opposite to those typically seen after administration of selective serotonin reuptake inhibitors (see Detke et al., 1995; Mague et al., 2003). Specifically, SalvA dose dependently increased occurrences of immobility (F5,34 ⫽ 5.98, P ⬍ 0.01) and decreased occurrences of swimming behavior (F5,34 ⫽ 6.07, P ⬍ 0.01). There was no effect on climbing or diving behaviors (data not shown). Post hoc analyses revealed that SalvA significantly increased immobility at 0.25 mg/kg (P ⬍ 0.05, Newman-Keuls test), 0.5 mg/kg (P ⬍ 0.05), 1.0 mg/kg (P ⬍ 0.01), and 2.0 mg/kg (P ⬍ 0.01). Similarly, SalvA significantly decreased swimming behavior at all doses from 0.25 to 2.0 mg/kg (P ⬍0.01). In contrast, SalvA caused no treatment-related differences in locomotor activity in an open field at any of the doses tested, regardless of whether the

Fig. 2. Effects of SalvA on behavior in the FST and in an open field. Doses are expressed as milligram/kilogram. a, in the FST, SalvA increased occurrences of immobility and decreased occurrences of swimming, without affecting climbing (means ⫾ S.E.M.). ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01 compared with vehicle, Newman-Keuls tests, six to eight rats/group. b, SalvA did not affect locomotor activity (means ⫾ S.E.M.) at doses with prodepressant-like effects in the FST (8 –11 rats per group).

data were analyzed as 5-min bins (Fig. 2b) or as total activity over the 30-min test period (data not shown). As would be expected, activity levels decreased significantly in all groups over the course of the 30-min test period (F5,260 ⫽ 350.5, P ⬍ 0.01). In the ICSS assay, there was no effect of repeated testing with SalvA on thresholds or maximal rates at any of the doses tested (data not shown). Because there were no effects of repeated testing, data from the first and second test sessions were combined into single means for each condition. The effects of SalvA on ICSS thresholds depended upon treat-

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ment (F6,42 ⫽ 19.0, P ⬍ 0.01); the drug produced effects on behavior (Fig. 3a) that were opposite to those typically seen after administration of psychomotor stimulant drugs such as cocaine (see Mague et al., 2003). Treatment with vehicle tended to cause small nonsignificant increases in ICSS thresholds and decreases in maximum response rates in comparison with saline, suggesting nonspecific effects of DMSO. In comparison with vehicle, SalvA significantly increased

Fig. 3. Effect of SalvA on ICSS. a, treatment with vehicle (75% DMSO) tended to cause small nonsignificant increases in ICSS thresholds (mean ⫾ S.E.M.) over the course of the 1-h test sessions. SalvA significantly increased ICSS thresholds at the doses that produced prodepressant-like effects in the FST. ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01 compared with vehicle, Newman-Keuls tests, seven rats per group. b, in comparison with vehicle, none of the treatments affected response capabilities (maximal rates).

ICSS thresholds within the same range of doses that had behavioral effects in the FST: 0.5 mg/kg (P ⬍ 0.05), 1.0 mg/kg (P ⬍ 0.01), and 2.0 mg/kg (P ⬍ 0.01). SalvA also appeared to affect maximal response rates (F6,42 ⫽ 3.72, P ⬍ 0.01). However, post hoc analyses revealed that this effect was due to differences between saline treatment and SalvA at 1.0 and 2.0 mg/kg. There were no differences between maximum rates after vehicle and any dose of SalvA, again suggesting nonspecific effects of DMSO on behavior in this assay. In the in vivo microdialysis studies, systemic administration of SalvA produced effects on extracellular concentrations of DA in the NAc that depended upon an interaction between dose and time (F16,168 ⫽ 9.47, P ⬍ 0.01) (Fig. 4a). Injection of 1.0 mg/kg SalvA produced rapid decreases in DA that were detectable for the entire 2-h test period (P ⬍ 0.01 for 0 –100 min; P ⬍ 0.05 for 100 –120 min), whereas DMSO or 0.125 mg/kg SalvA had no effect at any time point. In contrast, none of the treatments affected extracellular concentrations of 5-HT at any of the time points tested (Fig. 4b). All ICSS electrodes were located within the medial forebrain bundle at the level of the lateral hypothalamus. The placements were indistinguishable from those depicted previously (Todtenkopf et al., 2004). Similarly, all microdialysis probes were located within the NAc (Fig. 5). As such, data from all rats were included in the analyses.

Fig. 4. Effect of SalvA on extracellular concentrations of DA and 5-HT in the NAc, as measured by in vivo microdialysis and HPLC. a, SalvA caused rapid and long-lasting decreases in extracellular concentrations of DA. ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01 compared with vehicle, Neuman-Keuls tests, eight rats per group. b, in contrast, the drug did not affect extracellular concentrations of 5-HT.

Depressive Effects of Salvinorin A

Fig. 5. Histological reconstruction of the areas sampled in the microdialysis studies. All probes were located in the NAc; the active portions of the membranes are depicted by the black lines.

Discussion Systemic administration of SalvA—a potent, efficacious, and highly selective ␬-opioid receptor agonist (Roth et al., 2002; Chavkin et al., 2004)— has profound behavioral effects in rats. SalvA increased the occurrences of immobility behavior in the FST, an assay often used in the study of depression (Cryan et al., 2002). This general effect is opposite to that seen with virtually all types of antidepressant treatments used in humans, including noradrenergic reuptake inhibitors and SSRIs (Porsolt et al., 1977; Detke et al., 1995; Mague et al., 2003). This finding suggests that SalvA produces prodepressant-like effects in the FST. The increase in immobility behavior was accompanied by a decrease in swimming be-

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haviors. This specific effect is opposite to that seen with SSRIs, which decrease immobility and increase swimming behaviors (Detke et al., 1995). Although an increase in swimming behaviors is associated with the serotonergic actions of SSRIs, the significance of a decrease in swimming behaviors is unknown. This pattern of results might be an early indicator that the effects of SalvA on behavior in the FST involve 5-HT systems in critical brain regions. Moreover, SalvA increased ICSS thresholds, suggesting that it reduced the rewarding impact of medial forebrain bundle stimulation. Considering that reduced sensitivity to rewarding stimuli in rodents reflects anhedonia—a hallmark sign of clinical depression—these ICSS data provide additional evidence that SalvA produces depressive-like effects. We have reported previously that U-69593 produces the same pattern of results in these tests (Mague et al., 2003; Todtenkopf et al., 2004), suggesting a general effect of ␬-agonists rather than a specific effect of SalvA. The potency and efficacy of SalvA in these tests are approximately equivalent to those seen with U-69593, although the present studies were designed to enable qualitative rather than quantitative comparisons between these agents. Considering that SalvA binds to few (if any) other types of receptors in the brain (Roth et al., 2002), these data suggest that selective stimulation of ␬-receptors can produce depressive signs, at least in rats. One concern when using the FST is that nonspecific treatment effects on activity levels could complicate data interpretation. We conducted locomotion studies in parallel with the FST studies to identify potentially confounding effects. To facilitate comparisons between the assays, the locomotion studies were conducted under conditions identical to those used in the FST studies, except during the second day of testing the rats were placed in activity chambers rather than being re-exposed to forced swimming. SalvA did not alter activity at any of the doses tested. Additionally, the methods we use to conduct the ICSS test also enable analyses of whether SalvA affects the performance capabilities of the rats. Although there were nominal decreases in response rates over the 1-h ICSS test session, this was a general effect of the 75% DMSO vehicle rather than a specific effect of SalvA. Thus it seems unlikely that nonspecific effects of SalvA on performance capabilities can explain the prodepressant-like effects of the drug in the FST or ICSS tests at the doses tested here. The mechanisms that mediate these putative prodepressant actions of SalvA are not known. One possibility is that SalvA, through selective actions at ␬-receptors, affects the function of DA systems. Indeed, previous reports in rodents indicate that systemic or intracerebroventricular administration of ␬-agonists (e.g., U-50488H or the dynorphin analog E-2078) decreases extracellular concentrations of DA within the NAc (DiChiara and Imperato, 1988; Spanagel et al., 1992; Devine et al., 1993; Maisonneuve et al., 1994). These effects may be mediated, at least in part, within the NAc itself (Donzanti et al., 1992). Within the NAc, ␬-receptors are located on the terminals of DA neurons (Shippenberg and Rea, 1997; Svingos et al., 1999), where they are in a position to regulate DA release. In addition, inhibitory actions within the midbrain may also contribute to decreased extracellular concentrations of DA within the NAc (Margolis et al., 2003). Regardless, our microdialysis data confirm previous observations that ␬-agonists reduce activation of the mesolimbic DA

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system and extend them to SalvA, which is more selective and efficacious than the other agents. In contrast to the effects of SalvA on extracellular DA in the NAc, the drug had no effects on 5-HT concentrations in this region. These data provide early evidence that the decreases in swimming activity in the FST do not reflect reductions in 5-HT activity within the NAc, although different effects in other brain regions (e.g., raphe nucleus) cannot be ruled out. Considered together, our data raise the possibility that decreases in DA in the NAc contribute to the depressive-like behaviors that were observed in the present studies. Indeed, it has been proposed previously (Wise, 1982) that decreased DA function in the NAc produces anhedonia. Interestingly, a recent report in which in vivo microdialysis was performed in mice suggests that SalvA reduces extracellular concentrations of DA within the caudate putamen, but not within the NAc (Zhang et al., 2005). It is not clear why stimulation of ␬-receptors in mice would produce a different pattern of results than those seen here with SalvA, or in previous studies with other ␬-agonists (DiChiara and Imperato, 1988; Spanagel et al., 1992; Donzanti et al., 1992; Devine et al., 1993; Maisonneuve et al., 1994). This discrepancy raises the possibility that rats and mice have different ␬-receptor distributions or different sensitivities to ␬-opioid modulation of mesolimbic DA function, or that the small size of the striatum in mice makes it difficult to resolve the caudate putamen from the NAc in microdialysis studies. Alterations in the function of ␬-opioid systems may be part of a complex process that leads to the development and expression of depression or other mood disorders. Factors that can trigger depression in humans and depressive-like behaviors in laboratory animals—including stress and drug withdrawal—increase the activity of CREB in the NAc (Pliakas et al., 2001; Barrot et al., 2002; Shaw-Lutchman et al., 2002; Chartoff et al., 2003). Increased CREB activity in the NAc, in turn, leads to elevated expression of the gene for dynorphin (Carlezon et al., 1998). The possibility that increased stimulation of ␬-receptors causes depressive-like behaviors is supported by the finding that selective ␬-antagonists produce antidepressant-like effects in models used to study depression (Pliakas et al., 2001; Newton et al., 2002; Mague et al., 2003; McLaughlin et al., 2003). These findings, together with other evidence, have raised the possibility that selective ␬-antagonists might have clinical utility as antidepressants (Pliakas et al., 2001; Mague et al., 2003) or treatments for drug addiction (Rothman et al., 2000). Under some circumstances, the ability of ␬-agonists to decrease DA function in the basal forebrain might also have clinical utility. For example, antipsychotic drugs—which are frequently used to treat mania—activate dynorphinergic neurons (Ma et al., 2003). Although SalvA produces hallucinogenic effects under some conditions (e.g., when smoked) (Roth et al., 2002; Chavkin et al., 2004; Bucheler et al., 2005), it is conceivable that administration of a derivative or related compound under more carefully controlled conditions might be useful for the treatment of disorders characterized by hyperfunction of dopamine systems (e.g., mania). Less selective ␬-agonists (e.g., enadoline, spiradoline) can produce measures of sedation and decrease the frequencies of tics in individuals with Tourette’s Syndrome (Chappell et al., 1993; Walsh et al., 2001). Regardless, the present studies confirm and extend

the idea that ␬-opioid receptors may play a key role in regulating complex mood states. References Barrot M, Olivier JD, Perrotti LI, DiLeone RJ, Berton O, Eisch AJ, Impey S, Storm DR, Neve RL, Yin JC, et al. (2002) CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli. Proc Natl Acad Sci USA 99:11435–11440. Baumann MH, Ayestas MA, Dersch CM, and Rothman RB (2001) 1-(m-Chlorophenyl)piperazine (mCPP) dissociates in vivo serotonin release from long-term serotonin depletion in rat brain. Neuropsychopharmacology 24:492–501. Bucheler R, Gleiter CH, Schwoerer P, and Gaertner I (2005) Use of nonprohibited hallucinogenic plants: increasing relevance for public health? A case report and literature review on the consumption of Salvia divinorum (Diviner’s Sage). Pharmacopsychiatry 38:1–5. Carlezon WA Jr, Pliakas AM, Parrow AM, Detke MJ, Cohen BM, and Renshaw PF (2002) Antidepressant-like effects of cytidine in the forced swim test in rats. 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Depressive Effects of Salvinorin A Roth BL, Baner K, Westkaemper R, Siebert D, Rice KC, Steinberg S, Ernsberger P, and Rothman RB (2002) Salvinorin A: a potent naturally occurring non-nitrogenous kappa opioid selective agonist. Proc Natl Acad Sci USA 99:11934 –11939. Rothman RB, Gorelick DA, Heishman SJ, Eichmiller PR, Hill BH, Norbeck J, and Liberto JG (2000) An open-label study of a functional opioid kappa antagonist in the treatment of opioid dependence. J Subst Abuse Treat 18:277–281. Shaw-Lutchman TZ, Barrot M, Wallace T, Gilden L, Zachariou V, Impey S, Duman RS, Storm D, and Nestler EJ (2002) Regional and cellular mapping of cAMP response element-mediated transcription during naltrexone-precipitated morphine withdrawal. J Neurosci 22:3663–3672. Shippenberg TS and Herz A (1987) Place preference conditioning reveals the involvement of D1-dopamine receptors in the motivational properties of mu- and kappaopioid agonists. Brain Res 436:169 –172. Shippenberg TS and Rea W (1997) Sensitization to the behavioral effects of cocaine: modulation by dynorphin and kappa-opioid receptor agonists. Pharmacol Biochem Behav 57:449 – 455. Spanagel R, Herz A, and Shippenberg TS (1992) Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. Proc Natl Acad Sci USA 89:2046 –2050. Svingos AL, Colago EE, and Pickel VM (1999) Cellular sites for dynorphin activation

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of kappa-opioid receptors in the rat nucleus accumbens shell. J Neurosci 19:1804 – 1813. Todtenkopf MS, Marcus JF, Portoghese PS, and Carlezon WA Jr (2004) Effects of kappa opioid ligands on intracranial self-stimulation in rats. Psychopharmacology 172:463– 470. Walsh SL, Strain EC, Abreu ME, and Bigelow GE (2001) Enadoline, a selective kappa opioid agonist: comparison with butorphanol and hydromorphone in humans. Psychopharmacology 157:151–162. Wise RA (1982) Neuroleptics and operant behavior: the anhedonia hypothesis. Behav Brain Sci 5:39 – 87. Wise RA (1998) Drug-activation of brain reward pathways. Drug Alcohol Depend 51:13–22. Zhang Y, Butelman ER, Schlussman SD, Ho A, and Kreek MJ (2005) Effects of the plant-derived hallucinogen salvinorin A on basal dopamine levels in the caudate putamen and in a conditioned place aversion assay in mice: agonist actions at kappa opioid receptors. Psychopharmacology 179:551–558.

Address correspondence to: Dr. Bill Carlezon, Department of Psychiatry, McLean Hospital, MRC 217, 115 Mill Street, Belmont, MA 02478. E-mail: [email protected]

Drug and Alcohol Dependence 85 (2006) 157–162

Pattern of use and subjective effects of Salvia divinorum among recreational users D´ebora Gonz´alez a , Jordi Riba b , Jos´e Carlos Bouso a , Gregorio G´omez-Jarabo a , Manel J. Barbanoj b,∗ a

C´atedra de la Fundaci´on Cultural F´orum Filat´elico de Psicobiolog´ıa y Discapacidad, Departamento de Psicolog´ıa Biol´ogica y de la Salud, Facultad de Psicolog´ıa, Universidad Aut´onoma de Madrid, Madrid, Spain b Centre d’Investigaci´ o de Medicaments, Institut de Recerca, Servei de Farmacologia Cl´ınica, Hospital de la Santa Creu i Sant Pau, Departament de Farmacologia i Terap`eutica, Universitat Aut`onoma de Barcelona, Barcelona, Spain Received 9 March 2006; received in revised form 10 April 2006; accepted 12 April 2006

Abstract Backgroud: Salvia divinorum is a member of the Lamiaceae family and contains the psychotropic diterpene and kappa-opioid receptor agonist salvinorin-A. Originally a shamanic inebriant used by the Mexican Mazatec Indians, the plant and its preparations are becoming increasingly popular among non-traditional users. Methods: Demographic data and information on pattern of use and subjective effects were obtained by means of self-report questionnaires from a sample of 32 recreational users of salvia and other psychedelics. Results: Involvement with salvia appeared to be a recent phenomenon. Smoking the extract was the preferred form of administration. Subjective effects were described as intense but short-lived, appearing in less than 1 min and lasting 15 min or less. They included psychedelic-like changes in visual perception, mood and somatic sensations, and importantly, a highly modified perception of external reality and the self, leading to a decreased ability to interact with oneself or with one’s surroundings. Conclusions: Although some aspects of the subjective effects reported were similar to high doses of classical psychedelics with serotonin-2A receptor agonist activity, the intense derealization and impairment reported appear to be a characteristic of salvia. The observed simultaneous high scores on the LSD and PCAG subscales of the Addiction Research Center Inventory (ARCI) have been previously reported for other kappa-opioid agonists, and support kappa receptor activation as the probable pharmacologic mechanism underlying the modified state of awareness induced by salvia. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Salvia divinorum; Pattern of use; Subjective effects; Retrospective assessment

1. Introduction Salvia divinorum (Lamiaceae) is a psychotropic mint whose leaves are used for medicinal and religious purposes by Mazatec shamans in the Mexican state of Oaxaca (Wasson, 1962; Valdes et al., 1983). The Mazatecs, who call the plant “ska pastora” or “ska Maria pastora”, meaning “leaves of the shepherdess” or “leaves of Mary the shepherdess”, traditionally ingest the plant as a water infusion or by eating the fresh leaves (Wasson, 1962; Valdes et al., 1983). Early ethnological research found ∗ Corresponding author at: Centre d’Investigaci´ o de Medicaments, Institut de Recerca, Hospital de la Santa Creu i Sant Pau, St. Antoni Maria Claret, 167, Barcelona 08025, Spain. Tel.: +34 93 291 90 19; fax: +34 93 291 92 86. E-mail address: [email protected] (M.J. Barbanoj).

0376-8716/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.drugalcdep.2006.04.001

that the Mazatecs regard the psychotropic effects elicited by the plant as weak and use it only in substitution of the psilocybincontaining mushrooms when these are scarce (Wasson, 1962). However, the plant’s apparently weak potency could be due to limited absorption of the active principle when ingested orally (Ott, 1995). Despite its initial reputation as a lesser drug, interest for salvia has greatly increased in recent years among recreational users for the modified state of awareness it can elicit. The use of salvia has spread to Europe and North America in a similar fashion to other natural drugs, as the DMT-containing ayahuasca did a decade ago (Riba and Barbanoj, 2005). However, unlike many ayahuasca users, current non-traditional users of salvia have accessed the plant and its preparations outside a religious context, mainly through “smart shops” and internet websites selling

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psychotropic plants and extracts, paraphernalia and dietary supplements (Dennehy et al., 2005). The term “smart shop” originated in The Netherlands and describes stores where natural psychoactive drugs such as ephedra, mescaline-containing cacti, psilocybian mushrooms and salvia extracts are sold. Such stores can also be found in Spain but their activities have been restricted since a decree was issued prohibiting the sale of a large number of plants, including salvia (see http://www.boe.es, number 32, 6 February 2004). S. divinorum owes its psychoactive properties to salvinorinA, its main active principle. This compound is a neoclerodane diterpene which was first isolated and identified by Ortega et al. (1982), and shortly after by Valdes et al. (1984). Recent pharmacological research has found it to be a highly selective full agonist of the kappa-opioid receptor (Roth et al., 2002; Butelman et al., 2004; Chavkin et al., 2004). Salvinorin-A is the only nonnitrogenous natural compound known to date to exert agonistic activity at these sites. Furthermore, in contrast with the classical psychedelics, salvinorin-A does not interact with the serotonin2A receptor, but presumably induces its psychotropic effects through activation of the kappa-opioid receptor. Contrary to what was initially assumed, salvinorin-A can be quite powerful. Inhalation of the vaporized active principle has been found to be active in doses as low as 200 ␮g (Siebert, 1994), in the same range as LSD. Recreational users have developed methods of administration that appear to lead to intense psychoactivity. These include chewing the leaves and retaining the juices in the mouth to allow absorption through the mucosa and obtaining concentrated extracts that can be administered either sublingually, applied to the buccal mucosa, or smoked (Siebert, 1994; Ott, 1995). The subjective effects described in self-experiments and case reports range widely, from increased relaxation, to laughter, colored visions, out-of-body experiences and loss of consciousness (Siebert, 1994; B¨ucheler et al., 2005; Dennehy et al., 2005). In the present study we aimed to obtain systematic information on the pattern of use and the nature of the subjective effects elicited by salvia in recreational users. Self-report questionnaires were administered to the participants to obtain demographic and subjective effect data. 2. Methods

pleasant and unpleasant after-effects, and any potential problems they might have experienced derived from salvia use. Information on salvia-induced subjective effects was obtained by means of the retrospective assessment of drug effects when they last took salvia.

2.2. Subjective effect measures Retrospective assessment of the subjective effects induced by salvia was conducted by means of self-assessment questionnaires. The following questionnaires were administered: The Hallucinogen Rating Scale or HRS (Strassman et al., 1994) measures psychedelic-induced subjective effects and includes 71 items distributed into six scales: Somaesthesia, reflecting somatic effects including interoceptive, visceral and tactile effects; affect, sensitive to emotional and affective responses; volition, indicating the volunteer’s capacity to willfully interact with his/her “self” and/or the environment; cognition, describing modifications in thought processes or content; perception, measuring visual, auditory, gustatory and olfactory experiences; finally intensity, which reflects the strength of the overall experience. The range of scores for all scales is 0–4. In the present study, a Spanish version of the questionnaire was administered (Riba et al., 2001a). The HRS has proven sensitive to various psychedelics such as intravenous DMT (Strassman et al., 1994), oral psilocybin (Gouzoulis-Mayfrank et al., 1999) and ayahuasca (Riba et al., 2001b, 2003). The ARCI (Martin et al., 1971) consists of five scales or groups: MBG, morphine-benzedrine group, measuring euphoria and positive mood; PCAG, pentobarbital-chlorpromazine-alcohol group, measuring sedation; LSD, lysergic acid diethylamide scale, measuring somatic-dysphoric effects; BG, the benzedrine group, measuring intellectual energy and efficiency, and the A scale, an empirically derived scale measuring amphetamine-like effects. The range of scores is 0–16 for MBG, −4 to 11 for PCAG, −4 to 10 for LSD, −4 to 9 for BG, and 0–11 for A. A validated Spanish version was administered (Lamas et al., 1994). The State-Trait Anxiety Inventory-S (STAI-S) is a brief 20-item self-rating scale for the assessment of state anxiety (Spielberger et al., 1970). A validated Spanish version was administered (Seisdedos, 2002). The normative data for the Spanish adaptation differs from the original data for the American version. The reported mean (S.D.) values reported for State anxiety in the normal population are 20.54 (10.56) for male adults and 23.30 (11.93) for female adults (Seisdedos, 2002). The Altered States of Consciousness Questionnaire (“Aussergew¨ohnliche Psychische Zust¨ande”, APZ) developed by Dittrich (1998). It includes 72 items distributed in three subscales: Oceanic Boundlessness (“Ozeanische Selbstentgrenzung”, OSE), measuring changes in the sense of time, derealization and depersonalization; Dread of Ego-Dissolution (“Angstvolle IchAufl¨osung”, AIA) measuring thought disorder and decreased body and thought control associated with arousal and anxiety and Visionary Restructuralization (“Vision¨are Umstrukturierung”, VUS) referring to visual phenomena, such as illusions, hallucinations and synesthesia and to changes in the significance of objects. The range of scores is 0–13 for OSE, 0–22 for AIA, and 0–14 for VUS. A Spanish version of the questionnaire previously used in clinical studies involving psychedelic drugs was administered (Riba et al., 2002).

2.1. Sample 2.3. Statistical analysis The sample was recruited by direct approach by the first author, who also conducted the interviews. Potential participants had to have used salvia at least once in their lifetime. Given the infrequent nature of the behavior under study, adaptive sampling was used with participants referring to acquaintances who had also had experience with the drug (Thompson and Collins, 2002). Several leads were followed, so participants did not belong to a single social network. After initial contact with the first author, participants were given the forms, which they took away, filled out and later returned to the investigator. Anonymity of the information was guaranteed and all the participants gave their written consent to participate. The study was approved by the ethics committee at the Hospital de Sant Pau in Barcelona. Participants had not taken part in any clinical study conducted by our group and did not receive any payment for their participation in the present survey. Demographic information was collected from the participants, together with information on drug use history and salvia use history, route of administration,

The data presented in the present paper are descriptive in nature and accordingly, descriptive statistics are provided in Section 3. Percentages are reported for categorical variables and means and standard deviations for continuous variables obtained from subjective effect questionnaires. Given the small sample size, no inferential statistics were used to find differences associated with gender or route of administration.

3. Results 3.1. Demographic characteristics of the sample A total of 32 salvia users were recruited, 18 (56%) of whom were male and 14 (44%) were female. The mean age of the sam-

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ple was 25 years (S.D.: 4.32; range: 18–40 years). Education level was high with 23 (72%) of the sample having completed high school and 7 (22%) having obtained a university degree. At the time of the survey, 22 (69%) were attending university. Seventeen participants (53%) were full-time students, 6 (19%) combined studies with part-time jobs, 7 (22%) worked exclusively, and 2 (6%) were unemployed. 3.2. History of drug use (other than salvia) Except for two participants, all those in the study (93.7%) had a drink containing alcohol weekly. The average number of alcoholic drinks per week among the drinkers was 3.13 (S.D.: 2.69, range: 1–14). More than four-fifths of the participants (84.4%) were smokers, with a mean number of 14 cigarettes per day (S.D.: 6.74, range: 1–25). Except for one participant, all participants (96.9%) consumed cannabis at least once a week. The average number of cannabis joints was 21.32 per week (S.D.: 15.68, range: 2–70). They also had wide experience with other drugs; ecstasy had been used by 88%, cocaine by 84%, amphetamines 69%, opiates 56%, benzodiazepines 36%, and GHB 9%. Ninety-four percent of the volunteers had at some time used a psychedelic/hallucinogen, the most frequent being psilocybian mushrooms (78% of all participants), followed by LSD (63%), ketamine (34%), ayahuasca (28%), Amanita muscaria (13%), peyote (6.3%) mescaline (3%) and Datura stramonium (3%). Ten volunteers (31%) reported having consumed “other psychedelics” not listed in the questionnaire. Specified were: 2C-B (four volunteers), San Pedro (one volunteer) and Argyreia nervosa (one volunteer). 3.3. History and pattern of use of salvia Participants appeared to have first experienced salvia only recently, with 88% having used it for the first time in the last year. The average number of times the drug was consumed was 2 (range: 1–5). The source of the salvia was a “smart shop” in 88% of the cases and in the remaining 12% it had been obtained from a friend, without further specifying the source. All participants had consumed salvia as an extract and three (9%) had also used the leaves. Commercially available extracts usually consist of ground salvia leaves impregnated with salvia tincture, so that the final product may contain 5, 10 or 20 times the original salvinorin-A concentration. Regarding the preferred route of administration, 75% reported having smoked the extract, 22% reported combining sublingual and smoked administration and 3% (one subject) reporting smoking the leaves and the extract combined. As to the smoking technique, all volunteers reported using a bong or a pipe. No participant reported smoking it in the form of cigarrettes or mixing salvia with tobacco or marijuana. When asked about the psychotropic potency of salvia, 75% of the participants described the experience elicited by salvia from “intense” to “very intense” or “extremely intense”, with only 19% as “moderate” and 6% describing it as “slight”.

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Participants were asked to state the best and the worst aspects of their salvia experiences. These are listed in Table 1. The most commonly cited positive effects were the “trip” the drug elicits (41%), followed by its euphoric (28%) and dissociative effects (19%). Among the worst aspects, its short duration (38%) was the most frequently cited. Sixteen percent of the volunteers mentioned the lack of control over the experience and 13% the unpleasant after-effects as the worst aspect of salvia. Thirteen percent of the volunteers could find no negative aspect related to the experience. Fourteen volunteers (44%) reported having experienced some degree of malaise, hang-over or “comedown” immediately after the acute effects of salvia. These effects are also listed in Table 1 and essentially describe physical and mental tiredness. All volunteers unanimously agreed that these unpleasant effects were no longer present 1 day after salvia use, and that they had never experienced any mid-term unpleasant sensations they could attribute to salvia. Only one volunteer commented on having had problems with studies, work or relatives due to the use of salvia. He complained that friends who do not habitually use psychotropic substances were worried about his experimenting with drugs. Twenty participants (63%) commented that the effects of salvia were similar to those of other drugs. Subjects in this subgroup cited the following drugs, from most to least frequent: psilocybian mushrooms (55%), ayahuasca (20%), ketamine (20%), LSD (20%), marijuana (20%), MDMA (15%), opium (15%), poppers (15%), 2C-B (15%), Amanita muscaria (10%) and DMT (5%). Finally, when asked if they would like to take salvia regularly, only 44% of the subjects responded affirmatively. 3.4. Retrospective assessment of the most recent salvia consumption Participants responded to the subjective effect questionnaires recalling the effects they had experienced when they last took salvia. Fifty-six percent of the participants had used salvia for the last time within the preceding month, and 38% had last used salvia between the preceding month and the preceding year. Only 6% of the participants had used salvia more than a year ago. The preparation or part of the plant they had used on this last occasion was the extract in 91% of the cases and the leaves in 6% of the cases, while 3% declared having used a combination of smoked leaves plus smoked extract. Regarding the route of administration, 72% had smoked the extract, whereas 19% had combined smoking the extract and placing the extract sublingually. Two volunteers (6%) had smoked the leaves and one volunteer (3%) had combined smoking both the leaves and the extract. As to the intensity of the experience, all participants declared having experienced psychotropic effects; these were “slight” for 6% of volunteers, “moderate” for 22% of the sample, “intense” for 12%, “very intense” for 41% and “extremely intense” for 19%. The onset of effects was found to be “instantaneous” by 31% of the volunteers, “less than a minute” by 57% of the volunteers,

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Table 1 Volunteers’ written descriptions of the best and worst aspects of salvia and any unpleasant after-effect Best things about using salvia

n

Worst things about using salvia

n

Unpleasant after-effects

n

The “trip”, entering another reality Laughter, happiness, well-being

13 9

Short duration Lack of control over the experience

12 5

4 4

Separation from body, dissociation Visual effects Rapid onset of effects Its great potency Relaxation Perceptual modifications The “high” Loss of consciousness Novelty Pleasant after-effects Mental clarity Escape Auditory effects Dizziness

6 5 3 3 2 2 2 2 2 1 1 1 1 1

Tiredness Heaviness of head, like after smoking many marihuana joints Dizziness Physically exhausted Grogginess Mental slowness

Unpleasant after-effects None Unpleasant physical effects Excessively intense Effects are unreliable Onset too rapid

4 4 3 2 1 1

3 3 1 1

n: number of subjects reporting a specific effect.

“from 1 to 5 min” by 6% of the volunteers. Only one volunteer (3%) declared that “from 5 to 15 min” had elapsed and another (3%) declared that “half an hour had elapsed”. Separating by route of administration, the onset of effects after smoking the extract was found to be “instantaneous” or “less than a minute” according to 91% of participants who chose this route. Only 67% of those participants who combined sublingual extract plus smoked extract described the onset with one of these two categories.

Table 2 Mean (S.D.) scores obtained for the HRS, ARCI and APZ questionnaire subscales HRS

Scores

Somaesthesia Affect Perception Cognition Volition Intensity

1.42 (0.62) 1.66 (0.53) 1.53 (0.88) 1.32 (0.70) 1.98 (0.55) 2.50 (0.53)

ARCI

Scores

A BG MBG PCAG LSD

4.41 (1.81) −0.34 (1.64) 5.75 (3.06) 2.75 (3.38) 4.25 (2.43)

APZ

Scores

OSE AIA VUS

6.09 (3.44) 6.28 (4.30) 4.78 (3.99)

A: amphetamine scale; BG: benzedrine group; MBG: morphine-benzedrine group; PCAG: pentobarbital-chlorpromazine-alcohol group; LSD: lysergic acid diethylamide scale. OSE: Oceanic Boundlessness; AIA: Dread of EgoDissolution VUS: Visionary Restructuralization.

The duration of effects was described as “less than a minute” by 6% of participants, “between 1 and 5 min” by 60% of participants, “between 5 and 15 min” by 19%, “between 15 and 30 min” by 9% of participants. Only one volunteer (3%) described the duration to be “between 30 min and 1 h” and another (3%) described duration “between 1 and 2 h”. Separating by route of administration, 70% of those who had smoked the extract chose the options “less than a minute” or “between 1 and 5 min”, compared to 50% who combined sublingual plus smoked administration. Effects lasting longer than 5 min were described by 13% of participants who smoked the extract, and by 33% of participants who combined sublingual plus smoked. 3.4.1. HRS, ARCI and APZ questionnaires. Table 2 shows mean scores and standard deviations for the different subscales of these three questionnaires. 3.4.2. STAI-S. A mean (S.D.) score of 27.3 (8.5) was obtained for the STAI-S questionnaire. Separated by gender, scores of 26.9 (1.6) were obtained for male participants and 27.8 (2.8) for female participants. 4. Discussion Results from the present study show that awareness and involvement with salvia appears to be a recent phenomenon. Most participants had had their first contact with salvia during the last year, and had consumed it on average only on two occasions, mainly smoking the extract, which almost all participants had acquired in “smart shops”. It is worth mentioning here that the survey was conducted during the second half of 2003 and the first-half of 2004. In February 2004 a decree from the Spanish government prohibited the sale of salvia in the country (www.boe.es, number 32, 6 February 2004), but the product was still available for some months after that date. It is likely that Spanish users will now turn to internet sites or to “smart shops”

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in other countries, such as The Netherlands, in order to purchase the product. Although the effects of salvia were compared by the participants to those of other psychedelics, they differed in various aspects, particularly their extremely short duration. The effects seem to be by far the shortest amongst perception-modifying drugs, surpassing intravenous DMT (Strassman et al., 1994). Other important qualitative differences found are discussed below. Scores on the HRS subscales confirm the psychedelic-like effects of salvia. Mean scores on all but one subscale (cognition) were higher than the values our group had obtained in two clinical trials in which we evaluated the effects of fully psychotropic doses of ayahuasca equivalent to 0.50–1.0 mg DMT/kg body weight (Riba et al., 2001b, 2003) and fell between the scores obtained for intravenous doses of 0.2 and 0.4 mg DMT/kg body weight (Strassman et al., 1994). Interestingly, the score in the volition subscale, which reflects the subject’s degree of incapacitation, is the highest ever observed by our group in clinical (Riba et al., 2001b, 2003) and in survey studies (Riba et al., 2001a) and is even larger than that recorded by Strassman and colleagues after the highest intravenous DMT dose they administered (Strassman et al., 1994). The pattern of scores on the ARCI shows high values for the MBG and LSD subscales. We have also observed high scores on these subscales following ayahuasca (Riba et al., 2001b, 2003) and they highlight the coexistence of somatic and dysphoric effects with positive mood. A high score in the A scale and a low score in the BG are also typical of the psychedelics. Although these drugs display stimulant-like properties, they do not lead to high scores in the BG scale, which measures subjectivelyperceived intellectual efficiency. However, what is remarkable about salvia is the score obtained in the PCAG subscale. The score is unusually high for a psychedelic. High scores on the PCAG subscale have usually been reported in individuals experiencing “fatigue, weakness and sluggishness” after sedatives, such as alcohol, benzodiazepines and the opiate pentazocine (Arasteh et al., 1999). Scores on the APZ-OSE subscale provide insight into the high degree of derealization experienced by the participants, in line with the most frequently cited positive aspect of the drug, i.e. the sensation of entering another reality. The score obtained is higher than that observed by our group after the administration of an ayahuasca dose corresponding to 0.8 mg DMT/kg body weight (Riba et al., 2002). The APZ-AIA and APZ-VUS were also higher than in the mentioned study, pointing out the high intensity of the derealization and visionary phenomena induced by salvia. Scores on the STAI indicated levels of state anxiety above the normative mean both for male and female subjects. The obtained values fall between percentiles 70 and 75 for the males and percentiles 65 and 70 for the females (Seisdedos, 2002). These results indicate that the experience induced by salvia causes a certain degree of anxiety. Taking into consideration these STAI scores, elevations in the PCAG can be interpreted as reflecting an incapacitating rather than an anxiolytic effect. This interpretation is in line with the decreased ability to interact with them-

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selves or their surroundings reflected by the high HRS-Volition score and the marked degree of derealization and anxious depersonalization reflected by the APZ-OSE and APZ-AIA subscales, respectively. Thus, the pattern of responses obtained for salvia with the self-assessment instruments administered would reflect a psychedelic effect profile accompanied by a highly modified perception of external reality and a decreased ability of the individual to interact with themselves or their surroundings. An interesting aspect of the subjective effect profile of salvia is the simultaneous high scores on the LSD and PCAG scales observed. This is not a characteristic feature of the classical psychedelics displaying serotonin-2A agonist activity. However, this unusual pattern combining modifications in somaticdysphoric effects and sedation/impairment has been reported for agonists of the opioid kappa receptor. Thus, pentazocine (Arasteh et al., 1999; Zacny et al., 1998) and enadoline (Walsh et al., 2001) have been shown to elevate scores in the LSD and PCAG scales. At high doses, these drugs can cause modifications in visual perception and depersonalization (Walsh et al., 2001), which has led some authors to describe kappa receptor agonism as capable of inducing “psychotomimetic” effects (Pfeiffer et al., 1986; Walsh et al., 2001). The present results constitute a preliminary approach to the subjective effects of salvia. The investigation has several limitations associated with its naturalistic and exploratory nature. Information was obtained from a small sample of experienced psychedelic/hallucinogen users. These volunteers were regular users of other psychoactive agents such as cannabis and had experimented with rarely used drugs like ayahuasca. The investigators had no control over the salvia doses consumed, and the possibility of an interaction with the participants’ daily cannabis use cannot be ruled out. The pattern of subjective effects observed may therefore be difficult to extrapolate to the general population or to other drug users unfamiliar with psychedelics/hallucinogens. Also, the retrospective assessment performed does not substitute for the immediate assessment of the psychotropic effects of salvia, ideally in the context of clinical trials administering known doses of the drug and implementing optimal designs. To sum up, smoking extracts of salvia appears to be the most common form of use of the drug among recreational users. In the sample studied, this form of administration led to a very fast onset of effects which were intense but short-lived. The psychotropic effects reported bear similarities to those induced by the classical psychedelics regarding changes in perception, mood and somatic sensations. However, the increased derealization observed and the consequent decrease in the ability to interact with themselves and their surroundings appears to be particularly high for salvia. Although the perception- and realitymodifying potency seems higher, the profile of subjective effects induced by salvia is compatible with that of other kappa agonists, thus supporting the activation of this receptor as the drug’s mechanism of action in humans. However, considering the limitations associated with field investigations, the reported results should be considered as preliminary. Carefully planned clinical studies are warranted to further elucidate the pharmacology of salvia in humans.

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Acknowledgements The authors wish to thank Araceli Cabrero for her help in questionnaire scoring and elaboration of the data bases. This research was supported by internal funds. References Arasteh, K., Poudevida, S., Farre, M., Roset, P.N., Cami, J., 1999. Response patterns of the Spanish version of the 49-item short form of the Addiction Research Center Inventory after the use of sedatives, stimulants and opioids. Drug Alcohol Depend. 55, 117–125. B¨ucheler, R., Gleiter, C.H., Schwoerer, P., Gaertner, I., 2005. Use of nonprohibited hallucinogenic plants: increasing relevance for public health? A case report and literature review on the consumption of Salvia divinorum (Diviner’s Sage). Pharmacopsychiatry 38, 1–5. Butelman, E.R., Harris, T.J., Kreek, M.J., 2004. The plant-derived hallucinogen, salvinorin A, produces kappa-opioid agonist-like discriminative effects in rhesus monkeys. Psychopharmacology (Berl) 172, 220–224. Chavkin, C., Sud, S., Jin, W., Stewart, J., Zjawiony, J.K., Siebert, D.J., Toth, B.A., Hufeisen, S.J., Roth, B.L., 2004. Salvinorin A, an active component of the hallucinogenic sage Salvia divinorum is a highly efficacious kappa-opioid receptor agonist: structural and functional considerations. J. Pharmacol. Exp. Ther. 308, 1197–1203. Dennehy, C.E., Tsourounis, C., Miller, A.E., 2005. Evaluation of herbal dietary supplements marketed on the internet for recreational use. Ann. Pharmacother. 39, 1634–1639. Dittrich, A., 1998. The standardized psychometric assessment of altered states of consciousness (ASCs) in humans. Pharmacopsychiatry 31 (Suppl. 2), 80–84. Gouzoulis-Mayfrank, E., Thelen, B., Habermeyer, E., Kunert, H.J., Kovar, K.A., Lindenblatt, H., Hermle, L., Spitzer, M., Sass, H., 1999. Psychopathological, neuroendocrine and autonomic effects of 3,4-methylenedioxyethylamphetamine (MDE), psilocybin and d-methamphetamine in healthy volunteers. Psychopharmacology 142, 41–50. Lamas, X., Farr´e, M., Llorente, M., Cam´ı, J., 1994. Spanish version of the 49item short form of the Addiction Research Center Inventory. Drug Alcohol Depend. 35, 203–209. Martin, W.R., Sloan, J.W., Sapira, J.D., Jasinski, D.R., 1971. Physiologic, subjective, and behavioral effects of amphetamine, methamphetamine, ephedrine, phenmetrazine, and methylphenidate in man. Clin. Pharmacol. Ther. 12, 245–258. Ortega, A., Blount, J.F., Manchand, P.S., 1982. Salvinorin, a new transneoclerodane diterpene from Salvia divinorum (Labiatae). J. Chem. Soc., Perkin Trans. 1, 2505–2508. Ott, J., 1995. Ethnopharmacognosy and human pharmacology of Salvia divinorum and salvinorin A. Curare 18, 103–129. Pfeiffer, A., Brantl, V., Herz, A., Emrich, H.M., 1986. Psychotomimesis mediated by kappa opiate receptors. Science 233, 774–776.

Riba, J., Barbanoj, M.J., 2005. Bringing ayahuasca to the clinical research laboratory. J. Psychoactive Drugs 37, 219–230. Riba, J., Rodriguez-Fornells, A., Barbanoj, M.J., 2002. Effects of ayahuasca on sensory and sensorimotor gating in humans as measured by P50 suppression and prepulse inhibition of the startle reflex, respectively. Psychopharmacology (Berl) 165, 18–28. Riba, J., Rodriguez-Fornells, A., Strassman, R.J., Barbanoj, M.J., 2001a. Psychometric assessment of the Hallucinogen Rating Scale. Drug Alcohol Depend. 62, 215–223. Riba, J., Rodriguez-Fornells, A., Urbano, G., Morte, A., Antonijoan, R., Montero, M., Callaway, J.C., Barbanoj, M.J., 2001b. Subjective effects and tolerability of the South American psychoactive beverage Ayahuasca in healthy volunteers. Psychopharmacology (Berl) 154, 85–95. Riba, J., Valle, M., Urbano, G., Yritia, M., Morte, A., Barbanoj, M.J., 2003. Human pharmacology of ayahuasca: subjective and cardiovascular effects, monoamine metabolite excretion, and pharmacokinetics. J. Pharmacol. Exp. Ther. 306, 73–83. Roth, B.L., Baner, K., Westkaemper, R., Siebert, D., Rice, K.C., Steinberg, S., Ernsberger, P., Rothman, R.B., 2002. Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist. Proc. Natl. Acad. Sci. USA 99, 11934–11939. Siebert, D.J., 1994. Salvia divinorum and salvinorin A: new pharmacologic findings. J. Ethnopharmacol. 43, 53–56. Seisdedos, N., 2002. STAI Cuestionario de ansiedad estado-rasgo. Adaptaci´on espa˜nola del cuestionario y redacci´on del manual. TEA, Madrid. Spielberger, C.D., Gorsuch, R.L., Lushene, R.E., 1970. Manual for the StateTrait Anxiety Inventory. Consulting Psychologists Press, Palo Alto, California. Strassman, R.J., Qualls, C.R., Uhlenhuth, E.H., Kellner, R., 1994. Dose-response study of N N-dimethyltryptamine in Humans. II. Subjective effects and preliminary results of a new rating scale. Arch. Gen. Psychiatry 51, 98–108. Thompson, S.K., Collins, L.M., 2002. Adaptive sampling in research on riskrelated behaviors. Drug Alcohol Depend. 68 (Suppl. 1), S57–S67. Valdes III, L.J., D´ıaz, J.L., Paul, A.G., 1983. Ethnopharmacology of Ska Maria Pastora (Salvia divinorum Epling and J´ativa-M). J. Ethnopharmacol. 7, 287–312. Valdes III, L.J., Butler, W.M., Hatfield, G.M., Paul, A.G., Koreeda, M., 1984. Divinorin A, a psychotropic terpenoid, and divinorin B from the hallucinogenic Mexican mint Salvia divinorum. J. Org. Chem. 49, 4716– 4720. Walsh, S.L., Strain, E.C., Abreu, M.E., Bigelow, G.E., 2001. Enadoline, a selective kappa opioid agonist: comparison with butorphanol and hydromorphone in humans. Psychopharmacology (Berl) 157, 151–162. Wasson, R.G., 1962. A New Mexican Psychotropic Drug from the Mint Family, vol. 20. Botanical Museum Leaflets Harvard University, pp. 77–84. Zacny, J.P., Hill, J.L., Black, M.L., Sadeghi, P., 1998. Comparing the subjective, psychomotor and physiological effects of intravenous pentazocine and morphine in normal volunteers. J. Pharmacol. Exp. Ther. 286, 1197– 1207.

T

he hallucinogenic plant Salvia divinorum (i.e., “magic mint”) is a member of the Sage family that has been used for divination and shamanism by the Mazatecs. Over the past decade or so, S. divinorum has been increasingly used recreationally. The neoclerodane diterpene salvinorin A is the active component of S. divinorum, and recently, the κ opioid receptor (KOR) has been identified, in vitro and in vivo, as its molecular target. The discovery of KOR as the molecular target of salvinorin A has opened up many opportunities for drug discovery and drug development for a number of psychiatric and non-psychiatric disorders.

Timothy A. Vortherms1 and Bryan L. Roth1,2 1

Department of Pharmacology, School of Medicine and 2Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, and National Institute of Mental Health Psychoactive Drug Screening Program, Chapel Hill, NC 27599

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Introduction Salvia divinorum is a perennial herb of the Lamiaceae (mint) family that is indigenous to the Sierra Mazateca of Oaxaca, Mexico (1, 2) (Figure 1). Common names for the herb include ska Maria Pastora and la Maria, reflecting the Mazatec belief that Salvia is the incarnation of the Virgin Mary(1). S. divinorum has been traditionally used by Mazatec curanderos (i.e., folk healers) to produce hallucinogenic experiences essential for spiritual divination. S. divinorum has also been used in traditional healing practices for ailments as diverse as diarrhea, headache, and rheumatism, as well as the magical disease known as panzón de barrego (i.e., swollen belly), which is said to be caused by a curse from an evil sorcerer (1). S. divinorum was discovered by Wasson and Hofmann in 1962 and was subsequently described by Epling as a new species of Salvia (2). Traditionally, S. divinorum has been ingested by chewing fresh leaves as a quid or by drinking an extract made from freshly crushed leaves. Alternatively, S. divinorum can be taken by pyrolizing dried leaves and rapidly inhaling the resulting smoke (3). The hallucinatory effects are potent and intense, with extraordinarily rapid onset, and can last up to an hour (3, 4). Interestingly, the hallucinations produced by S. divinorum appear to be qualitatively different from other hallucinogens; users typically describe their experience of “entering another reality” and having a “separation from body,” subjective descriptions that are consistent with a “spatiotemporal dislocation” (5–7). Over the past decade, S. divinorum has become used for recreational purposes, particularly in the US and Europe. The use of S. divinorum as a legal hallucinogen has been facilitated by the availability of S. divinorum leaves and extracts through Internet suppliers. The use of S. divinorum has been unregulated, although several countries (e.g., Australia, Denmark, Italy, and Sweden) have recently classified S. divinorum as a controlled substance. In the US, S. divinorum is not listed under the Controlled Substances Act, although several states, including Missouri, Delaware, Louisiana, and Tennessee, have passed legislation controlling the use S. divinorum (see www.deadiversion.usdoj.gov). The active component of S. divinorum is salvinorin A, a neoclerodane diterpene (3) (Figure 1). The lipid-like salvinorin A molecule is chemically and structurally unique in that it represents the only known psychoactive diterpene and was the first non-nitrogenous hallucinogen to be identified. Smoking 200–500 µg of purified salvinorin A produces hallucinations that have been reported to be identical in nature to those observed following ingestion of fresh S. divinorum leaves (3). The effective dose of salvinorin A in humans is similar to that of the synthetic hallucinogens lysergic acid diethylamide (LSD) and 4-bromo-2,5-dimethoxy-phenylisopropylamine (DOB) (8). Initial attempts to identify the molecular target of salvinorin A were unsuccessful, despite binding probes against a number of molecular targets, including various serotonin (5-hydroxytryptamine; 5-HT) receptors (3). Subsequently, salvinorin A was submit-

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Figure 1. The magic mint and its products. A. Salvia divinorum is a member of the Lamiaceae (mint) family that has been traditionally used by Mazatec shamans for spiritual divination, and more recently S. divinorum has become increasingly used as a recreational hallucinogen. B. Salvinorin A is the active component of S. divinorum. C. Salvinorin B is a potential salvinorin A metabolite resulting from ester hydrolysis and is relatively inactive at KOR. Carbon atoms are shown in gray and oxygen atoms are shown in red.

Salvinorin A: From Natural product to Human Therapeutics

ted to the National Institutes of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP; see http://pdsp.med.unc.edu) and screened against a panel of fifty molecular targets, including cloned human G protein–coupled receptors, transporters, and ion channels, and for comparison, the prototypical hallucinogen LSD was simultaneously screened (9). Surprisingly, salvinorin A displayed significant binding at the Gαi-coupled κ opioid receptor (KOR), but did not bind to the cloned δ opioid receptor (DOR) or µ opioid receptor (MOR) at the concentration tested. Salvinorin A also failed to bind to the human 5-HT2A receptor, the principal molecular target of classical hallucinogens such as LSD (9, 10). Functional studies measuring inhibition of forskolin-stimulated cyclic AMP accumulation and [35S]GTPγS binding confirmed salvinorin A as a potent agonist at cloned KOR and at the native KOR expressed in guinea pig brain (9). Salvinorin A thus became the first identified naturally occurring, non-nitrogenous KORselective agonist with psychotomimetic properties (9). This discovery has paved the road for a new avenue of opioid research and has fueled the search for other diterpenes with similar pharmacological properties.

Isolation of Salvinorin A and Related Diterpenes from S. Divinorum

Although the psychotropic effects of Salvia divinorum have been widely known, the chemical component(s) responsible for these properties have not been extensively studied until quite recently. The first compounds isolated from S. divinorum leaves (Figure 2) were the neoclerodane diterpene salvinorin A and salvinorin B (11) [structurally identical to the terpenoids independently isolated and designated elsewhere as divinorin A and divinorin B (1, 12)]. Salvinorin A in the leaves of S. divinorum ranges in concentration from 0.89 to 3.70 mg/g dry weight (13), and salvinorin B, as well as other isolated diterpenes, are found at much lower concentrations (14–16). Salvinorin A was found to be a potent agonist at KOR, whereas salvinorin B, a potential metabolite of salvinorin A resulting from ester hydrolysis at the 2-acetoxy group, was inactive at KOR (9, 17). It has been suggested that rapid hydrolysis of salvinorin A resulting in the production of salvinorin B could contribute to the short duration of action in vivo (6). Chemical modifications at the C2 position O O O have been implemented as a potential means of increasing the metabolic stability R2 of salvinorin A (18) (Figure R1 H H O H H O H H O 3). Intriguingly, salvinorin A R1 RO is slightly more potent than R2 O O other KOR-selective agonists, such as U50488H and U69593, in functional assays O OCH3 O OR3 measuring potassium conducO OCH3 tance through G protein–reguR R R R R1 R2 1 2 3 1 Salvinorin A Ac 8 Divinatorin A lated K+ channels (17). OH H H 3 Salvinorin C OAc OAc 2 Salvinorin B H 9 Divinatorin B OH OH Me 4 Salvinorin D OAc OH Following the identifica10 Divinatorin C H OAc H 5 Salvinorin E OH OAc tion of salvinorin A and B, 11 Divinatorin D OH OAc Me 6 Salvinorin F OH H 12 Divinatorin E OH O Me 7 Salvinorin G O OAc several other diterpenes were isolated from the leaves of S. divinorum (Figure 2): salvinoMeO MeO rin C, which has weak affinity O O for KOR (19) and reportedly lacks psychotropic effects HO OMe HO OMe in humans (20); salvinorins HO HO D–G (15, 16, 21); divinatorins O H O O H H O H A–E (14, 21); and salvinicins O O O O A and B (22) (Figure 2). The chemical structures and pharO O macology of these salvinorin analogs have recently been O O OCH3 OCH3 characterized, and only salvinorin G (Ki = 418 ± 117 14 Salvinicin B 13 Salvinicin A nM) and divinatorin D (Ki = Figure 2. Structures of salvinorin A and B and related diterpenes isolated from Saliva divinorum. 230 ± 21 nM) exhibit meaOctober 2006 Volume 6, Issue 5

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O O

H

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R1 15 CH3CH2CO2 16 CH3C(O)N(CH3) 17 epi-(CH3)2CHN(H) 18 CH3OCH2O 19 PhCO2 20 4-BrPhCO2

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Figure 3. Structures of salvinorin A and representative derivatives. Representative modifications to the salvinorin A structure are shown for the indicated positions. Structural features of salvinorin A required for acitivty at KOR are shown in red, and elements shown in blue are not required for activity at KOR.

surable affinities for KOR (19, 21, 23). Salvinorin A is the only isolated neoclerodane diterpene known to exhibit high affinity for KOR. Interestingly, initial studies indicate that salvinicin A is a partial agonist at KOR, whereas salvinicin B demonstrates weak antagonist activity at MOR (22). These observations suggest that modification of the unique scaffold of salvinorin A may lead to the discovery of novel selective agonists and antagonists.

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Structure–Activity Relationships of Salvinorin A Derivatives The functional groups of salvinorin A have been extensively modified, and subsequent structure activity relationship (SAR) studies have begun to clarify the pharmacophore. The rapidly expanding library of salvinorin A analogs includes modifications and substitutions at the C1 (19), C2 (17, 18, 24–28), C4 (18, 19),

Salvinorin A: From Natural product to Human Therapeutics

Figure 4. Proposed KOR:salvinorin A binding complex. Views are presented through the helical bundles (A) or from the extracellular side of KOR looking into the binding pocket of KOR (B). Salvinorin A is stabilized in the binding pocket through hydrogen bonding and hydrophobic interactions with Y119 (helix 2), Y313 and Y320 (helix 7), I294 (helix 6) (shown in green), and the second extracellular loop of KOR (shown in yellow). For clarity, some helical residues are not shown; figures were constructed using PyMOL.

C17 (19), and C18 positions (18, 29), as well as the furan ring (19, 30) (Figure 3). Subsequent SAR studies have been consistent with the pharmacophore proposed by Munro and colleagues (19, 23). There are several reports exploring the effects of the 2-acetoxy group of salvinorin A on affinity and selectivity for KOR. In general, modifications in terms of size and electronegativity are not well tolerated at the C2 position (27). Interestingly, the propionate derivative retains high affinity for KOR (Figure 3, structure 15) but exhibits partial agonist activity (17). Further SAR studies at the C2 position reveal that the N-methylacetamide and 2-epi-isopropylamine derivatives (Figure 3, structures 16 and 17), which are predicted to have increased stability and aqueous solubility, are full agonists at KOR, with potencies comparable to salvinorin A (18). Additional work shows that substitution of the methoxymethyl group at the C2 position of salvinorin A (Figure 3, structure 18) results in a full agonist with a sevenfold increase in potency at KOR (27). Derivatization of salvinorin A has also identified the C2 position as a critical site for receptor subtype selectivity. In fact, the 2-benzoate derivative (structure 19) was the first neoclerodane diterpene identified with MOR agonist activity (26). The 4-bromo benzoyl derivative (structure 20) also exhibits high affinity for MOR, whereas the affinity for KOR is decreased more than 350-fold, thus significantly increasing the selectivity for MOR over KOR (28). Intriguingly, the C2 epimer of salvinorin A (not shown) has decreased activity at KOR, but also exhibits weak antagonistic activity at DOR, identifying the first neoclerodane diterpene with DOR antagonist activity (29). These observations suggest that further modification of the salvinorin A scaffold may result in derivatives with improved receptor affinity and selectivity. Modifications of salvinorin A at the C4 position reveal that the methyl ester group is required for KOR activity (Figure 3,

shown in red). The 18-hydroxy derivative (structure 21) fails to activate KOR but retains weak binding affinity (Ki = 347 ± 53 nM), perhaps representing antagonistic activity (19). Substitutions at the C18 position such as the dimethylamide derivative (structure 22), as well as other ester, amine, and ether substitutions, markedly decrease affinity for KOR (18, 29), confirming that the methyl ester group at the C4 position is a critical component of the pharmacophore. Removal of the lactone carbonyl from the C17 position (structure 24) has a nominal effect on the affinity and potency at KOR (19). Reduction of the C1 ketone to a hydroxy or acetoxy group significantly decreases binding affinity for KOR; however, removal of the ketone group (structure 26) causes only a fivefold decrease in binding affinity for KOR (19). The tetrahydro derivative (structure 25) displays a dramatic decrease (fortyfold) in affinity, but not potency, at KOR, suggesting that the furan ring is potentially part of the pharmacophore (19). Additional SAR studies reveal that epimerization at the C8 position (structure 23) results in a seventyfold decrease in binding affinity for KOR (19, 29). In summary, SAR studies suggest that the methyl ester at C4 and the furan ring at C12 are required for activity at KOR (Figure 3, shown in red), whereas the C17 lactone and C1 ketone are not as stringently required (19) (Figure 3, shown in blue).

Molecular Modeling and Mutagenesis Studies of the Salvinorin A:KOR Binding Complex There have been a limited number of receptor mutagenesis and molecular modeling studies probing the salvinorin A binding pocket of KOR (9, 31, 32). Site-directed mutagenesis of KOR October 2006 Volume 6, Issue 5

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reveals that salvinorin A is stabilized in the binding pocket by interactions with tyrosine residues in helix 2 (i.e., Y119) and helix 7 (i.e., Y313 and Y320) (32). Molecular modeling predicts that Y119 in helix 2 and Y320 in helix 7 stabilize binding through hydrogen bonds with the furan ring of salvinorin A, and Y313 in helix 7 is predicted to stabilize binding through hydrophobic interactions with the 2-acetoxy group of salvinorin A (32) (Figure 4). Mutation of Y139 in helix 3 and Y312 in helix 7 does not markedly alter the affinity of salvinorin A, suggesting that these residues are not involved in salvinorin A:KOR binding interactions (9, 32). In an elegant approach, Y119C, Y313C, and Y320C KOR mutants were exposed to a salvinorin A derivative containing a free sulfhydryl group (i.e., 2-thiosalvinorin B) (32). The affinity of 2-thiosalvinorin B was enhanced relative to salvinorin A at the Y313 mutant, suggesting that Y313 in helix 7 is in close proximity to the C2 position of salvinorin A (32). The key role of the conserved Y119, Y313, and Y320 residues in binding salvinorin A has been confirmed (31).

Pdyn CREB

D1

Gi KOR

Figure 5. Potential role of the KOR–dynorphinergic signaling complex in the treatment of mood disorders. This figure depicts the mesolimbic reward pathway that has been implicated in mood disorders. Dopaminergic neurons project from the ventral tegmental area (orange) to GABAergic neurons in the nucleus accumbens (blue). Synaptic dopamine (green triangles) release can lead to activation of Gαs-coupled D1 dopamine receptors (green spheres) resulting in accumulation of cyclic AMP, and subsequent activation of the cAMP response element binding protein (CREB) that regulates transcription of dynorphin (blue rectangles), the endogenous KOR ligand (46). In this model, dynorphin activates the presynaptic KOR that modulates dopamine release in the nucleus accumbens. Stress-induced increases in CREB activity have been associated with depressive-like behaviors in animal models, similar to the behavioral effects observed with administration of KOR-selective agonists such as salvinorin A, which decrease extracellular concentrations of dopamine within the nucleus accombens. These CREB-induced depressive behaviors can be attenuated with KOR-selective antagonists (47), further implicating a role for the KOR-dynorphinergic signaling pathway in the treatment of depressive-like behaviors.

In Vivo Pharmacology of Salvinorin: Implications for Human Therapeutics Recently, there have been several reports verifying that salvinorin A exerts its effects via KOR activation in vivo. Thus, intraperitoneal injections of salvinorin A (1.0 – 4.0 mg/kg) results in a dose- and time-dependent increase in tail-flick latencies in mice, with the maximal response occurring within ten minutes of salvinorin A administration (33, 34). Thereafter, the antinociceptive effects of salvinorin A diminish quite rapidly and return to baseline within thirty minutes of drug administration (34), which may reflect rapid metabolism of salvinorin A in vivo (6, 48). These

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antinociceptive effects were also observed using the hotplate assay, in which increased latencies occur at a salvinorin A dose of 1.0 mg/kg (34). Salvinorin A effects on tail-flick latencies are abolished following pretreatment with the KOR-selective antagonist nor-binaltorphimine, demonstrating the selectivity of salvinorin A for the KOR in vivo (33, 34). Salvinorin A also exhibits dose- and timedependent antinociceptive effects in the chemo-nociceptive acetic acid abdominal constriction assay (34). The antinociceptive effects of salvinorin A and the 2-propionate derivative (Figure 2, structure 15) have also been examined in wild-type and a novel strain of KOR knockout mice (34). In these studies, salvinorin A and 2-salvinorinyl propionate produced

Salvinorin A: From Natural product to Human Therapeutics

concentration- and time-dependent antinociception in wild-type mice but not in the KOR knockout mice. Similar to other KORselective agonists (36), salvinorin A and the 2-propionate derivative reduce rectal body temperature in wild-type mice, but this effect is absent in KOR knockout mice (35). Salvinorin B tested at the same concentrations failed to produce antinociception or hypothermic effects in wild-type mice, which is consistent with in vitro observations demonstrating that salvinorin B is inactive at KOR (9, 17). In addition to the recognized potential for antinociceptive therapy, it has recently been suggested that S. divinorum may provide a pharmacological basis for the treatment of diarrhea (37). Specifically, salvinorin A, as well as extract prepared from S. divinorum leaves, can inhibit myenteric cholinergic transmission in guinea pig ileum. The inhibitory effects of salvinorin A on ileum contraction are blocked by the non-selective opioid receptor antagonist naloxone and the KOR-selective antagonist nor-BNI. Taken together, these behavioral observations suggest that salvinorin A exerts its effects via activation of KOR in vivo. There is considerable evidence supporting a role for the KOR:dynorphinergic signaling complex in the regulation of mood disorders (9, 38, 39). Indeed, KOR agonists induce depressivelike behaviors in animal models, whereas KOR antagonists have antidepressant-like effects in vivo (40). These observations have led, in part, to the hypothesis that modulation of KOR signaling pathways will be useful for the treatment of depressive behaviors (41) (Figure 5). There is also significant evidence to support the involvement of KOR signaling pathways in the dependence of cocaine [for review, see (49)].To this end, several studies have examined whether salvinorin A can elicit the same behavioral responses as other synthetic KOR-selective ligands. Intraperitoneal injections of salvinorin A (0.25 – 2.0 mg/kg) in rats result in a dose-dependent increase in immobility in the forced swim test without altering locomotor activity in an open field (41). The same concentration range increases the threshold for intracranial self-stimulation and decreases extracellular dopamine concentrations in the nucleus accumbens (41). These observations are consistent with effects of the KOR-selective agonist U69593 (40) and support the hypothesis that the KOR system may play a critical role in depressive-like behaviors. It is noteworthy, however, that there has been at least one report demonstrating the usefulness of S. divinorum in the treatment of refractory depression (42). Ultimately, clinical trials will be needed to resolve the potential role for selective KOR agents in treating mood disorders. Additional studies in mice reveal that salvinorin A decreases extracellular dopamine levels in the caudate putamen, but not in the nucleus accumbens (43). The effect can be abolished by pretreatment with the KOR-selective antagonist nor-BNI (43). Salvinorin A also leads to conditioned place aversion and a decrease in locomotor activity (43), which is consistent with the observation that salvinorin A disrupts climbing behavior in an inverted screen task (44). These studies suggest that salvinorin A

causes some level of sedation and motor incoordination. Butelman et al. also have demonstrated that salvinorin A exerts its effects via activation of KOR in vivo. In non-human primates, salvinorin A produces a discriminative stimulus similar to the KOR-selective agonist U69593, whereas the NMDA antagonist ketamine is not generalized by U69593-trained subjects; the effect of salvinorin A can be blocked by a KOR-selective antagonist (45). Taken together, these animal studies are consistent with in vitro pharmacology studies demonstrating that salvinorin A is a highly potent and selective KOR agonist, which likely accounts for the effects of the drug in humans.

Conclusions Salvinorin A represents the first known non-nitrogenous KOR selective agonist and the first non-alkaloidal hallucinogen. Salvinorin A is a potent KOR agonist in vitro and in vivo, suggesting that the hallucinogenic effects produced by salvinorin A are mediated by activation of KOR. Modification of the salvinorin A scaffold has also led to the development of MOR-selective agonists. Thus, the chemical structure of salvinorin A may be manipulated for the design of novel receptor-specific ligands. Further studies with kappa agonists and antagonists will likely provide insight into the therapeutic potential of the KOR–dynorphinergic system in the treatment of psychiatric disorders associated with hallucinations, such as schizophrenia and Alzheimer’s disease, as well as various mood disorders. doi:10.1124/mi.6.5.7

Acknowledgments This work was supported by NIDA grants RO1 DA017204 (BLR) and NO1 MH80004 for the NIMH-PDSP (BLR). Timothy Vortherms has been awarded a postdoctoral National Research Service Award from NIDA (1F32 DA022188). The authors would like to thank Dr. Richard Westkaemper for providing the salvinorin A:KOR binding model and Daniel Siebert for the Saliva divinorum photograph. The authors would also like to thank Ryan Strachan for careful review and helpful comments with this paper. References 1.

Valdes, L.J., 3rd, Diaz, J.L., and Paul, A.G. Ethnopharmacology of ska Maria Pastora (Salvia divinorum, epling and jativa-m). J. Ethnopharmacol. 7, 287–312 (1983).

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Woods, J.H. Kappa-opioid receptor-mediated effects of the plant-derived hallucinogen, salvinorin A, on inverted screen performance in the mouse. Behav. Pharmacol. 16, 627–633 (2005). 45. Butelman, E.R., Harris, T.J., and Kreek, M.J. The plant-derived hallucinogen, salvinorin A, produces kappa-opioid agonist-like discriminative effects in rhesus monkeys. Psychopharmacology (Berl) 172, 220–224 (2004). 46. Carlezon, W.A., Jr., Thome, J., Olson, V.G., Lane-Ladd, S.B., Brodkin, E.S., Hiroi, N., Duman, R.S., Neve, R.L., and Nestler, E.J. Regulation of cocaine reward by CREB. Science 282, 2272–2275 (1998). 47. Mague, S.D., Pliakas, A.M., Todtenkopf, M.S., Tomasiewicz, H.C., Zhang, Y., Stevens, W.C., Jr., Jones, R.M., Portoghese, P.S., and Carlezon, W.A., Jr. Antidepressant-like effects of kappa-opioid receptor antagonists in the forced swim test in rats. J. Pharmacol. Exp. Ther. 305, 323–330 (2003). 48. Schmidt, M.D., Schmidt, M.S., Butelman, E.R., Harding, W.W., Tidgewell, K., Murry, D.J., Kreek, M.J., and Prisinzano, T.E. Pharmacokinetics of the plant-derived kappa-opioid hallucinogen salvinorin A in nonhuman primates. Synapse 58, 208–210 (2005). 49. Hasebe, K., Kawai, K., Suzuki, T., Kawamura, K., Tanaka, T., Narita, M., and Nagase, H. Possible pharmacotherapy of the opioid kappa receptor agonist for drug dependence. Ann. N Y Acad. Sci. 1025, 404–413 (2004).

Bryan L. Roth, MD, PhD, is a Professor in the Department of Pharmacology, School of Medicine and Division of Medicinal Chemistry and Natural Products, School of Pharmacy at the University of North Carolina at Chapel Hill. He is also Director of the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP). His research interests include GPCR structure and function, and he is actively involved in drug discovery using the resources of the NIMH-PDSP. Address correspondence to BLR. E-mail bryan_ [email protected]; fax 919-843-5788. Timothy A. Vortherms, PhD, received his doctoral degree from the Department of Medicinal Chemistry and Molecular Pharmacology at Purdue University in 2004. He is currently a postdoctoral fellow and has received a Ruth L. Kirschstein National Research Service Award to study the molecular determinants for salvinorin A interactions with κ opioid receptors.

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0022-3565/07/3201-300–306$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics JPET 320:300–306, 2007

Vol. 320, No. 1 112417/3166012 Printed in U.S.A.

Effects of Salvinorin A, a ␬-Opioid Hallucinogen, on a Neuroendocrine Biomarker Assay in Nonhuman Primates with High ␬-Receptor Homology to Humans Eduardo R. Butelman, Marek Mandau, Kevin Tidgewell, Thomas E. Prisinzano, Vadim Yuferov, and Mary Jeanne Kreek Laboratory on the Biology of Addictive Diseases, The Rockefeller University, New York, New York (E.R.B., M.M., V.Y., M.J.K.); and Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, Iowa (K.T., T.E.P.) Received August 11, 2006; accepted October 20, 2006

ABSTRACT This study focused on the in vivo effects of the ␬-opioid hallucinogen salvinorin A, derived from the plant Salvia divinorum. The effects of salvinorin A (0.0032– 0.056 mg/kg i.v.) were studied in a neuroendocrine biomarker assay of the anterior pituitary hormone prolactin in gonadally intact, adult male and female rhesus monkeys (n ⫽ 4 each). Salvinorin A produced dose- and time-dependent neuroendocrine effects, similar to the synthetic high-efficacy ␬-agonist U69,593 ((⫹)-(5␣,7 ␣,8␤)-N-methyl-N[7-(1-pyrrolidiniyl)-1-oxaspiro[4.5]dec-8yl]-benzeneacetamide), but of shorter duration than the latter. Salvinorin A was approximately equipotent to U69,593 in this endpoint (salvinorin A ED50, 0.015 mg/kg; U69,593 ED50, 0.0098 mg/kg). The effects of i.v. salvinorin A were not prevented by a small dose of the opioid antagonist nalmefene (0.01 mg/kg s.c.) but were prevented by a larger dose of nalmefene (0.1 mg/kg); the latter

Salvinorin A, a diterpenoid, is the main active compound from the leaves of the hallucinogenic plant, Salvia divinorum (Valdes, 1994). S. divinorum preparations were originally used in ethnomedical practice by the Mazatec people of Oaxaca, Mexico, but have recently become widely commercially available. There are emerging reports of use of such salvinorin A-containing products as hallucinogens, mainly by the smoking route (http://biopsych.com/cpdd/CPDD04_PDFs/ CPDD04_981339497336.pdf; Gonzales et al., 2006). A recent study determined that salvinorin A was a highly This work was supported by National Institute on Drug Abuse Grants DA017369 (to E.R.B.), DA05130 and DA00049 (to M.J.K.), and DA08151 (to T.E.P.). The present studies were reviewed by the Rockefeller University Animal Care and Use Committee and the Guide for the Care and Use of Animals (National Academy Press, Washington, DC, 1996). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.112417.

nalmefene dose is sufficient to produce ␬-antagonist effects in this species. In contrast, the 5HT2 receptor antagonist ketanserin (0.1 mg/kg i.m.) did not prevent the effects of salvinorin A. As expected, the neuroendocrine effects of salvinorin A (0.0032 mg/kg i.v.) were more robust in female than in male subjects. Related studies focused on full-length cloning of the coding region of the rhesus monkey ␬-opioid receptor (OPRK1) gene and revealed a high homology of the nonhuman primate OPRK1 gene compared with the human OPRK1 gene, including particular C-terminal residues thought to be involved in receptor desensitization and internalization. The present studies indicate that the hallucinogen salvinorin A acts as a high-efficacy ␬-agonist in nonhuman primates in a translationally viable neuroendocrine biomarker assay.

selective agonist at ␬-opioid receptors (Roth et al., 2002). Salvinorin A was approximately equipotent and equieffective in vitro to arylacetamide ␬-agonists such as U69,593, in the stimulation of guanosine 5⬘-O-(3-thio)triphosphate binding, or the inhibition of adenylate cyclase (Roth et al., 2002). In another signal transduction system (potentiation of G protein-coupled inwardly rectifying potassium channel currents), salvinorin A appeared to be an “ultrahigh” efficacy agonist (Chavkin et al., 2004). Salvinorin A was also found to have a lesser propensity to cause ␬-receptor desensitization and internalization in vitro, compared with arylacetamide ␬-agonists (Wang et al., 2004). It is unknown whether this in vitro profile confers salvinorin A with unique properties as a ␬-agonist in vivo, possibly underlying its hallucinogenic effects. There are some studies of the effects of salvinorin A in vivo, mostly in rodents. Salvinorin A caused ␬-receptor

ABBREVIATIONS: U69,593, (⫹)-(5␣,7 ␣,8␤)-N-methyl-N-[7-(1-pyrrolidiniyl)-1-oxaspiro[4.5]dec-8yl]-benzeneacetamide; ANOVA, analysis of variance; OPRK1, ␬-opioid receptor gene; PCR, polymerase chain reaction. 300

Neuroendocrine Effects of Salvinorin A

mediated place aversion and decreases in striatal dopamine dialysates in mice, similarly to synthetic ␬-agonists (Zhang et al., 2004, 2005). Salvinorin A also caused depressive-like behavioral effects and reduced dopamine dialysate levels in nucleus accumbens in rats (Carlezon et al., 2006). Salvinorin A produced ␬-receptor-mediated sedation/motor incoordination in mice (Fantegrossi et al., 2005). Salvinorin A may produce brief antinociceptive effects under certain conditions in rodents but is devoid of antipruritic effects, typically observed with ␬-agonists (Ko et al., 2003; Wang et al., 2004; Ansonoff et al., 2006; McCurdy et al., 2006). Salvinorin A was generalized by nonhuman primates trained to discriminate U69,593 in an operant assay (Butelman et al., 2004). Few studies have addressed in vivo the apparent efficacy of salvinorin A or that have endpoints that may be easily adapted to humans, thus having translational value. Serum prolactin levels have been used in nonhuman primates to study the potency, receptor selectivity, and apparent efficacy of ␬-agonists in vivo (other compounds, including ␮-opioid agonists, also cause prolactin release) (Bowen et al., 2002; Butelman et al., 2002). This neuroendocrine biomarker assay has also been used in clinical populations in the study of ␬-opioid effects of the neuropeptide dynorphin A (Kreek et al., 1999; Bart et al., 2003). These studies document the effects of salvinorin A in this biomarker assay and are consistent with the high efficacy ascribed to salvinorin A at ␬-receptors, based on in vitro studies. Nonhuman primates, such as Macaca mulatta (used herein) may be valuable models for translational studies of ␬-opioid function. Studies suggest that there are differences in rodent versus human or nonhuman primate ␬-receptor populations, in terms of neuroanatomical localization, relative Bmax, and neurobiological interactions (Mansour et al., 1988; Rothman et al., 1992; Berger et al., 2006). In addition, comparative studies in cloned human and rat ␬-receptors have detected differences in agonist-induced desensitization and internalization, and these could be ascribed to interspecies differences in protein structure at the C terminus of the receptor (e.g., at the 358-amino acid residue position; Li et al., 2002; Liu-Chen, 2004). To determine whether this nonhuman primate species shares these critical amino acid residues with human ␬-receptors, we present information on full-length cloning of the coding region of the M. mulatta ␬-receptor.

Materials and Methods Experimental Subjects in Neuroendocrine Studies. Captivebred, gonadally intact rhesus monkeys (M. mulatta; four male and four female; age range, 8 –11 years old approximately; weight range, 5.8 –12.5 kg) were used. Monkeys were singly housed in a room maintained at 20 to 22°C with controlled humidity, and a 12-/12-h light/dark cycle (lights on at 7:00 AM). Monkeys were fed approximately 11 jumbo primate chow biscuits (PMI Feeds, Richmond, VA) daily, supplemented by appetitive treats, and multivitamins plus iron. An environmental enrichment plan was in place in the colony rooms. Water was freely available in home cages, via an automatic waterspout. Procedure for Neuroendocrine Experiments. Chairtrained monkeys were tested after extensive prior exposure to the experimental situation. Monkeys were chaired and transferred to the experimental room between 10:00 AM and 11:00 AM on each

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test day. An in-dwelling catheter (24 gauge; Angiocath; Becton Dickinson, Sandy, UT) was placed in a superficial leg vein and secured with elastic tape. An injection port (Terumo, Elkton, MD) was attached to the hub of the catheter; the port and catheter were flushed (0.3 ml of 50 U/ml heparinized saline) before use and after each blood sampling or i.v. injection. Approximately 15 min following catheter placement, two baseline blood samples of approximately 2 ml were collected, 5 min apart from each other (defined as ⫺10 and ⫺5 min, relative to the onset of dosing), and kept at room temperature until the time of spinning (3000 rpm at 4°C) and serum separation. Serum samples were then kept at ⫺40°C until the time of analysis, typically within 2 weeks of collection. The samples were analyzed in duplicate with a standard human prolactin immunoradiometric kit (DPC, Los Angeles CA), following manufacturer’s instructions. There is high protein homology between human and rhesus monkey prolactin, and antibody crossreactivity between human and rhesus monkey prolactin has also been reported (Brown and Bethea, 1994; Pecins-Thompson et al., 1996; Ordog et al., 1998). The reported sensitivity limit of the present assay was 0.1 ng/ml; each individual kit was calibrated with known standards, in the range 2 to 200 ng/ml. The intra- and interassay coefficients of variation with this kit in the laboratory were 2 and 9%, respectively. Monkeys were tested in a time course procedure. Following baseline sample collection, a single agonist (salvinorin A or U69,593) injection was administered, followed by sampling at 5, 15, 30, 60, 90, and 120 min after administration. Unless otherwise stated, agonists were injected by the i.v. route. In antagonism experiments, a single dose of antagonist (s.c. nalmefene or i.m. ketanserin) was administered 30 min before salvinorin A, followed by testing as above. Each experiment was typically carried out in four males; selected experiments were carried out in four females in follicular phase (days 2–12 of each cycle of approximately 28 days, as defined by the onset of visible bleeding). Consecutive experiments in the same subject were separated by at least 96 h. Design of Neuroendocrine Studies. Time course studies were carried out with salvinorin A and U69,593 (0.0032– 0.056 mg/kg i.v.; typically n ⫽ 4) and vehicle. For salvinorin A and U69,593, the largest dose was only studied in three of four subjects. The fourth subject was not administered the largest dose for safety reasons, based on greater sensitivity to untoward effects of the compounds (e.g., tremors). In other studies, the opioid antagonist nalmefene (0.01 or 0.1 mg/kg s.c.) was administered as a pretreatment before the largest salvinorin A dose at which all subjects were studied (0.032 mg/kg). A similar pretreatment study was completed with the 5HT2 antagonist ketanserin (0.1 mg/kg i.m.), before salvinorin A (0.032 mg/kg). Female subjects were studied at the 0.0032 mg/kg i.v. dose, a dose that results in robust prolactin elevation in females but not in males. Female subjects were also studied after s.c. administration of salvinorin A (0.032 mg/kg), with and without nalmefene (0.1 mg/kg s.c.) pretreatment. Data Analysis. Prolactin values are presented as mean ⫾ S.E.M., after subtraction of individual mean preinjection baselines for each session (⌬ nanograms per milliliter). Dose-effect curves are also presented, as collated from a time of peak prolactin release caused by salvinorin A or U69,593 (15 min post-i.v. injection). Linear regression was used to calculate ED50 values from individual data points above and below the 50% level of effect. Significant differences in a parameter (e.g., log ED50 values) were considered to occur if there was a lack of overlap in their 95% confidence limits. Unless otherwise stated, experiments were carried out with n ⫽ 4. Repeated measures ANOVA was followed by post hoc tests [using either GraphPad Prism (GraphPad Software Inc., San Diego, CA) or SPSS-Sigmastat (SPSS Inc., Chicago, IL)]; the level of significance (␣) was set at p ⫽ 0.05. Drugs. Salvinorin A was extracted from commercially obtained S. divinorum leaves (Ethnogens.com, Berkeley, CA) in the laboratory of Dr. T.E. Prisinzano, as described previously (Tidgewell

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et al., 2004; Harding et al., 2006). In brief, dried S. divinorum leaves (1.5 kg) were ground to a fine powder and percolated with acetone. The acetone extract was concentrated under reduced pressure to afford a crude green gum, which was subjected to repeated column chromatography on silica gel with elution, using a mixture of EtOAc/hexanes to afford salvinorin A (thin-layer chromatography) and other minor diterpenes. The melting point, 1 H NMR, and 13C spectra of salvinorin A were in agreement with previous reports (Ortega et al., 1982; Valdes et al., 1984). Salvinorin A solutions for injection were prepared daily in ethanol/ Tween 80/sterile water (1:1:8, v/v; maximum concentration in this vehicle was 0.2 mg/ml). Nalmefene HCl (Baker Norton, Miami, FL) was dissolved in sterile water; U69,593 (Pharmacia-Upjohn, Kalamazoo, MI) was dissolved in sterile water with the addition of 1 drop of lactic acid. All of the above drug doses are expressed as milligrams per kilogram of the forms indicated above. Ketanserin tartrate (Sigma, St. Louis, MO) was dissolved in 5% dimethyl sulfoxide in sterile water (v/v) and was injected i.m. The ketanserin dose is expressed as the base, for consistency with prior publications (Fantegrossi et al., 2002). All drugs were injected in volumes of 0.05 to 0.1 ml/kg whenever possible. Cloning and Sequencing of M. mulatta ␬-Opioid Receptor (OPRK1) cDNA. The coding region of the OPRK1 gene was obtained by PCR amplification of M. mulatta brain cDNA (obtained from BioChain, Hayward, CA) with the forward primer 5⬘-TCCTCGCC TT CCTGCTGCA-3⬘, located 30 nucleotides upstream of ATG codon, and the reverse primer 5⬘-TCAGACTGC AGTAGTATC-3⬘, located 69 nucleotides downstream of the termination codon. The primer design was based on the human OPRK1 sequence (GenBank accession no. NM_000912). The final product, approximately 1260 bp in size, was purified using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA) and cloned in pCR II plasmid (Invitrogen, Carlsbad, CA). The clones were sequenced in both directions using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an ABI Prism 3700 capillary sequencer. Single Nucleotide Polymorphism Analysis. Genomic DNA was isolated from peripheral white blood cells, obtained by venipuncture from 14 subjects in the colony, including all eight subjects used in the present neuroendocrine studies (Versagene kit; Gentrasystems, Minneapolis, MN). The C terminus of the M. mulatta OPRK1 was amplified using forward primer 5⬘-ATTCTCTACGCCTTTCTTGAT-3⬘, located 160 bp upstream of the termination codon, and reverse primer 5⬘-TCAGACTGCAGTAG TATC-3⬘, located 69 bp downstream of the termination codon. PCR products, 257 bp in size, were sequenced to identify single nucleotide polymorphisms, as described above.

Results Baseline Prolactin Values and Effects of Vehicle Administration Preinjection prolactin values in males were relatively consistent and exhibited small decreases over time, after i.v. vehicle administration. Thus, mean preinjection values were 15.5 ng/ml (S.E.M. ⫽ 4.2); these values decreased gradually over a 120-min session, following i.v. administration of vehicle (1:1:8 ethanol/Tween 80/sterile water v/v; 0.16 ml/kg; see Fig. 1). Female subjects in follicular phase had similar preinjection baselines (mean ⫽ 15.1 ng/ml; S.E.M. ⫽ 3.0), and also exhibited a gradual decrease in prolactin levels over the 120-min experiment following i.v. vehicle. Effects of Salvinorin A or U69,593 Male Subjects. Intravenous salvinorin A and U69,593 (0.0032– 0.056 mg/kg) caused robust dose- and time-dependent increases in prolactin levels (Fig. 1). Salvinorin A effects were observable by 5 min after i.v. administration, peaked at 15 min after administration, and declined gradually by 120 min. A two-way (time ⫻ dose) repeated measures ANOVA for i.v. salvinorin A (5–120 min and 0.0032– 0.032 mg/kg vehicle) revealed a main effect of time [F(5,15) ⫽ 13.00], dose [F(3,9) ⫽ 12.48], and their interaction [F(15,45) ⫽ 9.70]. The largest salvinorin A dose (0.056 mg/kg) was not included in this analysis because one of the subjects could not be studied at this dose, for safety reasons. Newman-Keuls comparisons at different times post-salvinorin A revealed significant differences for all salvinorin A doses (except the smallest dose, 0.0032 mg/kg) versus vehicle at 5, 15, and 30 min. At 60 min, only the largest salvinorin A (0.032 mg/kg) was different from vehicle. By 90 and 120 min after salvinorin A, no significant differences were detected with Newman-Keuls comparisons. U69,593 effects were similar to those of salvinorin A, with longer duration of action, as suggested by prolactin elevations persisting at the end of the 120-min study period (Fig. 1). A two-way (time ⫻ dose) repeated measures ANOVA for i.v. U69,593 (5–120 min and 0.0032– 0.032 mg/kg and vehicle) revealed a main effect of time [F(5,15) ⫽ 15.73], dose [F(3,9) ⫽ 32.99], and their interaction [F(15,45) ⫽ 16.10]. The largest U69,593 dose (0.056 mg/kg) was not included in the analysis because one of the subjects could not be tested at this dose (the same subject as in the salvinorin A studies).

Fig. 1. Time course effects of i.v. salvinorin A (left) and i.v. U69,593 (right) on serum prolactin levels in male subjects (0.0032– 0.056 mg/kg; n ⫽ 4, except at the largest dose, which was n ⫽ 3). Abscissae, time in minutes from i.v. injection. Ordinates, serum prolactin levels, expressed as change from individual preinjection baseline (⌬ nanograms per milliliter). Data are mean ⫾ S.E.M.; in cases where no error bars are visible, these fall within the symbol for each data point.

Neuroendocrine Effects of Salvinorin A

Newman-Keuls comparisons at different times post-U69,593 revealed significant differences for all salvinorin A doses (except the smallest dose, 0.0032 mg/kg) versus vehicle at 5, 15, 30, 60, and 90 min. At 120 min after U69,593, only the largest U69,593 dose (0.032 mg/kg) was significantly different from vehicle. Dose-effect curves for salvinorin A and U69,593 were plotted at 15 min after i.v. administration (a time of peak effect) and exhibit approximately equal maximum effect and potency (Fig. 2). Clear maximum “plateau” effects were not observed at the largest doses studied in each subject, and this limited the quantitative determination of maximum plateau by nonlinear regression. Larger doses than those used herein (i.e., 0.032 for one subject and 0.056 for the other three) were not probed, due primarily to solubility limitations. Intravenous potency was quantified by linear regression and did not differ significantly between salvinorin A and U69,593 [ED50 for salvinorin A ⫽ 0.015 mg/kg (95% confidence limit ⫽ 0.0048 – 0.050); ED50 for U69,593 ⫽ 0.0098 mg/kg (95% confidence limit ⫽ 0.0041– 0.020)]. Subcutaneous Administration of Salvinorin A. The effects of a probe dose of salvinorin A (0.032 mg/kg) were also studied by the s.c. route in male subjects and resulted in much smaller prolactin release than that observed by the i.v. route and a slower onset. For example, the peak effect after s.c. salvinorin A (0.032 mg/kg) was observed at 60 min postinjection and reached a maximum mean of 31.4 ⌬ng/ml (S.E.M. ⫽ 11.6) (Fig. 3; note y-axis break added for illustration). A one-way repeated measures ANOVA for time (including mean preinjection baseline and 5–120 min after salvinorin A administration) was significant [F(6,18) ⫽ 7.27]. NewmanKeuls comparisons revealed that 0.032 mg/kg s.c. salvinorin A produced a prolactin increase compared with preinjection baseline only at 60, 90, and 120 min. Antagonism Experiments. In separate studies, nalmefene (0.01 or 0.1 mg/kg) was administered as a pretreatment to salvinorin A (0.032 mg/kg i.v.), a dose that produced maximal or near-maximal prolactin release in all subjects. The smaller nalmefene pretreatment dose did not cause significant antagonism of salvinorin A, whereas the larger nalmefene dose (0.1 mg/kg) robustly antagonized the effects of salvinorin A (Fig. 4). A two-way repeated measures ANOVA [time ⫻ pretreatment condition (no pretreatment versus nalmefene 0.01 or 0.1 mg/kg)] revealed significant

Fig. 2. Dose-effect curve for the effects of i.v. salvinorin A and i.v. U69,593 on serum prolactin levels in male subjects (data are obtained from 15 min after administration of each dose; see Fig. 1). Abscissa, dose of salvinorin A or U69,593. Ordinate, serum prolactin levels, expressed as change from individual preinjection baseline (⌬ nanograms per milliliter). Other details as in Fig. 1.

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Fig. 3. Time course effects of salvinorin A (0.0032 mg/kg) administered by the i.v. or s.c. route on serum prolactin levels in male subjects (n ⫽ 4 each). Abscissa, time in minutes from injection. Ordinate, serum prolactin levels, expressed as change from individual preinjection baseline (⌬ nanograms per milliliter; note axis break). Other details as in Fig. 1.

effects of time [F(5,15) ⫽ 11.78] and pretreatment condition [F(2,6) ⫽ 11.49] and their interaction [F(10,30) ⫽ 8.54]. Newman-Keuls comparisons revealed that only the larger nalmefene pretreatment dose (0.1 mg/kg) was significantly different from the “no pretreatment” condition. Antagonism surmountability experiments were not attempted for practical reasons, primarily solubility limitations for salvinorin A. In a separate pretreatment study with the 5-HT2 antagonist ketanserin (0.1 mg/kg i.m.), no antagonism of the same probe dose of salvinorin A (0.032 mg/kg i.v.) was observed (Fig. 4). Female Subjects. Salvinorin A (0.0032 mg/kg i.v.) was studied in follicular phase females (n ⫽ 4). This salvinorin A dose, which produced only slight effects in males (above), produced larger prolactin elevations for this assay in the female subjects (see Fig. 5; with comparison to male subjects). Pilot studies with larger salvinorin A i.v. doses (0.032 mg/kg) revealed even greater neuroendocrine effects. To probe the effects of salvinorin A route of administration, a larger salvinorin A dose (0.032 mg/kg) was studied by the s.c. route. In females, s.c. salvinorin A produced robust prolactin release from 15 min after administration, and this effect persisted for at least 120 min (see Fig. 6). This effect of salvinorin A was prevented by nalmefene (0.1 mg/kg s.c. 30 min pretreatment). In this case also, the effects of salvinorin A were more robust in females than in males (compare Figs. 3 and 6). Cloning of OPRK1 and Genotyping of C-Terminal Sequence. The cloned cDNA contained 30 bp of the 5⬘untranslated region, 1140 bp of the coding region, and 87 bp of the 3⬘-untranslated region. The obtained full-length coding sequence for rhesus monkey OPRK1 was compared with the published sequence for the human OPRK1. There were 21 nucleotides and 6 amino acid residues that differed between the rhesus monkey and human OPRK1 (see Fig. 7). Two predicted amino acid residue changes are located in the N terminus, and two others are in the C terminus of the rhesus monkey OPRK1, compared with the human OPRK1 (Fig. 7). It is noteworthy that a proposed phosphorylation site serine residue (S358) in the C terminus in the human OPRK1 is conserved in the rhesus monkey OPRK1 (Liu-Chen, 2004). Sequence analysis of the C terminus of genomic DNA from 14 rhesus monkeys in the colony (including all eight subjects used in the present neuroendocrine studies, four male and four female) confirms the presence of this S358 residue in all

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Fig. 4. Effects of nalmefene (0.01 or 0.1 mg/kg s.c.) or ketanserin (0.1 mg/kg i.m.) pretreatment to the effects of salvinorin A (0.032 mg/kg i.v.) in male subjects (n ⫽ 4 each). Abscissae, time in minutes from i.v. injection (point above N or K are samples obtained 20 min after administration of nalmefene or ketanserin alone, respectively). Ordinates, serum prolactin levels, expressed as change from individual preinjection baseline (⌬ nanograms per milliliter). Other details as in Fig. 1.

Fig. 5. Effects of i.v. salvinorin A (0.0032 mg/kg) or vehicle in male or female subjects (n ⫽ 4 each). Other details as in Fig. 1.

Fig. 6. Effects of s.c. salvinorin A (0.032 mg/kg) alone or after pretreatment with nalmefene (0.1 mg/kg s.c.) in female subjects. Point above N represents sample obtained 20 min after nalmefene alone. Other details as in Fig. 1.

subjects; no polymorphisms were detected in this region overall.

Discussion The main aim of these studies was to examine the neuroendocrine effects of the widely available hallucinogen salvinorin A in an assay shown to be a useful biomarker for

␬-opioid agonist effects in rhesus monkeys. A related aim of these studies was to determine the similarity of the rhesus monkey ␬-receptor sequence, given reports of relevant interspecies differences between human ␬-receptors and common experimental rodent species such as rat (Liu-Chen, 2004). Salvinorin A was reported to be a selective ␬-agonist, with potentially unique pharmacodynamic effects (Roth et al., 2002; Chavkin et al., 2004; Wang et al., 2004). In the present studies, i.v. salvinorin A caused robust dose-dependent prolactin release in male rhesus monkeys. Salvinorin A was approximately equipotent and equieffective to the synthetic high-efficacy ␬-agonist U69,593, similar to initial in vitro reports (Roth et al., 2002). As expected from prior studies in humans, probe experiments with salvinorin A in gonadally intact female monkeys revealed quantitatively greater effects (Kreek et al., 1999). A probe experiment revealed that the neuroendocrine effects of a probe salvinorin A dose (0.032 mg/kg) were significantly greater by the i.v. than the s.c. route in males. Reasons for this are unclear but may be related to pharmacokinetic factors, possibly limiting bioavailability by the s.c. route (Schmidt et al., 2005). As mentioned above, salvinorin A is a highly selective agonist at ␬-receptors; however, it is known that other compounds, including ␮-agonists, can also cause prolactin release in mammals. We therefore carried out antagonism studies with the clinically available compound nalmefene, which acts as a ␮-opioid antagonist in rhesus monkeys at small doses (e.g., 0.01 mg/kg), and acts as both a ␮- and ␬-antagonist at relatively larger doses (e.g., 0.1 mg/kg) (France and Gerak, 1994; Butelman et al., 2002). In these studies, the smaller dose of nalmefene mentioned above did not prevent the effects of salvinorin A, whereas the larger dose of nalmefene fully prevented such effects. Taken together with previous data (France and Gerak, 1994; Butelman et al., 2002), these studies are consistent with mediation by ␬-receptors in the neuroendocrine effects of salvinorin A. Salvinorin A is distinct from classic hallucinogens such as d-lysergic acid diethylamide, in that it does not bind to the 5-HT2A receptor (Roth et al., 2002). We wanted to determine whether the present neuroendocrine effects of salvinorin A could be indirectly mediated by 5-HT2 receptors. In a probe experiment, the 5-HT2 antagonist ketanserin (0.1 mg/kg) did not block the neuroendocrine effects of salvinorin A under the

Neuroendocrine Effects of Salvinorin A

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Fig. 7. Amino acid coding sequence for OPRK1 cloned from cDNA. Rhesus monkey sequence is compared with published sequence for human OPRK1 (GenBank accession no. NM_000912), and rat OPRK1 (GenBank accession no. NM_017167) (Yakovlev et al., 1995).

present conditions. This dose of ketanserin was sufficient to block the reinforcing effects of the stimulant/hallucinogen methylenedioxymethamphetamine (Ecstasy) in this species (Fantegrossi et al., 2002); methylenedioxymethamphetamine is also known to cause prolactin release in humans (Grob et al., 1996). Overall, this experiment supports the conclusion that salvinorin A produces this neuroendocrine effect through ␬- and not 5-HT2 receptors in primates. Cloning of the rhesus monkey OPRK1 gene revealed greater predicted homology to human ␬-receptor (374 of 380 residues; 98.4% homology) compared with that of other experimental species previously reported (for review, see LiuChen, 2004). Rhesus monkey OPRK1, as determined from cDNA and confirmed by genotyping the present subjects, exhibit the S358 residue in the C terminus, which is present in human OPRK1. Studies indicate that this residue is of critical importance for the maintenance of adaptations including receptor desensitization and internalization (LiuChen, 2004). Interestingly, this residue is not conserved in rat OPRK1, and this may underlie the lesser propensity for such adaptations in rat OPRK1 in vitro. Overall, these initial studies suggest that rhesus monkey OPRK1 may have greater functional similarity to human OPRK1 than those of other experimental species. This is the first report, to our knowledge, of full-length cloning of a nonhuman primate ␬-receptor. As expected based on studies of ␮-receptors, nonhuman primates may provide valuable insights into species differences that may occur with other experimental subjects such as rodents (Miller et al., 2004). In summary, the widely available hallucinogen salvinorin A produced effects consistent with high-efficacy agonist actions at ␬-receptors, in a neuroendocrine biomarker of translational value. This confirms the ␬-receptor as the primary site of action in vivo of this unique hallucinogen. These are, to

our knowledge, the first data on the neuroendocrine effects of salvinorin A in any species. Salvinorin A’s effects in this assay are consistent with reports of fast onset and relatively short duration of salvinorin A-containing preparations in humans (http://biopsych.com/cpdd/CPDD04_PDFs/ CPDD04_981339497336.pdf; Gonzales et al., 2006). Acknowledgments

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diterpene from Salvia divinorum (Labiatae). J Chem Soc Perkin Trans I 1:2505– 2508. Pecins-Thompson M, Brown NA, Kohama SG, and Bethea CL (1996) Ovarian steroid regulation of tryptophan hydroxylase mRNA expression in rhesus macaques. J Neurosci 16:7021–7029. Roth BL, Baner K, Westkaemper R, Siebert D, Rice KC, Steinberg S, Ernsberger P, and Rothman RB (2002) Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist. Proc Natl Acad Sci USA 99:11934 –11939. Rothman RB, Bykov V, Xue BG, Xu H, DeCosta BR, Jacobson AE, Rice KC, Kleinman JE, and Brady LS (1992) Interaction of opioid peptides and other drugs with multiple kappa receptors in rat and human brain: evidence for species differences. Peptides 13:977–987. Schmidt MD, Schmidt MS, Butelman ER, Harding WW, Tidgewell K, Murry DJ, Kreek MJ, and Prisinzano TE (2005) Pharmacokinetics of the plant-derived hallucinogen salvinorin A in nonhuman primates. Synapse 58:208 –210. Tidgewell K, Harding WW, Schmidt M, Holden KG, Murry DJ, and Prisinzano TE (2004) A facile method for the preparation of deuterium labeled salvinorin A: synthesis of [2,2,2-2H3]-salvinorin A. Bioorg Med Chem Lett 14:5099 –5102. Valdes LJ (1994) Salvia divinorum and the unique diterpene hallucinogen, salvinorin (divinorin) A. J Psychoactive Drugs 26:277–283. Valdes LJ, Butler WM, Hatfield GM, Paul AG, and Koreeda M (1984) Divinorin A, a psychotropic terpenoid, and divinorin B from the hallucinogenic Mexican mint, Salvia divinorum. J Org Chem 49:4716 – 4720. Wang Y, Tang K, Inan S, Siebert D, Holzgrabe U, Lee DYW, Huang P, Li JG, Cowan A, and Liu-Chen LY (2004) Comparison of pharmacological activities of three distinct k-ligands (salvinorin A, TRK-820 and 3FLB) on kappa opioid receptors in vitro and their antipruritic and antinociceptive activities in vivo. J Pharmacol Exp Ther 312:220 –230. Yakovlev AG, Krueger KE, and Faden AI (1995) Structure and expression of a rat kappa opioid receptor. J Biol Chem 270:6421– 6424. Zhang Y, Butelman ER, Schlussman SD, Ho A, and Kreek MJ (2004) Effects of the kappa opioid agonist R-84760 on cocaine-induced increases in striatal dopamine levels and cocaine-induced place preference in C57BL/6j mice. Psychopharmacology 173:146 –152. Zhang Y, Butelman ER, Schlussman SD, Ho A, and Kreek MJ (2005) Effects of the plant-derived hallucinogen salvinorin A on basal dopamine levels in the caudate putamen and in a conditioned place aversion assay in mice: agonist actions at kappa opioid receptors. Psychopharmacology 179:551–558.

Address correspondence to: Dr. Eduardo R. Butelman, The Rockefeller University, Box 171, 1230 York Avenue, New York NY 10021. E-mail: [email protected]

Psychopharmacology (2007) 190:441–448 DOI 10.1007/s00213-006-0639-1

ORIGINAL INVESTIGATION

Hallucinatory and rewarding effect of salvinorin A in zebrafish: κ-opioid and CB1-cannabinoid receptor involvement Daniela Braida & Valeria Limonta & Simona Pegorini & Alessia Zani & Chiara Guerini-Rocco & Enzo Gori & Mariaelvina Sala

Received: 23 May 2006 / Accepted: 3 November 2006 / Published online: 12 January 2007 # Springer-Verlag 2007

Abstract Rationale The hallucinatory effect and potential abuse of salvinorin A, the major ingredient of Salvia divinorum, has not been documented in animals. Objective The effects of salvinorin A on the zebrafish (Danio rerio) model, through its swimming behavior and conditioned place preference (CPP) task, was studied. Materials and methods Swimming activity was determined in a squared observational chamber after an i.m. treatment of salvinorin A (0.1–10 μg/kg). For the CPP test, zebrafish were given salvinorin A (0.2 and 1 μg/kg) or vehicle and evaluated in a two-compartment chamber. Results Salvinorin A (0.1 and 0.2 μg/kg) induced accelerated swimming behavior in comparison with vehicle, whereas a “trance-like” effect, at doses as 5 and 10 μg/kg, was obtained. Pretreatment with the κ-opioid antagonist, nor-binaltorphimine (nor-BNI; 10 mg/kg) and the cannabinoid type 1 (CB1) antagonist, rimonabant (1 mg/kg), blocked salvinorin A-induced both stimulating and depressive effects obtained at a dose of 0.2 and 10 μg/kg, respectively. In the CPP test, salvinorin A (0.2 and 0.5 μg/kg) produced an increase in the time spent in the drugassociated compartment. A dose of 1 μg/kg produced no effect, whereas a dose of 80 μg/kg induced aversion. D. Braida (*) : V. Limonta : S. Pegorini : A. Zani : M. Sala Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy e-mail: [email protected] C. Guerini-Rocco : E. Gori Behavioural Pharmacology and Drug Dependence Center, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy

Pretreatment with nor-BNI or rimonabant fully reversed the reinforcing properties of salvinorin A (0.5 μg/kg). Conclusions Taken together, these results indicate that salvinorin A, as is sometimes reported in humans, exhibits rewarding effects, independently from its motor activity, suggesting the usefulness of the zebrafish model to study addictive behavior. These effects appear mediated by activation of both κ-opioid and cannabinoid CB1 receptors. Keywords Salvia divinorum . Zebrafish . Conditioned place preference . Swimming behavior . SR 141716A . Nor-binalthorphimine

Introduction Salvia divinorum, a member of the Lamiaceae family, has been used for many years by the Mazatec Indians of northeastern Oaxaca, Mexico, primarily for its psychoactive effects (Wasson 1962, 1963; for reviews, see Valdes et al. 1983; Sheffler and Roth 2003). The active ingredient of S. divinorum, salvinorin A, is a non-nitrogenous neoclerodane diterpene, which represents the most potent naturally occurring hallucinogen until now known (Valdes et al. 1984; Siebert 1994). Both S. divinorum and salvinorin A have been used recreationally for their hallucinogenic properties (Giroud et al. 2000). Salvinorin A induces in humans an intense, short-lived hallucinogenic and positive effects (changes in depth perception, increase in sensual and aesthetic appreciation, creative dream-like experience). All these effects are distinct from those of the classical hallucinogens, such as lysergic acid diethylamide (LSD), psilocybin, and mescaline (Siebert 1994). However, depending on the dose and the route of administration, the

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effects are sometimes extremely negative (overly-intense experiences, fear, terror and panic, increased perspiration and possible difficulty integrating experiences), and people have no desire to repeat the experience (Turner 1996). Users, particularly teenagers, claim the drug is not addictive, but its potential abuse is not known. Salvinorin A is a potent and highly selective κ-agonist, with greater efficacy than that of the synthetic κ-agonist U-50488 and U-69593, as revealed by binding studies (Roth et al. 2002; Sheffler and Roth 2003; Chavkin et al. 2004). In vivo studies revealed that salvinorin A is a relatively low toxic substance after acute or chronic treatment in mice and rats even when given at doses many times greater than those used by humans (Mowry et al. 2003). In a range of doses between 0.5 and 4.0 mg/kg, an antinociceptive activity in the tail-flick and hot-plate test, through the activation of κopioid subtype receptor, has been found (McCurdy et al. 2006). Salvinorin A (0.5–2 mg/kg) produces pro-depressant-like effects in the forced swimming test, increasing the occurrence of immobility and decreasing swimming behavior (Carlezon et al. 2006). Until now, the potential abuse of salvinorin A has been poorly investigated. Salvinorin A (0.001–0.032 mg kg−1 sc−1) had discriminative stimulus effects similar to those of κ-agonist U-69593 in rhesus monkey (Butelman et al. 2004). Treatment with salvinorin A (1 and 3 mg/kg i.p.) in C57BL/6J mice produced conditioned place aversion and decreased the dopamine levels in the caudate putamen but not in the nucleus accumbens (Zhang et al. 2005). This effect was completely blocked by the pre-injection with the κ-opioid receptor antagonist nor-binaltorphimine (nor-BNI) at a dose of 10 mg/kg. Recently, some authors have been developing a rapid method to assess reinforcing properties of cocaine (Darland and Dowling 2001) in zebrafish using a conditioned place preference (CPP) paradigm. The zebrafish (Danio rerio) is a small freshwater teleost rapidly emerging as an important model organism in genetics and developmental neurobiology. Although zebrafish enjoy widespread use in biomedicine (e.g. Shin and Fishman 2002), their usefulness, in studying neurological disorders, remains unexplored. During the recent years, zebrafish has been also used in neurobehavioral toxicology (Dlugos and Rabin 2003; Anichtchik et al. 2004; Bretaud et al. 2004) as a model system for the study of human diseases and behavioral disorders for drug potential therapeutic application (Dooley and Zon 2000; Levin and Chen 2004; Zon and Peterson 2005; Giacomini et al. 2006; Levin et al. 2006). In the study of drugs of abuse, the use of a zebrafish as a model and subsequent genetic studies could provide important insights into the molecular and cellular mechanisms underlying addiction. The nervous system of zebrafish is more comparable to that of humans compared to Drosophila. Treatment with ethanol to adult zebrafish lead to dose-

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dependent modifications of locomotor activity (Gerlai et al. 2000) and tolerance as has been observed in mammals as well as Drosophila (Scholz et al. 2000). Moreover, ethanolinduced hyperlocomotion can be blocked by the addition of a dopamine antagonist, suggesting the involvement of the brain dopamine system (Lockwood et al. 2004). Most validation of the zebrafish model, in studying the process of addiction in vertebrates, comes from the use of CPP test with cocaine (Darland and Dowling 2001) and amphetamine (Ninkovic et al. 2006). The aim of the present work was to assess the suitability of using the zebrafish as a model system to investigate the behavioral effects of salvinorin A. In particular, the effect of an acute i.m. injection of a wide range of doses on swimming behavior, using a square observation chamber, was tested. Because a conditioned place aversion was obtained in mice with doses (1–3.2 mg/kg) higher than those effective in the smoked S. divinorum in humans (200–500 μg/human that is about 3–7.5 μg/kg; Valdes 1983; Siebert 1994), low doses of salvinorin A were investigated in the CPP paradigm to determine its potential rewarding properties. As a comparison, cocaine and spiradoline, a κ-opioid agonist, were employed. To investigate the mechanism of action, the κopioid receptor antagonist, nor-BNI, was given before salvinorin A at a dose (10 mg/kg) that has been reported to reverse salvinorin A-induced aversion and dopamine extracellular levels in the caudate putamen in mice (Zhang et al. 2005). Moreover, because an interaction between both κopioid and cannabinoid systems on self-administration has been recently demonstrated (Mendizabal et al. 2005), a possible involvement of the CB1-cannabinoid receptor on salvinorin A effect was studied using rimonabant at a dose (1 mg/kg) able to antagonize Δ9-tetrahydrocannabinolinduced CPP and self-administration (Braida et al. 2004).

Materials and methods Subjects The experimental protocol was approved by the Italian Governmental Decree No. 13/2004-A. All efforts were made to minimize the number of animals used and their discomfort. Adult zebrafish (D. rerio) were kept at approximately 28.5°C on a 14-h light/10-h dark cycle, and behavioral testing took place during the light phase between 900 and 1400 hours. Tank water consisted of deionized H2O and sea salts (0.6 g/10 l of water; Instant Ocean, Aquarium Systems, Sarrebourg, France). The home tanks with groups of adult fish were maintained with constant filtration and aeration. Fish were fed daily with brine shrimp and flake fish food. All the fish were drug naive, and each fish was used only once. There were 10–30 fish per group.

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Treatment Body weight was measured according to Novak et al. (2005). Fish were removed from their tank using a net, momentarily blot dried on gauze, and placed in a container containing tank water, positioned on a torn digital balance. Fish weight was determined as the weight of the container plus the fish minus the weight of the container before the fish was added. The mean of three measurements was recorded. Fish were injected i.m. in the caudal musculature with a volume depending on the weight of the fish (2 μl/g) using a Hamilton syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland).

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anced. On the third day, the fish were free to access to two sides for 15-min and the time spent in each compartment was recorded. The change in preference, obtained by subtracting baseline percentage from the final value, reflected reinforcing or aversive properties. Drugs

Immediately after treatment, each subject was placed in a square observation chamber (10×10 cm) containing home tank water filled at a level of 12 cm. Each fish was observed for 30 s, immediately after injection, every 5 min, for a total of six observation bins, over a 30-min period. Swimming activity was evaluated using a score scale applied to Siamese fighting fish by Abramson and Evans (1954) and adapted by us to zebrafish: 0—“trance-like” effect, horizontal motionless position on bottom tank maintained for 2–3 min at a time at the peak of the narcosis and broken by a very brief change of position by means of a slight stimulus; 2—slowed swimming, normal body position; 4—normality state; 6—accelerated swimming, normal body position; 8—frenetic swimming, with the body suspended in the vertical or some angled from the vertical; 10—frenetic circling behavior.

Salvinorin A (Daniel Siebert, The Salvia divinorum Research and Information Center, Malibu, CA) was dissolved in a vehicle containing cremophor, ethanol, and saline (1:1:8) and injected i.m. in a range of doses between 0.1 and 80 μg/kg. N-piperidino-5-(4-chlorophenyl) 1-(2,4-dichlorophenyl)-4 methyl pyrazole 3-carboxamide (rimonabant), kindly supplied by Synthelabo–Sanofi Recherche, Montpellier, France (1 mg/kg), was dissolved in cremophor, ethanol, and saline (1:1:18). As κ-opioid agonist, (±)-(5α,7α,8β)-3,4-dichloroN-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro [4.5]dec-8-yl] benzeneacetamide mesylate (spiradoline) was dissolved in saline and given i.m. at a dose of 1 mg/kg. Nor-BNI 2HCl (10 mg/kg) and cocaine HCl (Sigma, Chemical, St. Louis, MO, USA; 20 mg/kg) were dissolved in physiological saline. κ-Opioid and cannabinoid CB1 receptor antagonists were given 2 h and 15 min before salvinorin A, respectively. Cocaine HCl (Sigma), used as drug reference, was given at a dose of 20 mg/kg. This dose of cocaine was within the range of doses shown to be effective within the place preference design in rats (Busse et al. 2004). The dose of spiradoline was chosen on the basis of a previous study in rats (Smith et al. 2003) and our pilot experiments in zebrafish. The doses of the drugs were calculated as salt. All drugs were prepared fresh daily.

Conditioned place preference

Statistics

The fish were tested in a two-chamber tank (10-cm wide × 20-cm long × 15-cm high) according to Darland and Dowling (2001), with slight modifications. The tank was divided into two halves (10×10 cm) containing distinct visual cues (two black pois) with a perforated wall that allowed complete, albeit somewhat impeded, movement. On the first day, after an initial introduction to the apparatus, the fish were tested for baseline preference by calculating the percent time spent on a given side during a 15-min trial (preconditioning phase). The fish that displayed abnormal behavior in the apparatus, such as deficient or excessive swimming or a baseline preference greater than 70%, were not tested further (rarely more than 3 or 4 of 20 fish tested from a given family). The fish were then treated i.m. with the drug and then restricted to the least preferred side for 30 min. The next day, fish receiving vehicle were confined in the opposite compartment for 30 min. Drug-texture pairings were always counterbal-

The data were expressed as mean±SEM. Swimming activity, evaluated as mean score of six observation bins, was analyzed by Kruskal–Wallis test followed by Dunn’s post-hoc test. Data from CPP test were assessed by oneway analysis of variance (ANOVA) for multiple comparisons followed by Tukey’s post-hoc test. All statistical analyses were done using software Prism, version 4 (GraphPad, San Diego, CA, USA). The accepted level of significance was P 98%. All chemicals and solvents were American Chemical Society analytical grade or HPLC grade. InVitroSomes™, human recombinant cytochrome P450 enzymes were purchased from InVitro Technologies (Baltimore, MD). Human UGT2B7 Supersomes™ enzymes were purchased from BD Biosciences Discovery Labware (Woburn MA). MDCK-MDR1 cells were provided by Dr.Peter W. Swaan (University of Maryland). DMEM, phosphate buffered saline, nonessential amino acid, fetal bovine serum (FBS), L-glutamine, pencillin G-streptomycin sulfate antibiotic mixture and trypsin (0.25%)-EDTA (1mM) were purchased from Invitrogen Laboratories (Carlsbad, CA). Polymixin, amphotericin, heparin, and dextran were purchased from the Sigma Chemical Co. (St.Louis, MO). Twelve well transport plates (cell culture treated) were purchased from Corning Costar (Cambridge, MA). 2.2.SalvinorinA-stimulated P-gp ATPase activity

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In order to assess whether or not Salvinorin A was a P-gp substrate, we determined its ability to stimulate ATPase activity. Salvinorin A stimulated P-gp ATPase activity was estimated by Pgp-GIO assay system (Promega, Madison, WI). This method relies on the ATP dependence of the light-generating reaction of firefly luciferase where ATP consumption is detected as a decrease in luminescence. In a 96 well plate recombinant human P-gp were incubated with Pgp-GIO assay buffer™ (20 μL), verapamil (200 μM), sodium orthovanidate (100 μM), Salvinorin A (2.5 to 100 μM). Each compound was loaded into four individual wells. Verapamil served as a positive control while sodium orthovanadate was used as a P-gp ATPase inhibitor. In the presence of sodium orthovanadate ATP consumption by P-gp is negligible and without sodium orthovanadate, P-gp consumes ATP to a greater or lesser extent than the control, dependent on the effect of the test compounds. The reaction was initiated by addition of MgATP (10 mM), stopped 40 min later by addition of 50 μl of firefly luciferase reaction mixture (ATP detection reagent) that initiated an ATP-dependent luminescence reaction. Signals were measured 100 min later by Lmax® luminometer (Molecular Devices Corporation, Sunnyvale, CA) and converted to ATP concentrations by interpolation from a luminescent ATP standard curve. The rate of ATP consumption (pmol/min/μg protein) was determined as the difference between the amount of ATP in absence and presence of sodium orthovanadate. Salvinorin Astimulated P-gp ATPase activity was reported also as fold-stimulation relative to the basal Pgp ATPase activity in the absence of compound (control). 2.3. In vitro cell culture studies: MDCK-MDR1 cells MDCK-MDR1 cells were cultured in Dulbecco’s modified eagle serum (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100U/mL of pencillin and streptomycin. The cells were plated onto 12 well Costar TranswellR inserts (0.4μM pore size, 1cm2 surface area) at a density of 425,000 cells/cm2. The cells were cultured and maintained in DMEM supplemented with 10% FBS, 2% L-glutamine, 1% non essential amino acid, 1% pencillinstreptomycin under standard conditions of 5% CO2, 37 ± 0.5 °C and 95% humidity until Eur J Pharm Biopharm. Author manuscript; available in PMC 2010 June 1.

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confluence was reached on day four. The medium was changed every day after seeding and confluent monolayers were used for transport studies outlined below. Monolayer integrity was checked by measuring the transepithelial resistance (TEER) and [14C]-mannitol permeability. P-gp functional confirmation was determined by [14C]paclitaxel efflux values. 2.4. Transport study MDCK-MDR1 transport studies were performed as previously described in our laboratory [26]. All transport experiments were performed at 37°C in phosphate buffered saline (PBS). Salvinorin A (5 μM), radiolabeled marker ([14C]mannitol, [14C]paclitaxel) or blank buffer were added to either the apical or basolateral side. Cell monolayers were continuously agitated on a plate shaker during transport experiments (60-70 rpm). For examination of transport in the apical to basolateral (A→B) direction, the transwell inserts were moved to wells with fresh PBS. At time t = 0, 0.5 mL of Salvinorin A solution was added to the apical side of the monolayer. Inserts were moved to new wells at times between 10 and 120 minutes. For examination in the basolateral to apical (B→A) direction, Salvinorin A (1.5 mL) was initially added to the basolateral side at time t = 0. At various time intervals between 10-120 min, samples were collected from the apical side and replaced with fresh, pre-warmed PBS. Samples were analyzed by a UV-HPLC method for Salvinorin A. Apparent permeability coefficients were determined for each transport study. Radioactive compounds were analyzed by scintillation counter (Beckman Coulter LS 6500).

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2.5.In vitro metabolism screening with various human CYP450 isoforms and UGT2B7

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A screening phenotyping approach was used to identify the isozyme(s) responsible for its metabolism using various cytochrome (CYP) isoforms including CYP2D6, CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2E1, CYP3A4 and CYP3A5. InVitroSomes™, used in this study, are single enzyme systems that express a specific CYP isoform (e.g., CYP2D6) and yeast CYP-reductase co-expressed in Saccharomyces cerevisiae. In VitroSomes™ are ideal for use in drug metabolism studies [27] particularly in specific CYP pathway identification (reaction phenotyping) and CYP inhibition screening. InVitroSomes™ expressing the aforementioned isoforms were incubated with Salvinorin A (0, 50 μm or 5 μm), and TE (500 mM Tris, 10 mM EDTA) reaction buffer. TSE (500 mM Tris, 2 M NaCl, 10 mM EDTA) reaction buffer was used for CYP2B6, CYP3A4, and CYP3A5 instead of TE buffer. This solution was incubated with shaking for 5 minutes at 37°C. At the end of this period, 20 μL of NADPH (5 mg/mL) was added to each sample to start the reaction. This was followed by one hour incubation at 37°C. At the end of this second incubation period, 100 μL of cold acetonitrile was added to stop the reaction. All samples were analyzed for Salvinorin A using our validated UV-HPLC method. Preliminary studies were performed to find the optimal incubation time which was used in subsequent studies. Salvinorin A (50 μM) was incubated with CYP2D6 at different incubation times (10, 30 and 60 min) at 37°C. Significant decrease was observed at 60 min, and this incubation time period was used for subsequent studies. In vitro metabolism studies were also conducted to evaluate the role of UGT in Salvinorin A’s metabolism. Supersomes™ were used for possible glucuronidation activity. The reaction mixture (0.2 mL) containing 1 mg/mL protein (UGT2B7), 1mM uridine diphosphoglucuronic acid (UDPGA), 10 mM magnesium chloride, 0.025 mg/mL alamethicin and 0, 10 or 50 μM of Salvinorin A in 50 mM tris (pH 7.5) was incubated at 37°C for one hour. Two hour incubation time was used for 0 and 5 μM of Salvinorin A. After the incubation period, the reaction was stopped by the addition of 100 μL acetonitrile and centrifuged (10,000 rpm) for 15 minutes. All samples were analyzed using an UV-HPLC method.

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2.6. Pharmacokinetic studies

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2.6.1. Animals—Male Sprague Dawley rats weighing 275 to 300 g were purchased from Harlan Laboratories (Indianapolis, IN). The animals were housed in an AAALAC accredited facility run on a 12 hour light and dark cycle. The animals were allowed unrestricted access to food and water. The protocol for the animal studies was approved by the School of Pharmacy, University of Maryland IACUC. 2.6.2. Dosing and sampling—To evaluate the in vivo pharmacokinetic and brain distribution of Salvinorin A, a single dose study was performed in adult male Sprague Dawley rats. Salvinorin A was administered as a single i.p. dose of 10 mg/kg (in Cremophor EL: ethanol, 70%:30%). A destructive sampling study design was followed where cohorts of three animals were euthanized by CO2 asphyxiation at pre-dose and at the following time points post dosing: 5, 10, 15, 30, 60, 90, 120, and 240 minutes. Blood samples were collected by cardiac puncture using pre-heparinized syringes and immediately transferred into tubes containing acetonitrile to inhibit possible esterase metabolism. Blood samples were centrifuged at 3500 rpm for 10 minutes and plasma separated. Brain tissue was immediately excised, blotted dry and weighed. All samples were stored at -80°C until analyzed. 2.7. Quantification of Salvinorin A

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2.7.1. UV-HPLC systems to quantifying Salvinorin A in in vitro samples—A UVHPLC method was used to quantify the Salvinorin A concentrations in the transport and in vitro metabolism studies. The chromatographic conditions consisted of a Waters Symmetry (C18 5 μm, 150 × 4.6 mm) column plus Supelguard 5 μm LC-18, 2 cm guard column. The mobile phase (acetonitrile: water, 55:45 v/v) was filtered through a 0.45 μm nylon filter and degassed under ultrasound and vacuum for 15 min and pumped at a flow rate 1 mL/min over a 20 min period. The injection volume was 200 μL. Salvinorin A was quantified at a UV wavelength of 210 nm and its retention time was 12 min. The sensitivity limit for Salvinorin A using this method was 100 ng/mL.

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2.7.2. LC/MS/MS analytical method to quantifying Salvinorin A in biological matrix—An LC/MS/MS analytical method was used to quantify Salvinorin A in plasma and brain. Four mL of hexane was added to 0.4 mL of plasma, vortexed (1 min) and centrifuged at 10,000 rpm for 10 min. The supernatant was evaporated to dryness at 40°C under a gentle stream of nitrogen and reconstituted with 100 μL of mobile phase. Brain tissue was homogenized, and diluted with an equal volume of PBS. Four mL of hexane was added to the brain homogenate (0.8 mL), vortexed for 2 minutes and centrifuged at 10,000 rpm for 10 min. The organic phase was transferred to a clean test tube, evaporated under nitrogen and reconstituted with 100 μL of mobile phase. The supernatant was transferred to a microvial and thirty μL were injected onto the LC/MS/MS system. The LC/MS/MS system consisted of a quattro micro triple quadrapole mass spectrometer (Micromass-Waters, Millford, Boston) operated in the positive ion mode with an ESI-probe. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The source was operated at 140 degree and nitrogen was used as the nebulizer gas and argon was used as the collision gas set at 10 psi. The cone voltage was 45 V, capillary voltage was 3.5 V, and the entrance and exit voltages were -5 and 1 respectively. The HPLC system consisted of a Waters 2695 quaternary system, Xterra MS C18 (2.5 μm, 2.1 × 50 mm,) column and the mobile phase was composed of acetonitrile and water (55:45, v/v). The mobile phase was pumped at flow rate of 0.2 mL/min and the injection volume was 30 μL. Following HPLC separation, the Salvinorin A peak area corresponding to 433.5-373 parent-daughter transition and the internal standard peak (BZT) peak area corresponding to 342.5-201.1 parent daughter transition were quantitated. The retention time for Salvinorin A was 2.8 min and 1.6 min for

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the internal standard. The total run time was five minutes. The plasma and brain calibration curves were linear in the range of 7.5 to 500 ng/mL (r2 ≥ 0.999) and 7.5 to 200 ng/mL (r2 ≥ 0.997) for plasma and brain, respectively. 2.8. Data analysis 2.8.1. ATPase assay—Basal P-gp activity, test compound stimulated P-gp activity and fold stimulation by a test compound were calculated according to the following equations:

Eq.(1)

Eq.(2)

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Where ATPvanadate is the number of non consumed (total) pmoles of ATP in the presence of sodium orthovanadate. ATPcontrol is the number of non-consumed pmoles of ATP in presence of the assay buffer. ATPcompound is the number of non consumed pmoles of ATP in presence of a test compound.

Eq.(3)

2.8.2 Permeability calculations and cell culture data analysis—The calculation of apparent permeability (Papp), for transport studies across cell monolayers was determined from the following equation:

Eq.(4)

NIH-PA Author Manuscript

where Papp is the permeability, Vr is the receiver compartment volume, dCr is the receiver compartment concentration at the end of the interval, dt is the time of the interval, A is the area of the filter, and Cd is the donor compartment concentration at the start of the interval. All experiments were performed in triplicate and data from the transport experiments are presented as mean ± standard deviation (SD). The efflux ratio (R) was calculated according to the following equation:

Eq.(5)

2.8.3. In vitro metabolism data analysis—In vitro metabolism data were converted to the % remaining of Salvinorin A as below:

Eq.(6)

Eur J Pharm Biopharm. Author manuscript; available in PMC 2010 June 1.

Teksin et al.

Page 7

NIH-PA Author Manuscript

where Ctreated is enzyme treated Salvinorin A concentration, Cinitial is initial Salvinorin A concentration. All metabolism experiments for each enzyme were done in triplicate and data was presented as mean ± SD. Data were statistically compared by Student’s t-test and significance set at p10,000

>10,000

300 ± 23

7a

>10,000

>10,000

75 ± 3

7b

>10,000

>10,000

81 ± 6

8a

777 ± 40

>5,000

6.7 ± 0.32

8b

>5,800

>5,000

1,233 ± 33

9a

>3,000

>5,000

5,261 ± 680

9b

3,523 ± 389

>5,000

7,438 ± 764

10c

ND

ND

41 ± 1

11c

ND

ND

171 ± 7

12c

1,540 ± 140

ND

ND

14

>3,000

>3,000

59 ± 4

a

Receptor binding was performed in CHO cells which express the human MOP, DOP, or KOP receptors. All results are N = 3.

b

Data from reference 21.

c ND indicates that Ki was found to be >10,000 nM in a range finding study.

NIH-PA Author Manuscript Medchemcomm. Author manuscript; available in PMC 2012 December 1.

Prevatt-Smith et al.

Page 16

Table 2

NIH-PA Author Manuscript

[35S]GTP-γ-S functional assay for compounds 3, 4, 5, 7,8a, and 14. Cmpd

ED50 ± SDa (κ) nM

Emax ± SDb (κ)

1c

40 ±10

120 ± 2

3

6±1

118 ± 2

4

0.65 ± 0.17

127 ± 5

5

60 ± 6

109 ± 3

7a

1220 ± 230

112 ± 8

7b

690 ± 80

103 ± 4

8a

150 ± 14

101 ± 3

14

1934 ± 239

113 ± 5

a

ED50 = Effective dose for 50% maximal response.

b

Emax is % at which compound stimulates [35S]GTP-γ-Sbinding compared to (−)-U50,488 (500 nM) at KOP receptors.

c Data from reference 21.

NIH-PA Author Manuscript NIH-PA Author Manuscript Medchemcomm. Author manuscript; available in PMC 2012 December 1.

Journal of Psychopharmacology http://jop.sagepub.com/

Salvia divinorum use and phenomenology: results from an online survey HR Sumnall, F Measham, SD Brandt and JC Cole J Psychopharmacol 2011 25: 1496 originally published online 11 October 2010 DOI: 10.1177/0269881110385596 The online version of this article can be found at: http://jop.sagepub.com/content/25/11/1496

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Original Paper

Salvia divinorum use and phenomenology: results from an online survey HR Sumnall1, F Measham2, SD Brandt3 and JC Cole4

Journal of Psychopharmacology 25(11) 1496–1507 ! The Author(s) 2011 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0269881110385596 jop.sagepub.com

Abstract Salvia divinorum is a hallucinogenic plant with ethnopharmacological and recreational uses. It differs from classic serotonergic hallucinogens such as LSD and psilocin in both phenomenology and potent agonist activity of the active component salvinorin A at k-opioid receptors. Awareness of S. divinorum has grown recently, with both an increase in its public representation and concern over its potential harmful effects. This discussion is particularly relevant as S. divinorum is legal to use in many countries and regions and easily available through online retailers. Drawing upon previous investigations of S. divinorum and other hallucinogens, this study surveyed 154 recent users and questioned them on their use behaviours, consequences of use and other attitudinal measures. Although reporting an extensive substance use history, and considering the limitations of online surveys, there was little evidence of dysfunctional S. divinorum use, and few reports of troubling adverse consequences of use. Furthermore, there was no evidence that users exhibited increased schizotypy. Respondents reported that S. divinorum produced mixed hallucinogenic and dissociative effects, which lends support to assertions that it phenomenologically differs from other hallucinogens with primary serotonergic activity. The functions of use changed with greater experiences with the drug, and although many respondents reported use of S. divinorum as an alternative to illegal drugs it, was apparent that legal proscription would be unlikely to dissuade them from use. These results are discussed with reference to psychopharmacologically informed public health responses to substance use.

Keywords Drug effects, hallucinogens, Salvia divinorum, subjective experiences

Introduction Salvia divinorum (S. divinorum) has traditional uses as an entheogen and as an ethnopharmacological treatment (Ott, 1995), although it is better known in the developed world as a recreational hallucinogen (Khey et al., 2008). The active component salvinorin A is a potent neoclerodane diterpene hallucinogen with selective agonist activity at k-opioid receptors (and peripheral actions on cholinergic transmission), distinguishing it from the classic serotonergic hallucinogens such as LSD and psilocin (see Butelman et al., 2007; Capasso et al., 2006; Ortega et al., 1982; Roth et al., 2002). Further indirect actions on dopaminergic, noradrenergic, and endocannabinoid systems have also been characterized (Braida et al., 2008, Grilli et al., 2009; Zhang et al., 2005). The drug is active in humans in doses around 200 mg when administered through vaporization (thus avoiding hepatic first-pass metabolism), and is orally active when held in the mouth for >10 min (Siebert, 1994). One interesting feature of salvinorin A and its naturally occurring derivatives is the lack of nitrogen, and it would appear that none of the currently identified plant constituents are alkaloids. Other compounds isolated from the plant include Salvinorins B–I (Lee et al., 2005; Munro and Rizzacasa 2003; Shirota et al., 2006; Valde´s et al., 1984, 2001), divinorin F, salvidivins A–D (Shirota et al., 2006), divinatorins A–F (Bigham et al., 2003; Munro and Rizzacasa, 2003) and salvinicins A and B (Harding et al.,

2005). Little is known about whether they would be centrally active in humans, but the salvinorin A nucleus provides a structural template for a large number of chemically altered entities (for example, see Beguin et al., 2006, 2008, 2009). 2-Methoxymethyl-salvinorin B, for example, is a more potent k-opioid receptor agonist than salvinorin A, and shows longer-lasting behavioural activity in murine tests of ambulation and nociception (Wang et al., 2008). To date there have been only a few national estimates of S. divinorum use prevalence in the general population. In the USA, the National Survey on Drug Use and Health estimated that about 1.8 million persons aged 12 or older used

1 Centre for Public Health, Liverpool John Moores University, Liverpool, UK. 2 Department of Applied Social Science, Lancaster University, Lancaster, UK. 3 School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK. 4 School of Psychology, University of Liverpool, Liverpool, UK.

Corresponding author: Dr Harry Sumnall, Centre for Public Health, Liverpool John Moores University, Henry Cotton Building, Webster Street, Liverpool L3 2ET, UK Email: [email protected]

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S. divinorum in their lifetime, and approximately 750,000 did so in the previous year (SAMHSA, 2008). Other local and population-specific studies have been conducted. For example, lifetime prevalence was estimated to be 28.7% and lastyear prevalence of 8.8% in readers of the UK dance music magazine Mixmag; modal frequency of use was monthly (Winstock, personal communication). In university students in Florida (where S. divinorum was legal at the time of the study), 11% of males and 4% of females reported a lifetime use (Khey et al., 2008). A similar study in California estimated last-year prevalence at 4.4% (Lange et al., 2008). Regression analysis in another US college sample showed users were most likely to be young white males with a high prevalence of cannabis use (Miller et al., 2009). Over half of respondents in an internet-based survey reported reduction or cessation of use in the previous 12 months, most commonly citing dislike of the subjective effects or a loss of interest in S. divinorum (Biglete et al., 2009). In this study, age of initiation was related to use function, with young adults reporting using for fun, whilst older adults cited ‘spiritual’ reasons (not defined by the authors, but see Saunders et al., 2000, and Wilber, 2006, for popular discussions). In retrospective surveys of the subjective effects of use, participants typically report a small number of lifetime uses (n < 20), and a range of hallucinogen/psychedelic-like experiences (Baggott et al., 2004). Interestingly, however, these are reported to be qualitatively distinct from those produced by serotonergic hallucinogens such as LSD and psilocin. For example, the derealization and physical impairment (similar to that produced by NMDA receptor antagonists) produced by S. divinorum at typical doses is thought to be unique, as this effect is only seen at relatively higher doses with other hallucinogens (Arthur, 2008; Ball, 2007; Dalgarno, 2007; Gonza´lez et al., 2006; Lange et al., 2010; Pendell, 1995). Data on the potential adverse effects and toxicity of S. divinorum are limited. Over 10 years, 37 cases were reported to the California Poison Control System after intentional exposure to S. divinorum (Vohra et al., 2011). Just under half of these were associated with ingestion of S. divinorum alone, and the most common symptoms were confusion or disorientation, hallucinations, dizziness, and gastrointestinal disturbances. All patients recovered after appropriate intervention. In rats, no significant effects were seen on heart rate, body temperature, or galvanic skin response after chronic salvinorin A administration, although an increase in pulse pressure was recorded (Mowry et al., 2003). Furthermore, toxicity was not apparent in mice after chronic administration of 400–6400 mg/kg (around 1–16 times the typical human dose) once daily over 2 weeks (Mowry et al., 2003). One clinical case report described a 15-year-old male with a recent history of S. divinorum use presenting with paranoia, de´ja` vu and blunted affect shortly after self-administration of cannabis (Singh, 2007). An 18-year-old female was admitted to psychiatric services with acute onset of agitation, disorganization, and hallucinations shortly after smoking cannabis (Paulzen and Gru¨nder, 2008). It transpired that her partner had added S. divinorum to the herbal cannabis smoking mixture. A 21-year-old man also presented with symptoms of acute psychosis and paranoia, including echolalia and psychomotor agitation (Przekop and Lee, 2009).

Despite antipsychotic treatment the authors noted that the patient did not exhibit improvement at 4 months’ follow-up. In all three cases, the authors exclusively attributed these symptoms to S. divinorum because of purported links between k-opioid receptor agonist activity and changes in dopaminergic transmission, with psychomimetic symptomatology (Pfeiffer et al., 1986). However, with respect to this latter case, it should be noted that chronic psychotic episodes after hallucinogen use are rare (Strassman, 1984), and the authors provided no information on treatment adherence during this period. In contrast, daily low-dose self-medication for depression with orally administered S. divinorum leaves has been reported, apparently with the full remission of symptoms (Hanes, 2001). Understanding of the behavioural pharmacology of salvinorin A is growing. A study investigating the human pharmacokinetics of smoked salvinorin A had to be abandoned after the two volunteers became too intoxicated to provide blood samples, although it appeared in urine up to 1.5 h after administration, suggesting rapid elimination (Pichini et al., 2005). In non-human primates, the elimination half-life of salvinorin A was 56.6  24.8 minutes after a bolus intravenous (i.v.) administration that was predicted to have the same disposition as the smoked drug (Schmidt et al., 2005). Sex-dependent pharmacokinetics were also noted, suggesting the possibility of differences in pharmacology. Salvinorin A produced dose-dependent k-opioid receptor agonist-like response after drug discrimination training with U69,593 in both rats and rhesus monkeys, supporting the role of this receptor in the production of behavioural/subjective effects (Baker et al., 2009; Butelman et al., 2004; Wilmore-Fordham, 2007). However, salvinorin A did not substitute for the 5-HT2A receptor agonist hallucinogen DOM in rhesus monkeys (Li et al., 2008). As with other k-opioid receptor agonists, administration of salvinorin A attenuated cocaine seeking in rats (Morani et al., 2009) and produced a conditioned place aversion in mice at high doses (1–3.2 mg/kg) (Zhang et al., 2005). This latter effect was similar to that produced by mescaline (Cappell and LeBlanc, 1971), but not LSD (Meehan and Schechter, 1998), and was associated with a decrease in dopamine concentration in the caudate putamen. However, in rats, 0.05–160 mg/kg subcutaneous (s.c.) salvinorin A produced a conditioned place preference, and 0.01–1 mg intracerebroventricular (i.c.v.) infusions were self administered (Braida et al., 2008). Place preference was also observed in zebra fish (Braida et al., 2007). These findings suggest that the rewarding effects of salvinorin A may be dose, species, and model specific. In mice, antinociception, sedation, and motor incoordination effects have been observed (Fantegrossi et al., 2005; McCurdy et al., 2006), and in the forced swim test rats treated with high doses of salvinorin A showed increased immobility and decreased swimming, suggesting pro-depressant like effects (Carlezon et al., 2006). However, at lower doses (0.25–2 mg/kg), rats exhibited both anxiolytic and antidepressant effects (Hanes, 2001), again suggesting behavioural effects are dose dependent (Braida et al., 2009). Although not a new phenomenon (Hoffmann, 1980), the increased awareness of the use of S. divinorum has led to both public health and legislative concerns (Bu¨cheler et al., 2005). Federal legislation against S. divinorum exists only in some

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Journal of Psychopharmacology 25(11)

countries, and there is also legislation in some USA states, although at the time of writing the UK’s Advisory Council on the Misuse of Drugs (ACMD) is considering providing recommendations for Government on its legal status. This concern has partly been driven by perceived ease of access to S. divinorum and other drugs through the internet and city centre retailers (Dennehy et al., 2005; Halpern and Pope, 2001; Hoover et al., 2008; Siemann et al., 2006), and also by popular representations of use in the media, particularly through new media such as the online YouTube video site (Lange et al., 2010). Other authors have suggested that such powerful, but legal, recreational drugs are popular as they allow intoxication without the need for otherwise law-abiding citizens to engage with criminal markets (Hammersley, 2010; Measham et al., 2010). This study aimed to provide further clarification of the subjective effects of S. divinorum, use patterns, and experience of adverse effects in order to inform psychopharmacologically based public health discussions. The present study explored multidimensional attitudes regarding S. divinorum which provided a more complete cultural understanding than that reported by Gonza´lez et al. (2006). We were also interested in whether the legality of S. divinorum, and as a consequence relative ease of availability, was a motivating factor for use. Furthermore, considering the case reports cited above describing psychosis after acute administration, we analysed reporting of schizotypy in the sample to investigate whether S. divinorum users had increased risk of psychosis (Williams et al., 1996).

Methods

individuals had submitted more than one survey. The Severity of Dependence Scale (SDS) (Gossop et al., 1995) was included to assess dependence upon S. divinorum. Although this scale has not been previously validated for S. divinorum it was believed that this would provide important preliminary information on the likelihood of use disorders. Furthermore, the SDS yields robust assessments on a range of abused drugs. The next section requested information on S. divinorum purchasing patterns, including those formulations usually purchased, sources of purchases, and reasons for use (e.g. ‘interest in drug-induced states of consciousness’; ‘curiosity’). Participants were then asked to think about their most recent (representative) S. divinorum experiences (for example, length of experience, circumstances surrounding use), and were presented with a list of 31 statements that described typical subjective effects of classical hallucinogens and related drugs. Items were generated from earlier informal interviews with hallucinogen users, personal communications with colleagues, and also adapted from literature describing the acute and immediate recreational effects of S. divinorum (Albertson and Grubbs, 2009; Dalgarno, 2007; Gonza´lez et al., 2006). Further items were adapted from the Psychedelic Experience Questionnaire (Pahnke and Richards, 1966) and the ecstasy effect experiences questionnaire (Sumnall et al., 2006). Participants were asked to indicate how often they experienced each particular effect or event listed after taking S. divinorum by selecting a number along a fivepoint Likert scale. Finally, the questionnaire included the cognitive–perceptual subscale of the schizotypy personality questionnaire (SPQ) (Raine, 1991). In normal populations, the mean score of the subscale is 11.7  7.4 (Raine, 1992).

Subjects

Statistical analyses

Participants were recruited by advertisements posted on internet sites discussing S. divinorum and other substance use, online retailers, and social networking sites (Facebook, MySpace). Cards advertising the study were also provided to internet and ‘head’/smart shops retailers in the North West of England to include with S. divinorum purchases. As prevalence is relatively low compared with other recreational drugs, convenience sampling was deemed appropriate for this research. The study was advertised as an investigation of the effects of S. divinorum and was only open to those who reported using S. divinorum at least once in their lifetime. All potential volunteers were provided with detailed information about the study and were assured that their responses would remain confidential. The ethics committee at Liverpool John Moores University gave their approval for this research study and all subjects were required to give informed consent after reading a description of the investigation.

Preliminary data screening reduced the number of scale variables included in subsequent analyses. Briefly, we identified and removed items with limited range (i.e. all points on the scale not used) and/or with high/low standard deviation. Other items were considered for removal if they yielded statistically significant skewness and kurtosis distribution scores, or if they did not significantly correlate at 1% or 5% significance levels, along with items correlating too highly with many other items to avoid multicollinearity. This resulted in the exclusion of two items (‘On salvia I found it hard to take on ordinary social roles’; ‘On salvia I thought more in images than in abstract thoughts’). The remaining variables were entered into a principal components analysis (PCA) with Scree plot criterion to determine the number of components to be entered into oblique direct oblimin rotation. Before final analysis of the extracted components, the anti-image matrix was examined to enable removal of partially correlated items. Factor-based scale scores were generated and subscales were explored as a function of use intention, patterns of drug use and demographics using a variety of statistical techniques. SPSS v18.0 was used for all analysis; significance was set at p < 0.05.

Questionnaire design Volunteers were required to complete a single online questionnaire hosted by Bristol Online Surveys (http://www. survey.bris.ac.uk/). The questionnaire asked for participant demographic information and a detailed history of use of a wide variety of substances. The time of survey submission and patterns of answers were inspected to reduce the chance that

Results In total, 209 people began the survey, and 155 completed it (74.2% completion rate). Reasons for non-completion

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are unknown. Non-completers were equally likely as completers to be male, resident in the UK or USA, and report similar ages and drug use histories. Unfortunately not enough data were submitted to compare scale scores. Data from one participant who had completed the whole survey were excluded as deliberately misleading answers were provided. Of the 154 analysed datasets, 128 (83.1%) were from males and 26 (16.9%) females. The mean age of respondents was 24.7  8.7 years, and 135 (87.7%) self reported their ethnicity as Caucasian. In total, 59 (38.3%) had completed at least an undergraduate university degree, with the majority of others reporting either completing further, or some higher education. The modal occupation was student (n ¼ 54, 35.1%), and other respondents were either employed (n ¼ 67, 43.5%) or unemployed (n ¼ 13; 8.4%). The majority of respondents lived in the United States (n ¼ 92, 59.7%), followed by the United Kingdom (n ¼ 29; 18.8%).

Drug use history Table 1 shows substance use histories. No one reported use of naloxone, which was included to help verify accuracy of reporting. After S. divinorum, the most frequently reported substances were alcohol, cannabis, tobacco, and psilocybin mushrooms. Almost three-quarters of respondents reported use of S. divinorum in the previous year (73.4%), suggesting just over one-quarter had either ceased or reduced their use

after their initial use, and subjects estimated that they used it twice a month during regular use periods. On average, time since last use in those reporting use in the previous year was 10 days (range 1–30 days). The mean SDS score for S. divinorum in previous year users was 0.4  1.4 (range 0–10); five respondents scored above 4, suggesting the presence of a use disorder.

S. divinorum use history The age of first use of S. divinorum was 21.7  7.9 years (range 13–65 years). It was most frequently obtained from ‘head’/ smart shops (n ¼ 85), followed by online retailers (n ¼ 67), friends and relatives (n ¼ 37), cuttings from a live plant (n ¼ 15), and from illegal drug dealers (n ¼ 2). It was usually taken at home (74% of respondents) or outdoors (excluding music festivals) (20.8%). Of those who bought it from ‘head’/ smart shops, 84.6% reported that it was usually on clear display (as opposed to having to ask specifically for it). Table 2 shows the formulations usually purchased. Some 40 subjects (26%) reported that they used S. divinorum as an alternative to illegal drugs. Of these, 27.5% reported they did so because they did not wish to break the law; 27.5% because they wanted to try a new experience; 22.5% because they preferred natural products; 17.5% because it produced similar effects to illegal hallucinogens such as LSD and mushrooms; and 5% because it was considered easier to obtain than illegal drugs.

Table 1. Drug use characteristics in Salvia divinorum users. All values are mean  SD

Alcohol Amphetamine sulphate Anabolic steroids BZP3 Cannabis Cocaine (powder) Cocaine (crack) GHB4 Glue/solvents Heroin MDMA (Ecstasy) Ketamine LSD5 Methamphetamine Mushrooms6 Amyl nitrate ‘Poppers’ Salvia Divinorum Spice7 TFMPP8 Tobacco Tranquilisers9 Sildenafil (Viagra)

% reporting 1 use in lifetime (n ¼ 154)

% reporting use in previous year

Self-reported uses in typical month in previous year1

Days since last use2

95.5 40.3 2.6 13.6 95.5 48.7 12.3 8.4 9.1 14.3 63.6 28.6 54.5 14.3 80.5 24.0 100.0 20.3 4.5 85.7 33.8 9.7

89.6 21.4 1.3 6.5 84.4 20.1 1.9 1.9 1.9 8.0 39.0 12.3 33.1 5.2 42.9 6.5 73.4 16.2 2.6 56.5 16.9 8.0

7.7  7.4 9.4  10.9 6.5  7.8 3.2  4.7 15.5  11.4 4.3  6.2 9.5  0.7 4.0  2.2 2.3  2.3 5.1  5.5 1.9  1.9 2.6  1.9 1.5  1.3 3.7  3.9 2.0  2.9 4.4  7.7 1.8  1.9 3.8  3.8 0.7  0.5 18.0  12.8 – –

5.2  6.5 7.1  7.9 1.0  0.0 10.3  11.4 4.9  6.5 11.3  10.3 4.0  0.0 5.5  2.1 4.5  0.7 8.2  11.2 12.0  8.8 9.4  9.2 12.3  9.5 15.0  13.0 14.2  9.3 11.5  6.4 10.0  9.3 12.1  11.8 20.0  0.0 4.0  6.9 10.3  9.1 6.0  1.0

1

In those who reported use in the previous year; 2in those who reported use in the last month; 31-benzylpiperazine; 4g-hydroxybutyrate; 5Lysergic Acid Diethylamide; typically Psilocybe Semilanceata, Psilocybe Cubensis, and Psilocybe Mexicana; 7Spice is the generic name of a smoking mixture consisting of synthetic cannabinoids added to a herbal substrate; 81-(3-(Trifluoromethyl)phenyl) piperazine; 9any form of anxiolytic or hypnotic drug. 6

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Journal of Psychopharmacology 25(11)

If S. divinorum was made illegal in their country, 72.1% reported that they would continue using it, and 79.9% would continue to use if a supply could be guaranteed. There was no difference in lifetime and last-year drug use prevalence between those who reported using S. divinorum as an alternative to illegal drugs and those who did not (data not shown). Of the sample, 60.4% thought that it was either important or extremely important to them that S. divinorum was legal, 13.6% thought it was either slightly or not at all important, and 26% neither important nor unimportant. A third (33.2%) of respondents reported taking other drugs at the same time (  1–2 h) as S. divinorum, including alcohol (13.7% of respondents); cannabis (33.2%); and other hallucinogens (6.6%). Participants were asked to report S. divinorum use functions, differentiating between their first use, and more recent occasions (if different). These are shown in Table 3. There appeared to be changes in the proportion endorsing each use function as experience with S. divinorum increased. For example, whilst 13.6% reported use for personal ‘psychotherapy’ at initiation, this had increased to 61.1% at the most recent episode; conversely, endorsement of curiosity decreased from 82.5 to 29.2%. Comparing use behaviours in young (21 years, n ¼ 51) initiates, it was found that younger initiates were just as likely to use S. divinorum indoors (odds ratio (OR) ¼ 0.44, CI ¼ 0.17–1.15, p ¼ 0.08), and to purchase from an online or ‘head’/smart shops (OR ¼ 1.59, CI ¼ 0.75–3.37, p ¼ 0.23) as older initiates. Examining use functions, younger initiates were much more likely to report using S. divinorum at both the first and most recent episode ‘For fun’ (OR ¼ 7.01, CI ¼ 3.16–15.59, p < 0.001; OR ¼ 2.89, CI ¼ 1.23–6.80, p < 0.05, respectively). Differences in the likelihood of endorsement of other use functions were non-significant (data not shown). Respondents were asked to estimate the time course of their most recent S. divinorum experience. The total experience was estimated to last for 21.8  25.2 min; initial effects after ingestion were felt after 1.3  2.9 min; subsequent onset to peak subjective effects lasted for 2.3  10.1 min, and lasted for 8.4  8.7 min; S. divinorum effects took approximately 14.3  24.5 min to subside, and after effects 52.7  429.6 min (this large SD was attributed to one respondent who reported residual effects up to 80 h after administration).

A range of adverse effects was reported after administration of S. divinorum, including; excessively intense experience (reported by 51.9%); unexpected effects (46.1%); loss of control over the experience (42.2%); heaviness of head, like smoking too many cannabis joints (27.9%); unpleasant physical effects (27.3%); unreliable effects (27.3%); tiredness (24.7%); dizziness (22.1%); grogginess (21.4%); mental slowness (20.8%); physically exhaustion (17.5%); and unpleasant after effects (16.2%). Participants were presented with a range of S. divinorumrelated behaviours and first asked to rate acceptability and then to indicate whether they had ever undertaken it (Table 4). Subjects showed disapproval of a range of public and social use behaviours, especially those involving deception and social responsibilities.

Principal component analysis The Kaiser–Meyer–Olkin measure of sampling adequacy was 0.797, indicating the solution was robust (Hutcheson and Sofroniouo, 1999). The Bartlett’s test of sphericity was significant (p < 0.001), indicating the original correlation matrix was not an identity matrix. Items were removed from the solution if loadings were less than 0.40 on primary

Table 3. Endorsed Salvia use functions on first and recent occasions. Shown are percentages, totals >100% as participants could report more than one function % reporting Function

First use

Most recent use

As part of a personal ‘psychotherapy’ Curiosity For (self-defined) spiritual purposes For fun For social purposes Interest in drug-induced states of consciousness To enhance creativity To enjoy music To feel close to nature

13.6 82.5 49.4 48.7 11.7 81.2

61.1 29.2 87.0 56.5 7.1 61.7

11.0 5.8 11.7

19.5 17.8 23.4

Table 2. Preparations of Salvia usually purchased by the sample. Extracts (5–60) refer to ‘strength’ of preparations sold by retailers, although no units of measurement are provided. For example 1 usually refers to the natural potency of the plant (2.5 mg/g), whilst 10 would be ten times the potency of 1. These ‘doses’ are often subjective and are also partly determined by the age and water weight of the plant (Wolowich et al., 2006; Vohra et al., 2011) Extract

5

10

% (n ¼ 286 mentions) Dried leaf (n ¼ 66) Tincture (n ¼ 19) Other forms

18.2 8.0 28 g 56 g 54.5 21.2 2 mL 10 mL 42.1 47.3 Whole plants; cuttings; extraction using

20

40

60

Other

31.1 10.5 8.0 24.2 100 g 200 g Other 3.0 3.0 18.3 Other 10.6 whole leaf and acetone; fresh leaf; extracted Salvinorin A; pre-rolled joints

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Table 4. Perceived acceptability of different types of Salvia use behaviours Modal response (% reporting)

Behaviour Using Salvia in public places Posting videos of Salvia on YouTube Giving someone else an unexpected dose (e.g. telling them it was cannabis) Giving someone else an unexpectedly high dose Having a hidden negative motive for giving it to someone else (e.g. to make them panic) Having a neutral motive for giving it to someone else (e.g. so that they can experience the psychedelic effects) Driving shortly after use Combining Salvia with responsibilities (e.g. childcare, before going to work/college) Selling it on at a profit

Strongly disagree Strongly disagree Strongly disagree Strongly disagree Strongly disagree Agree (37.0%)

% reporting this activity (55.2%) (54.5%) (91.6%) (83.8%) (94.8%)

14.9 1.9 1.3 3.9 0.6 32.5

Strongly disagree (73.4%) Strongly disagree (72.7%) Neither agree nor disagree (36.4%)

3.2 3.2 5.8

Table 5. Salvia divinorum experiences questionnaire components. Survey respondents were requested to refer to their most recent use Item 1. Positive effects (Eigenvalue ¼ 6.89; 22.24% of variance;  ¼ 0.87) On Salvia I felt that there were no boundaries between inner and outer reality I had a noetic sense on Salvia; that is I instinctively understood the universe When on Salvia I felt a personal identification with whatever I was looking at; a sense of unity Salvia produced a sense of reverence in me Salvia made me feel beyond or outside of time I felt more connected to other people when I was on Salvia I have the sense that in order to describe parts of the Salvia experience I would have to use statements that appear to be illogical, involving contradictions and paradoxes On Salvia, wherever I looked was especially beautiful I experienced variations in the passing of time If I tried to smell something I could do so more vividly than when off Salvia I felt that my consciousness/mind was located outside my physical body Salvia made the temperature of things take on new qualities Auditory images (mental images that I created in response to things that I hear) were more vivid when I was on Salvia I felt changes in the perception of my size, weight, and posture when I was on Salvia My memory for otherwise forgotten things was strong on Salvia I felt that though I was still myself, at the same time I was also someone or something else 2. Negative intoxication effects (Eigenvalue ¼ 5.45; 17.59% of variance;  ¼ 0.70) I had a transcendental experience on Salvia. I felt detached from all problems, anxieties and human interactions When I had taken Salvia I felt an increased ease and enjoyment of talking to and understanding people When I was on Salvia I had strong feelings of caring or compassion for people who I was with On Salvia I could deliberately generate insights concerning myself, my personality, and my relationships with other people When I was on Salvia I found that I had problems remembering things I got anxious when I was on Salvia Salvia lowered my inhibitions so that I said and did things I’m normally too inhibited to do I had difficulty focusing upon one thing at a time when I was on Salvia After the Salvia high was over I became depressed or ‘burned out’ Salvia gave me headaches I experienced thoughts that I believed were not my own *Note negative loading.

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Item loading

Mean score (  SD)

0.73 0.67 0.64 0.64 0.63 0.58 0.58

31.7 2.39 1.96 1.73 2.14 3.14 1.23 2.90

(11.2) (1.53) (1.49) (1.43) (1.39) (1.15) (1.29) (1.25)

0.56 0.54 0.53 0.5 0.49 0.48

1.73 2.84 0.79 2.32 1.62 2.39

(1.34) (1.24) (1.09) (1.44) (1.43) (1.36)

0.46 0.44 0.40

2.72 (1.36) 1.39 (1.36) 1.47(1.38)

.60*

20.4 (7.0) 2.31 (1.39)

.59*

1.05 (1.19)

.50* .43*

1.34 (1.34) 1.61 (1.44)

0.65 0.59 0.56 0.51 0.47 0.42 0.40

1.91 1.47 0.87 1.77 0.57 0.50 1.77

(1.54) (1.38) (1.08) (1.43) (1.01) (0.94) (1.43)

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Journal of Psychopharmacology 25(11)

Less Equal More 100

% Endorsing

80

60

40

Viagra

Tobacco

Tranquilisers

Spice

TFMPP

Mushrooms

LSD

Methamphetamine

Ketamine

MDMA (Ecstasy)

Glue

Heroin

GHB

Cocaine (crack)

Cocaine (powder)

BZP

Cannabis

Anabolic Steroids

Alcohol

Amphetamine

0

Amyl nitrate ‘Poppers’

20

D

Figure 1. Perceived harmfulness of Salvia divinorum compared with other drugs. BZP, 1-benzylpiperazine; GHB, g-hydroxybutyrate; LSD, Lysergic Acid Diethylamide; Mushrooms are typically Psilocybe Semilanceata, Psilocybe Cubensis and Psilocybe Mexicana; Spice is the generic name of a smoking mixture consisting of synthetic cannabinoids added to a herbal substrate; TFMPP, 1-(3-(Trifluoromethyl)phenyl) piperazine; tranquilisers are any form of anxiolytic or hypnotic drug.

components or greater than 0.40 on a secondary component. Rotation retained two components (29 items) accounting for 40% of the total item variance. This was confirmed by visual inspection of the Scree plot. Positive effects consisted of 17 items, and Negative intoxication effects consisted of 12 items (Table 5). Backwards stepwise regression was used to identify predictors of positive and negative S. divinorum effect component scores. Predictors of positive effect scores (r2 ¼ 0.245, p < 0.001) were using S. divinorum outdoors (exp(b) ¼ 0.234, p < 0.05); younger age of initiation (exp(b) ¼ 0.315, p < 0.05); shorter time of S. divinorum effect onset (exp(b) ¼ 0.252, p < 0.05), and a greater length of time for acute effects to subside (exp(b) ¼ 0.355, p < 0.01). Predictors of negative effect scores (r2 ¼ 0.232, p < 0.001) were using S. divinorum indoors (exp(b) ¼ 0.269, p < 0.05); being of a younger age (exp(b) ¼ 0.332, p < 0.05), and reporting higher SPQ scores (exp(b) ¼ 0.403, p < 0.001). Regarding the perceived harmfulness of S. divinorum, respondents were asked to compare the relative harmfulness of their own type and pattern of S. divinorum use with that of

Table 6. Scores on the cognitive–perceptual components of the schizotypy personality (SPQ) questionnaire Odd beliefs or magical thinking Unusual perceptual experiences Ideas of reference Suspiciousness Total SPQ (cognitive–perceptual subscale)

2.1  2.4 2.8  2.3 3.2  2.6 1.9  2.2 9.9  7.5

other drugs (Figure 1). S. divinorum was perceived to be less harmful than all drugs apart from cannabis (Class B under the UK Misuse of Drugs Act 1971 since 2009) and mushrooms (all forms of psilocin are Class A under the Misuse of UK Drugs Act 1971 since 2005), which were viewed as equally harmful. Finally, data from the SPQ questionnaire are shown in Table 6. As can be seen, the mean sample score was consistent with general population mean of 11.7, indicating that this population was not experiencing schizotypal symptoms. However, there were small but significant correlations

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Sumnall et al.

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between S. divinorum effect component scores and total SPQ score (Positive effects r2 ¼ 0.282, p < 0.001; negative effects r2 ¼ 0.187, p < 0.001). There were no significant correlations between S. divinorum component scores and SPQ subscales, and there were no significant differences in subscale score between younger and older initiates (data not shown).

Discussion This study investigated the use behaviours and subjective experiences of S. divinorum in healthy adult subjects. Participants reported a range of drug use histories and experiences with S. divinorum. Use of other serotonergic hallucinogens (LSD, psilocybin mushrooms) was high compared with general population estimates (Hoare, 2009), but similar to other studies of S. divinorum users (Albertson and Grubbs, 2009; Gonza´lez et al., 2006). Therefore it would be useful to determine whether this population represents a distinct druguse typology using techniques such as latent class/profile analysis. Subjects also appeared to have a sense of social ‘responsibility’ regarding their use, suggesting the establishment of informal user group injunctive norms. These findings are important, as drug prevention and harm reduction advice is often delivered through social marketing techniques that rely on an understanding of the experiences and motivations of the target audience (Bennett and Henderson, 1999). PCA of responses to the survey of S. divinorum effects revealed two main components. Positive effects comprised perceptual and cognitive effects, whilst Negative effects included items related to social withdrawal, mental confusion, amnesia, and anxiety. Future research will allow for refining and improving component scale reliability. As expected (Schmidt et al., 2005), the S. divinorum experience was typically short (98% pure by HPLC. 2.7. Statistical analysis Values are expressed as mean ± S.E.M. As noted in the figure legends and in the result section, one-way analysis of variance was used followed by post hoc testing (Bonferroni, Dunnett and Tukey) for multiple comparisons. Two-tailed unpaired Student's t test analysis was performed for comparisons between two groups using

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Prism (GraphPad, San Diego, CA). A value of p  0.05 was considered statistically significant.

3. Results 3.1. KOR activation upregulates DAT 3.1.1. SalA increases DAT function via KOR activation Addition of SalA to EM4 cells coexpressing KOR and DAT produced a concentration dependent increase in ASPþ accumulation rate (F (3, 211) ¼ 11.50; p  0.0001) (Fig. 1A). Increased accumulation occurred within 2 min after ASPþ addition and persisted for at least

Fig. 1. SalA and other KOR agonists alter biogenic amine transporters function differentially via a BNIereversible mechanism. EM4 cells were transiently cotransfected with mycKOR plus YFP-DAT or myc-KOR plus hSERT or myc-KOR plus hNET. After 48 h, DAT, SERT and NET functions were measured as accumulation of ASPþ over time as described under Materials and Methods. A. Influence of SalA concentrations (1e10 mM) on ASPþ accumulation rate (AFU). *p < 0.05; **p < 0.001, (SalA 3 mM: n ¼ 47 and 10 mM: n ¼ 37 respectively) compared with vehicle control (One-Way ANOVA with Dunnett's multiple comparison test. B. Effect of DAT blocker GBR12909 on DAT mediated ASPþ accumulation. GBR12909 (10 mM) blocked DAT mediated ASPþ accumulation both in the presence and absence of SalA. ****p < 0.0001 (SalA: n ¼ 73) compared to corresponding Veh/Veh control (N ¼ 31). ^^p < 0.01 (GBR12909/Veh: n ¼ 160; GBR12909/SalA: n ¼ 118 and nontransfected cells: N ¼ 144) compared with Veh/Veh or Veh/SalA. The GBR12909 insensitive background signals were similar to those observed in nontransfected cells. C. Effect of D2 receptor antagonist L-741,626 on SalA induced increase in ASPþ accumulation. L-741,626 (10 mM) did not alter SalA induced increase in ASPþ accumulation in EM4 cells coexpressing DAT and KOR. ****p < 0.0001 compared with L-741,626/Veh (N ¼ 181). SalA failed to increase DAT-mediated ASPþ accumulation in EM4 cells expressing DAT only. ^^p < 0.01 (N ¼ 157) compared with Veh/SalA (N ¼ 149) or L-741,626/SalA (N ¼ 181) in cells coexpressing both DAT and KOR. ns, non significant, p ¼ 0.84, (Veh/SalA versus L-741,626/SalA). D. Effect of KOR agonists SalA (10 mM), U69,593 (10 mM) and U50,488 (10 mM) on DAT function. BNI (1 mM) prevents KOR evoked DAT upregulation. ***p < 0.001 (SalA: n ¼ 64; U69,593: n ¼ 35 and U50,488: n ¼ 55) versus corresponding Veh/Veh control; $$$p < 0.001 (n ¼ 60) versus Veh/SalA; ### p < 0.001 (n ¼ 42) versus Veh/U69,593 and ^^p < 0.001 (n ¼ 44) versus Veh/U50,488. E. Effect of SalA (10 mM, 10 min) on SERT and NET function. A between cell design was utilized to determine the effect of SalA. Background uptake values were corrected and normalized to SERT or NET expression levels. ***p < 0.001 (n ¼ 47); significantly different from Veh/Veh control; ^^p < 0.01 (n ¼ 16) significant different compared with Veh/SalA. Data are the mean ± S.E.M (One-Way ANOVA with Bonferroni multiple comparison test).

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10 min. At 10 mM SalA, the magnitude of stimulation of ASPþ accumulation rate varied between experiments with a minimum of ~20% to a maximum of ~60%. ASPþ uptake as well as SalA-mediated increase in the ASPþ accumulation was also blocked by DAT inhibitor GBR12909 (F (4,521) ¼ 213; p < 0.0001) (Fig.1B). GBR12909 insensitive ASPþ accumulations found in cells coexpressing DAT and KOR was similar to ASPþ accumulation in non-transfected cells (Fig. 1B). SalA was found to be a highly potent and selective KOR agonist. In order to validate whether SalA mediated stimulation of DAT activity was attributable to KOR, the effect of SalA was assessed in the presence of a selective KOR antagonist, nor-binaltorphimine (BNI). EM4 cells were preincubated for 10 min with BNI (1 mM) prior to the addition of vehicle or SalA (10 mM) followed by measuring ASPþ accumulation and was compared with vehicle treatment (Fig. 1D). While SalA increased DAT activity in the absence of BNI, it did not alter DAT activity in the presence of BNI (F (3,212) ¼ 129; p  0.0001). On the other hand, SalA increased ASPþ uptake both in the presence and absence of D2 receptor antagonist, L-741,626 (Fig. 1C). Furthermore, SalA treatment of EM4 cells expressing only the DAT did not produce any effect on DAT activity (Fig. 1C). These results indicate that SalA induced stimulation of DAT is KOR mediated.

requires Gi/Go and ERK1/2 activation. Pretreatment of EM4 cells coexpressing DAT and KOR with PTX (100 ng/ml for 16e24 h) prevented SalA-mediated increase in ASPþ uptake (Fig. 2A, F (3,268) ¼ 74.56, p ¼ 0.0001). Next we examined whether SalA

3.1.2. Other KOR agonists increase DAT function via KOR activation Next we sought to examine whether SalA mediated DAT upregulation is unique to SalA or general consequence of KOR activation. We tested the effect of other known KOR agonists including U69,593 and U50,488 on DAT-mediated ASPþ accumulation in EM4 cells coexpressing KOR and DAT. Similar to SalA, both U69,593 and U50,488 at 10 mM increased ASPþ accumulation (F (3, 3175) ¼ 29.12; p  0.0001) (Fig. 1B). However, preincubation of BNI (1 mM) for 10 min prior to the addition of vehicle or U69,593 or U50,488 completely blocked the stimulatory effect on DAT activity (F (5,270) ¼ 18.73; p  0.0001) (Fig. 1B), suggesting the involvement of KOR. Kinetic analysis of ASPþ uptake in EM4 cells coexpressing the DAT and KOR proteins showed that SalA (10 mM) treatment significantly increased the values of Vmax (vehicle: 1.09 ± 0.07 AFUs; SalA: 1.49 ± 0.89 AFUs; t ¼ 3.24, df ¼ 741; p < 0.001) and the values of Km (vehicle: 2.74 ± 0.55 mM; SalA: 5.31 ± 0.85 mM; t ¼ 2.15, df ¼ 741; p < 0.03). Thus, SalA increased the maximal velocity while decreasing the affinity. 3.1.3. Differential influence of SalA on serotonin and norepinephrine transporters We next asked whether SalA regulates other amine transporters. Treatment with SalA (10 mM, 10 min) significantly reduced SERTmediated ASPþ accumulation in EM4 cells coexpressing KOR and human SERT (t ¼ 9.85, df ¼ 44, p < 0.0001) (Fig 1C). BNI pretreatment blocked the decrease in SERT activity induced by SalA (t ¼ 8.31, df ¼ 26, p < 0.0001) (Fig 1C). Kinetic analysis of ASPþ uptake by SERT revealed a significant decrease in Vmax value (vehicle: 1.93 ± 0.47 AFUs; SalA: 0.86 ± 0.21 AFUs; t ¼ 2.08, df ¼ 510, p < 0.04) with no significant change in Km value following SalA treatment (vehicle: 9.46 ± 4.01 mM; SalA: 2.88 ± 2.89 mM; t ¼ 1.34, df ¼ 519; p ¼ 0.18). On the other hand, SalA (10 mM) and or BNI did not alter ASPþ accumulation in EM4 cells co-expressing KOR and NET (p > 0.1) (Fig 1C). These results collectively indicate that SalA exhibits differential effect on DAT and SERT while it has no influence on NET. 3.1.4. SalA-KOR mediated DAT upregulation is dependent on Gi/Go and ERK1/2 activation KOR is predominantly coupled to pertussis toxin (PTX) sensitive Gi/Go types of G proteins and triggers diverse signalling systems including ERK1/2 (Bruchas and Chavkin, 2010). Therefore, we examined whether SalA-KOR-mediated stimulation of DAT activity

Fig. 2. SalA upregulates DAT function in PTX sensitive and ERK1/2-dependent mechanism. EM4 cells co-expressing myc-KOR and YFP-DAT were pre-incubated with PTX (100 ng/ml-16-24 h) or PD98059 (10 mM-15 min) or vehicle prior to SalA (0; 10 mM) addition. ASPþ uptake or t-ERK1/2 and p-ERK1/2 or phospho-p38 MAPK and t-p38 MAPK levels were determined as described under Materials and Methods. A. PTX pretreatment prevents SalA e evoked DAT upregulation. *p < 0.01 (n ¼ 87); significantly different from Veh/Veh control; ^p < 0.01 (n ¼ 64) significant difference compared with Veh/SalA (One-Way ANOVA with Bonferroni multiple comparison test). B. Representative immunoblot showing phospho-ERK1/2, total ERK1/2, phospho-p38 MAPK and total p38 MAPK levels in vehicle or SalA or U69,593 or U50,488 treated cells. C. Pre-treatment of ERK1/2 inhibitor PD98059 but not p38 MAPK inhibitor SB203580 prevents SalA e induced DAT activity. ***p < 0.001 (n ¼ 89); significantly different from Veh/Veh control; ^^^p < 0.001 (n ¼ 71) significant difference compared with Veh/SalA. Data are the mean ± S.E.M (One-Way ANOVA with Bonferroni multiple comparison test).

B. Kivell et al. / Neuropharmacology 86 (2014) 228e240

treatment activates ERK1/2 and p38 MAPK in EM4 cells coexpressing DAT and KOR by measuring ERK1/2 and p38 MAPK phosphorylation. Five min incubation of EM4 cells with SalA (10 mM) produced a significant (t-test; p  0.0001; n ¼ 3) ~2.5-fold increase in pERK1/2 levels with out any changes in total ERK1/2 confirming ERK1/2 activation by SalA (Fig. 2B). Similar to SalA, 5 min exposure of U69,593 (10 mM, ~3 fold, t-test; p  0.005; n ¼ 3) or U50,488 (10 mM, ~3.5 fold t-test; p  0.008; n ¼ 3) significantly increased pERK1/2 levels (Fig. 2B) with out altering total ERK1/2 expression. However, parallel analysis of the level of phospho-p38 MAPK and total p38 MAPK showed no significant changes (F (5,30) ¼ 1.67, p ¼ 0.17) suggesting that KOR activation through SalA or U69,593 or U50,488 activates ERK1/2, but not p38 MAPK under the experimental conditions used. Incubation of cells with the ERK1/2 kinase inhibitor, PD98059 (10 mM), did not alter ASPþ uptake but prevented SalA-evoked increases in ASPþ accumulation (Fig. 2C; F (3,278) ¼ 42.23, p ¼ 0.001). In contrast, pretreatment of cells with the p38 MAPK inhibitor, SB203580 (3 mM; 5 min), was ineffective in attenuating SalA-evoked increases in ASPþ accumulation rate (% increase in slope: Veh/SalA ¼ 28.29 ± 2.9%; SB203580/ SalA ¼ 36.42 ± 6.6%; t ¼ 1.4; df ¼ 156, p > 0.15, n ¼ 39). Parallel analysis showed that, PD98059 specifically reduced the SalA induced pERK1/2 level with no effect on phospho-p38 MAPK or total ERK1/2 and or total p38 MAPK. These results are consistent with our observations that SalA triggers ERK1/2 activation and subsequently upregulates DAT but not p38 MAPK. 3.2. SalA upregulates DA clearance in rat striatum via ERK1/2 The above results (Figs. 1 and 2) revealed that SalA triggers DAT activity through KOR-linked ERK1/2 activation in heterologous coexpressing cell model. Next we sought to examine whether SalA upregulates DAT function in native tissue. Given the fact that KOR is expressed in striatal dopamine axons and varicosities (Svingos et al., 2001), RDE voltammetry and radiolabelled DA uptake was performed in mince preparations of striatal tissue or synaptosomes respectively, to determine the effect of KOR activation on DAT function. Analogous to our results using heterologous expression

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systems, SalA as well as U50,488 significantly increased DAT mediated DA-transport in a GBR12909-sensitive manner (Fig. 3A; F (2,30) ¼ 7.03, p ¼ 0.003) and was blocked by the presence of BNI (F (4,37) ¼ 7.06, p ¼ 0.0003) suggesting specific KOR-mediated effect on DAT. Furthermore, the ERK1/2 inhibitor PD98059 (10 mM) pretreatment (30 min) attenuated the SalA evoked DAT function Fig. 3B. PD98059 at 10 mM concentration did not affect dopamine clearance significantly (F (1,16) ¼ 1.3; p ¼ 0.3), but it attenuated the SalA-evoked increase in DA clearance (F (1,16) ¼ 5.1; p ¼ 0.04). 3.3. SalA increases DAT Vmax The kinetic parameters of the DAT (MichaeliseMenten constant, Km, and maximal velocity, Vmax) were determined in vehicle and SalA (10 mM; 5 min) treated striatal synaptosomes (Fig. 4). DATspecific DA transport was determined in the presence of the NET blocker nisoxetine (see details under methods and materials). SalA treatment increased the maximal velocity ( Vmax) significantly from 27.8 ± 4.4 to 45.10 ± 1.0 pmol/mg protein per minute (t ¼ 3.84, df ¼ 4; p < 0.009) with no significant changes in the Km (vehicle: 29.77 ± 1.24 nM; SalA: 35.11 ± 2.23 nM; t ¼ 2.09, df ¼ 4; p ¼ 0.105). 3.4. SalA upregulates DAT cell surface expression To determine whether up-regulation of DAT function is associated with increased DAT cell surface expression, surface biotinylation studies were conducted in EM4 cells coexpressing KOR and DAT (Fig. 5A). Consistent with the uptake data, SalA exposure of cells for 10 min significantly increased biotinylated DAT (% of vehicle, 214.3 ± 18.31%, p ¼ 0.0001). Treatment with SalA did not alter the total amount of DAT protein (Fig. 5A). Next, a rat striatal synaptosome preparation was used for biotinylation studies to examine whether SalA increases surface DAT in endogenously expressing preparations (Fig. 5B). Similar to the cell model, and consistent with the change in Vmax of DA clearance, SalA exposure of striatal synaptosomes resulted in a significant increase in surface DAT (% of vehicle, 183.7 ± 13.2%, p ¼ 0.0001) with out altering the total level of the DAT protein (Fig. 5B). Furthermore, pretreatment

Fig. 3. KOR activation by SalA and U50,488 increases DA clearance in striatum and SalA-mediated DAT upregulation is ERK1/2 dependent. Minced slice preparations from rat striatum were exposed to appropriate vehicle(s) or SalA or U50,488 and RDE voltammetry was performed to measure DA clearance as described under Materials and Methods. GBR12909 (100 nM) was used to determine the specific DAT mediated DA clearance in RDE experiments. DA uptake in the presence of GBR12909 was subtracted from the total clearance to yield specific DAT-mediated DA uptake. A. KOR antagonist BNI blocked SalA (10 mM) or U50,488 (10 mM) induced DAT-mediated DA clearance. Slices were pre-incubated with BNI (1 mM-5 min) prior to SalA or U50,488 (0; 10 mM) addition. Values (pmoles/s/g tissue) represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01 compared with Veh/Veh control. #p < 0.05 compared with Veh/SalA. ^p < 0.05 compared with Veh/U50,488. (One-Way ANOVA with Bonferroni multiple comparison test). B. ERK1/2 inhibitor PD98059 prevents SalA induced changes in dopamine clearance. Minced slice preparations from rat striatum were exposed to vehicle or PD98059 (10 mM) for 10 min prior to the addition of SalA (0; 10 mM). RDE voltammetry was performed to measure DA clearance as described under Materials and Methods. Values were expressed as percentage of uptake relative to the uptake observed in Veh/Veh treated slices and represent the mean ± SEM of three independent experiments. *p < 0.01 significant difference between Veh/Veh versus Veh/SalA; ^p < 0.04 significant difference between Veh/SalA and PD98059/SalA (One-Way ANOVA with Bonferroni multiple comparison test).

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Fig. 4. SalA increases DAT Vmax. DA uptake kinetic characteristics mediated by DAT in vehicle or SalA treated striatal synaptosomes. Synaptosomes (50 mg) were preincubated with the vehicle or SalA (10 mM) for 5 min at 37  C. After this treatment, uptake of DA was measured (5 min) over a range of 0.01e2 mM mixed with 50 nM nisoxetine (to block NET mediated DA uptake) and radiolabelled [3H]DA as described under Materials and Methods. In parallel, nonspecific uptake at each concentration of DA used (in the presence of 100 mM cocaine) was subtracted from total uptake. Values are the averages from three independent experiments, and the mean values ± SEM are given. Nonlinear curve fits of data for uptake used the generalized MichaeliseMenten equation (Prism).

of cells or striatal synaptosomes with the ERK1/2 inhibitor PD98059 (10 mM, 30 min) blocked the SalA induced increase in surface DAT and synaptosomes (in % of vehicle; cells: Veh/SalA: 214.3 ± 18.31%, PD98050/SalA: 122.6 ± 3.0%, p ¼ 0.0007; synaptosomes: Veh/SalA: 183.7 ± 13.2%, PD98050/SalA: 97.5 ± 14.4%, p ¼ 0.02). PD98059 at 10 mM concentration did not affect total and surface levels of DAT (Fig. 5A and B). We validated our surface biotinylation experiments for cellular integrity by determining the presence of intracellular calnexin in biotinylated fractions. Both in cells and synaptosomal experiments, less than 0.5% of total calnexin was present in biotinylated fractions suggesting that surface membranes were intact and intracellular proteins were not significantly biotinylated and contaminated/recovered with surface biotinylated proteins (data not shown). 3.5. DAT and KOR interaction 3.5.1. DAT and KOR exist in a physical complex Our previous studies demonstrated that DAT and presynaptic D2s-Receptor form a stable complex (Bolan et al., 2007). Furthermore, Lee et al. established a direct interaction of the N-terminus of DAT with the third intracellular loop of the D2 receptor (Lee et al., 2007). Given the fact that both DAT and KOR are coexpressed in presynaptic DA-terminals (Svingos et al., 2001), and KOR activation upregulates DAT surface levels, we postulated that DAT and KOR might co-exist in a complex. To determine whether KOR and DAT formed interacting complexes we used three approaches to investigate the DATeKOR association: co-immunoprecipitation, BRET and FRET in live cells to quantify the dynamics of DATeKOR association. First, co-immunoprecipitation experiments were carried out in cells coexpressing myc-KOR and FLAG-DAT. Immunoprecipitation of myc-KOR with anti-myc antibody followed by immunoblotting with anti-DAT antibody revealed a ~85 kDa band corresponding to mature, monomeric DAT in cells co-expressing both DAT and KOR proteins (Fig. 6A lane 2). No specific DAT band was observed when Protein A beads were used with nontransfected cellular extracts which indicates the specificity of the myc-antibody to the immunoprecipitated myc-KOR protein (Fig. 6A lane 3). Furthermore, no specific DAT band was observed in the

Fig. 5. SalA increases DAT cell surface expression and is ERK1/2 dependent. EM4 cells coexpressing myc-KOR and YFP-DAT or rat striatal synaptosomes were pre-treated with vehicle or PD98059 (10 mM) for 15 min prior to SalA (0; 10 mM, 5 min) followed by biotinylation. Isolation of biotinylated proteins, detection and quantification of DAT were performed as described under Materials and Methods. Western blots of DAT from total lysates and avidin bead eluates from striatal synaptosomes (A) and EM4 cells coexpressing KOR and DAT (B) are shown at the top. Quantified surface DAT band densities are shown at the bottom. ***p < 0.001 (N ¼ 9) significant changes in biotinylated DAT band densities compared with Veh/Veh control (n ¼ 8) or Veh/PD98059 (N ¼ 4) or PD98059/SalA (N ¼ 9) in EM4 cells and striatal synaptosomes (Veh/Veh control (n ¼ 11) or Veh/PD98059 (N ¼ 3) or PD98059/SalA (N ¼ 3). ^^p < 0.005 (Veh/Sal versus PD98509/SalA). Data are the mean ± S.E.M (One-Way ANOVA with Bonferroni multiple comparison test).

anti-myc-KOR immunocomplex isolated from the mixture of lysates that were prepared from cells expressing myc-KOR alone or FLAG-DAT alone (Fig. 6A lane 1). These results suggest that the formation of DATeKOR complexes requires the expression of DAT and KOR in the same cell and rules out a simply an artefact due to solubilization and immunoprecipitation processes. Due to the presence of IgG in the eluate from the immunocomplex and as a result of cross immunoreactivity with secondary antibody, IgG bands were detected. In addition, to determine whether DAT/KOR complexes also exist in native tissues, we blotted DAT immunoprecipitates from rat striatal synaptosomes for KOR (Fig 6B). Immunoblotting of DAT immunoprecipitates (obtained using two different DAT antibodies) with a polyclonal KOR antibody revealed the presence of a ~45 kDa band consistent with the expected size for KOR. In parallel experiments, the ~45 kDa band is not immunoprecipitated from striatal extract when an irrelevant IgG (with Protein A sepharose) is utilized for immunoprecipitations (Fig 6B). Second, we used BRET (Angers et al., 2000) to determine whether DAT and KOR proteins are in close proximity (30 min) relative to that of

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voltammetry (sec) may underlie the observed lack of effect in dorsal striatum. 4.4. KOR activation enhances DAT cell surface expression Changes in Vmax are often due to alterations in protein trafficking. Constitutive DAT trafficking between the membrane and intracellular compartments has been reported (Loder and Melikian, 2003). Changes in trafficking enable rapid regulation of dopamine transmission. Surface biotinylation studies revealed the enhanced surface DAT availability following SalA exposure in both cells and native tissue suggesting that SalA increase DAT function, in part, by promoting redistribution of DAT to the cell surface. Consistent with the blockade effect of ERK1/2 inhibition on SalA induced upregulation of DAT activity, inhibition of ERK1/2 prevented SalA triggered increase in surface DAT. However, level of SalA induced DAT surface enhancement is higher than uptake suggesting the involvement of multiple stages of transporter trafficking and catalytic activation and or due to different assays employed (uptake versus biotinylation-immunoblot). It is noteworthy that regulation of PKG and p38 MAPK-dependent SERT regulation has been associated with enhancement of surface SERT followed by catalytic activation (Zhu et al., 2004). We have demonstrated that activation of D3dopamine receptor regulates DAT trafficking at the level of DAT endocytosis e exocytosis e recycling in a biphasic manner to regulate surface DAT expression and function (Zapata et al., 2007). Further research is needed to fully identify additional cellular mechanisms involved in the regulation of SalA-KOR mediated DAT upregulation. 4.5. DAT and KOR exists in a physical complex Co-immunoprecipitation studies from transfected cells coexpressing KOR and DAT and striatal tissue suggest that KOR and DAT exist in a physical complex. Furthermore, BRET experiments revealed energy transfer between Luc-KOR and YFP-DAT suggesting that they are in close proximity. Importantly, energy transfer can arise from random interactions within the membrane when high expression levels and single acceptor/donor ratios are employed (James et al., 2006). Furthermore, FRET studies confirmed the presence of a physical complex of DAT with KOR in living cells. Taken together, these results demonstrate that KOR and DAT are in sufficient proximity to interact, although an indirect interaction cannot be excluded. Interestingly, SalA treatment enhanced DATeKOR assembly suggesting that activation of KOR promotes and/or regulate DATeKOR assembling. Recently it has been demonstrated that A3 adenosine receptor activation augments SERT-A3 adenosine receptor complex formation (Zhu et al., 2011). Activation of NK1R triggers NET redistribution through raftmediated translocation of NET-NK1R complexes and facilitates recruitment of signalling molecules (Arapulisamy et al., 2013). It is also possible that DAT activation/inhibition by DAT substrates and inhibitors respectively may regulate KOR-mediated DAT regulation and physical association of DAT and KOR. 4.6. Concluding remarks Phosphorylation of monoamine transporters is one cellular mechanism by which protein kinases regulate amine uptake. DAT contains putative consensus sites for several protein kinases including potential phosphorylation sites for ERK (Ser-13, Thr-53; Thr-595) (Gorentla et al., 2009). Mutation of Thr-53 eliminates ERK triggered DAT phosphorylation providing direct evidence that Thr-53 is a phosphorylation site for ERK (Gorentla et al., 2009) and also has functional significance (Foster et al., 2012). These findings

raise the possibility that ERK activation by SalA may promote DAT phosphorylation and this action may contribute to SalA-evoked DAT upregulation. Studies addressing this issue are currently in progress. Taken together, the results of the present study, demonstrating that SalA increases DAT function suggests that it may affect behaviour and dopamine neurotransmission by two distinct mechanisms, inhibition of release (Ebner et al., 2010) and ERK dependent upregulation of dopamine transport. Furthermore, the demonstration that KOR and DAT are in close proximity in both cells and native tissue (Svingos et al., 2001) provides a cellular basis by which SalA and synthetic KOR agonists (Thompson et al., 2000) regulate basal DAT function. Authorship contributions Participated in research design: Kivell, B., Chefer, V., Devi, L.A., Jayanthi, L.D., Sitte, H.H., Ramamoorthy, S, Shippenberg, T.S. Conducted experiments: Kivell, B., Chefer, V., Jaligam, V., Bolan, E., Simonson, B., Sundaramurthy, S., Rajamanickam, J., Ewald, A., Annamalai, B., Mannangatti, P., Gomes, I. Uzelac, Z. Contributed new reagents or analytic tools: Prisinzano, T. Performed data analysis: Kivell, B., Chefer, V., Gomes, I, Devi, L.A., Sitte, H.H., Ramamoorthy, S, Shippenberg, T.S. Wrote or contributed to the writing of the manuscript: Kivell, B., Chefer, V., Sitte, H.H., Ramamoorthy, S, Shippenberg, T.S. Acknowledgements This work was supported by the National Institutes of Health and National Institute on Drug Abuse Intramural Research Program (T.S.S), NIH grants, MH083928, MH091633 (S.R), DA018151 (T.E.P), DA019521, DA08863, GM071558 (L.A.D.), The Health Research Council of New Zealand (B.K) and Austrian Science Fund/FWF, P23658 (H.H.S). CCRP-YFP was kindly provided by Dr Michel Bouvier (University of Montreal). DAT constructs were provided by Dr. Jonathan Javitch. References Adkins, E.M., Samuvel, D.J., Fog, J.U., Eriksen, J., Jayanthi, L.D., Vaegter, C.B., Ramamoorthy, S., Gether, U., 2007. Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Biochemistry 46, 10484e10497. Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T., Saltiel, A.R., 1995. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270, 27489e27494. Amara, S., Kuhar, M., 1993. Neurotransmitter transporters e recent progress. Annu. Rev. Neurosci. 16, 73e93. Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., Bouvier, M., 2000. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. U. S. A. 97, 3684e3689. Arapulisamy, O., Mannangatti, P., Jayanthi, L.D., 2013. Regulated norepinephrine transporter interaction with the neurokinin-1 receptor establishes transporter subcellular localization. J. Biol. Chem. 288, 28599e28610. Bartholomaus, I., Milan-Lobo, L., Nicke, A., Dutertre, S., Hastrup, H., Jha, A., Gether, U., Sitte, H.H., Betz, H., Eulenburg, V., 2008. Glycine transporter dimers: evidence for occurrence in the plasma membrane. J. Biol. Chem. 283, 10978e10991. Beerepoot, P., Lam, V., Luu, A., Tsoi, B., Siebert, D., Szechtman, H., 2008. Effects of salvinorin A on locomotor sensitization to D2/D3 dopamine agonist quinpirole. Neurosci. Lett. 446, 101e104. Belcheva, M.M., Clark, A.L., Haas, P.D., Serna, J.S., Hahn, J.W., Kiss, A., Coscia, C.J., 2005. Mu and kappa opioid receptors activate ERK/MAPK via different protein kinase C isoforms and secondary messengers in astrocytes. J. Biol. Chem. 280, 27662e27669. Bohn, L.M., Belcheva, M.M., Coscia, C.J., 2000. Mitogenic signaling via endogenous kappa-opioid receptors in C6 glioma cells: evidence for the involvement of protein kinase C and the mitogen-activated protein kinase signaling cascade. J. Neurochem. 74, 564e573. Bolan, E.A., Kivell, B., Jaligam, V., Oz, M., Jayanthi, L.D., Han, Y., Sen, N., Urizar, E., Gomes, I., Devi, L.A., Ramamoorthy, S., Javitch, J.A., Zapata, A., Shippenberg, T.S.,

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Article pubs.acs.org/jnp

Open Access on 07/30/2015

Studies toward the Development of Antiproliferative Neoclerodanes from Salvinorin A Tamara Vasiljevik,†,‡ Chad E. Groer,† Kurt Lehner,† Hernan Navarro,§ and Thomas E. Prisinzano*,† †

Department of Medicinal Chemistry, School of Pharmacy, The University of Kansas, Lawrence, Kansas 66045, United States Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, North Carolina 27709, United States

§

S Supporting Information *

ABSTRACT: The success rate for central nervous system (CNS) drug candidates in the clinic is relatively low compared to the industry average across other therapeutic areas. Penetration through the blood−brain barrier (BBB) to reach the therapeutic target is a major obstacle in development. The rapid CNS penetration of salvinorin A has suggested that the neoclerodane nucleus offers an excellent scaffold for developing antiproliferative compounds that enter the CNS. The Liebeskind−Srogl reaction was used as the main carbon−carbon bond-forming step toward the synthesis of quinonecontaining salvinorin A analogues. Quinone-containing salvinorin A analogues were shown to have antiproliferative activity against the MCF7 breast cancer cell line, but show no significant activity at the κ-opioid receptors. In an in vitro model of BBB penetration, quinone-containing salvinorin A analogues were shown to passively diffuse across the cell monolayer. The analogues, however, are substrates of P-glycoprotein, and thus further modification of the molecules is needed to reduce the affinity for the efflux transporter.

I

third of the top-selling drugs in the world are natural products or natural product derivatives.4 Natural products as well as natural product derived entities have contributed to the development of various anti-infectives, anticholesteromics, and antitumor agents, most of which are still actively used in the clinic.6 Because of the vast structural diversity that nature has provided, it is safe to say that further investigation into natural products and their derivatives can provide us with novel drug entities and biological probes. Nakijiquinones are marine sesquiterpene quinones that were isolated from an Okinawan sponge of the Spongiidae family in the early 1990s.7 These natural products possess three distinct structural elements: a terpene core, an amino acid side chain, and a central p-quinoid moiety (examples in Chart 1).7−12 Upon their isolation, this family of quinones was shown to be the first naturally occurring inhibitors of the Her-2/Neu receptor tyrosine kinase.8 Cytotoxicity assays in murine leukemia and human epidermoid carcinoma cells demonstrated IC50 values for several of the isolated quinones in the range 0.5 to 6 μg/mL, making them compounds of pronounced interest.7,8,10 Recently, some clerodane diterpenes have also demonstrated cytotoxicity against several cell lines.13−16 Therefore, it is unclear whether the antiproliferative activity of the nakijiquinones is due to the quinone, the terpene core, or their combination. The terpene core of these molecules, however, offers few options for structural manipulation and compound optimization.

n 2010, it was estimated that approximately 688 000 people in the United States are living with primary tumors of the brain and central nervous system (CNS), 138 000 of which are living with malignant tumors and 550 000 with nonmalignant tumors.1 According to the Central Brain Tumor Registry of the United States (CBTRUS), approximately 69 720 new cases of primary tumors and nonmalignant brain and CNS tumors were expected to be diagnosed in 2013 in the United States. One of the most prevalent primary tumors in the CNS is gliomas. Seventy percent of all CNS gliomas are malignant, and this type of CNS cancer has been shown to be the most frequent and lethal of the cancers originating in the CNS, with a high rate of recurrence and mortality.2,3 Several therapeutic strategies are available; however, they are not efficient for every patient with recurrent glioblastoma, and prognosis remains uncertain. Thus, new agents and novel diagnostic tools are necessary for the improvement of the outcome for glioblastoma patients. The discovery of novel biological probes could help us gain a better understanding of this disease state, which could in turn assist in faster diagnosis of affected patients, providing them with a greater survival rate. The use of natural products has been documented in many ancient civilizations that were utilizing natural products in their ethnomedicinal traditions and spiritual practices. They represent secondary metabolites that are produced by various organisms in response to external stimuli such as temperature, growth, infection, and stress from competition.4 Natural products have played a major role in the discovery and development of drugs for the treatment of various human diseases.5 That natural products are well represented in the pharmaceutical industry is demonstrated by the fact that one© 2014 American Chemical Society and American Society of Pharmacognosy

Received: March 3, 2014 Published: July 30, 2014 1817

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Chart 1. Examples of Nakijiquinones Isolated from an Okinawan Sponge of the Spongiidae Family and Salvinorin A

One clerodane that has not been explored for its cytotoxic activity is salvinorin A.17−19 Previous studies have shown that salvinorin A, a potent κ-opioid receptor (KOP) agonist, is able to cross the blood−brain barrier (BBB) within 1 min of intravenous or inhaled administration,20−22 although it is a substrate for P-glycoprotein (P-gp).21 On the basis of its rapid CNS penetration, the neoclerodane nucleus offers an excellent scaffold for the development of CNS cancer probes that could be used as potential diagnostic tools. In addition, the salvinorin A nucleus offers additional opportunities for optimizing the activity of such biological probes due to the various chemical handles already present in the salvinorin A diterpene core. Therefore, we hypothesized that a combination of the salvinorin A nucleus and the nakijiquinone p-quinoid moiety will result in a novel scaffold that is expected to pass the BBB and have antiproliferative activity (Figure 1). As a first step, we evaluated a series of quinone-containing analogues similar in structure to the quinone moiety in the nakijiquinones. It was envisioned that these modifications would provide an initial proof of concept before embarking on a more complex synthetic undertaking. The resulting molecules showed no activity at the KOP, but exhibited antiproliferative activity against cancer cell lines. Finally, the salvinorin A quinones are predicted to rapidly cross the BBB by passive diffusion and are an important starting point for the development of novel probes for studying CNS cancers.

Figure 1. Design strategy.

■

RESULTS AND DISCUSSION Chemistry. The use of the Liebeskind−Srogl reaction as the main carbon−carbon bond cross-coupling step was based on the recent observation that the mild coupling conditions of this 1818

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Scheme 1a

Reagents and conditions: (a) NaIO4, RuCl3·3H2O, CCl4/CH3CN/H2O; (b) PhSH, CDMT, NMM, THF; (c) CuTC, Pd(dba)2, RB(OH)2, P(OEt)3, THF; (d) (R)-(+)-2-methyl CBS reagent, borane-dimethyl sulfide complex, toluene, −78 to −30 °C; (e) Et3SiH, BF3·OEt2, CH2Cl2, rt; (f) CAN, CH3CN, rt.

a

Scheme 2a

Reagents and conditions: (a) H2O2, H2SO4, MeOH; (b) K2CO3, MeI, acetone; (c) n-BuLi, B(OMe)3, THF, −78 °C to rt; (d) Br2, CH2Cl2, 0 °C; (e) n-BuLi, trimethyborate, THF, −78 °C.

a

reaction, which utilize bis(dibenzylideneacetone)palladium(0), copper(I) thiophene carboxylate, and triethylphosphite at ambient temperature, are well tolerated by the salvinorin A scaffold.23 As described previously, salvinorin A was isolated from Salvia divinorum leaves and subjected to oxidative degradation utilizing sodium periodate and ruthenium(III) chloride to yield the corresponding carboxylic acid in 74% yield. The resulting acid was then esterified with thiophenol to yield thioester 3 in 60% yield (Scheme 1).21 Thioester 3 was used as the key intermediate for the synthesis of our targeted quinones. The reaction of 3 with several different boronic acids yielded aromatic ketones 4−6 in 48%, 79%, and 90% yield, respectively. Ketones 4−6 were reduced using Corey−Bakshi−Shibata (CBS) conditions to yield benzylic alcohols 7−9 in 64−90%

yield. Deoxygenation of 7−9 was accomplished using trimethylsilane and boron trifluoride diethyl etherate to give methylene analogues 10−12. Finally, oxidation of 10−12 with ceric ammonium nitrate (CAN) gave quinones 13−15 in 28− 72% yield. Several of the necessary boronic acids were prepared following published reports.24,25 2,3,4,5-Tetramethoxyphenylboronic acid was synthesized following and slightly modifying conditions proposed by Tremblay and co-workers (Scheme 2).24 Oxidation of 2,3,4-trimethoxybenzaldehyde (16) with hydrogen peroxide afforded phenol 17 in 84% yield. Methylation of 17 with iodomethane under basic conditions gave 1,2,3,4-tetramethoxybenzene (18) in 67% yield. Treatment of 18 with n-butyllithium followed by trimethylborate 1819

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before a conclusion about an optimal substitution pattern for the quinone ring can be reached. Salvinorin A-derived analogues were tested in an in vitro model of BBB penetration (Table 3). The MDCK-MDR1 cell

gave boronic acid 19 in 52% yield. 2,4,5-Trimethoxyphenylboronic acid was prepared following conditions proposed by Sutherland and co-workers.25 The reaction of 1,2,4-trimethoxybenzene 20 with bromine gave bromobenzene 21 in 96% yield. Treatment of 21 with n-butyllithium followed by trimethylborate afforded boronic acid 22 in 38% yield. Biological Testing. Two natural product scaffolds were utilized to afford analogues in which the C-12 furanyl moiety of salvinorin A was replaced with functionalized p-benzoquinone groups, which were tested for activity at KOP as well as antiproliferative properties. Analogues 13−15 were subjected to a calcium mobilization assay, which is a functional assay that will determine their activity at the KOP (Table 1). Salvinorin A

Table 3. In Vitro Model of Brain Penetration in MDCKMDR1 Cells (% Transporta) pretreatment cmpd caffeine prazosin salvinorin A 14 15

Table 1. KOP Activity Using a Calcium Mobilization Assay cmpd

EC50 (nM)a

Emaxb

Salvinorin A 13 14 15

1.7 ± 0.6 2500 ± 900 >10 000c >10 000c

103 ± 2 27 ± 4 NDd NDd

Table 2. Antiproliferative Activity in MCF7 Breast Cancer Cell Line 0.04 2.57 >100c 6.54 2.70 5.84

± 0.02 ± 0.81 ± 1.57 ± 0.91 ± 0.50

% inhibitionb 81 85 NDd 77 86 83

2.4 1.8 3.2 1.5 3.4

73.0 35.5 54.2 47.2 38.4

± ± ± ± ±

17.8 2.1*** 3.4** 11.9** 5.3**

line (Madin-Darby canine kidney cells stably transfected with MDR1; MDR1, multidrug resistance gene 1) has been useful in determining whether compounds may passively diffuse across a cell monolayer and whether they may be substrates for P-gP, an important efflux transporter in the BBB and gene product of MDR1.27,28 Caffeine, which is not a P-gp substrate, shows high transport from the apical to the basolateral side of the cell monolayer with and without P-gp inhibition by verapamil (73.0% and 85.9% transport, respectively),29 indicative of passive diffusion and limited efflux by P-gp. In contrast, prazosin, a known P-gp substrate,30 shows significantly less transport from the apical to basolateral side of the membrane. Pretreatment with verapamil, however, significantly increases the transport of prazosin to the basolateral side, indicating that the control compound prazosin passively diffuses across the monolayer and is actively effluxed by P-gp. Salvinorin A and the two benzoquinoids 14 and 15 show similar results to prazosin, suggesting that they passively diffuse across the cell monolayer and are actively effluxed by P-gp, consistent with salvinorin A being a known P-gp substrate.21,31 Therefore, these benzoquinoids are predicted to rely heavily on passive diffusion to enter the brain, similar to salvinorin A.20−22 Since the short duration of action of salvinorin A is due in part to its affinity for the P-gp, manipulation of the salvinorin A scaffold may be necessary to reduce efflux, in order to pursue novel cancer therapies and/or diagnostic probes based on this approach. Alternatively, a targeting strategy may be utilized to direct the compounds to cancer cells. The similarity between two natural products, the plantderived diterpenoid salvinorin A and the marine sponge derived nakijiquinone family of natural products, prompted an idea that a combination of the two scaffolds may yield biological probes that could have potential in CNS-related cancers. Several compounds were synthesized with similarities to both scaffolds, i.e., salvinorin A analogues that possess a C-12 quinone instead of a furanyl moiety. These benzoquinoids exhibited negligible activity at the KOP receptors, but exhibited moderate antiproliferative activity due to the quinonoid moiety. Finally, the salvinorin A-derived benzoquinoids are predicted to passively diffuse across a membrane, but are subject to active efflux by P-gp. These promising results are indicative of the possibility for use of these salvinorin A-derived compounds as

showed full efficacy and potency (EC50 of 1.7 ± 0.6 nM), consistent with previous observations.26 Analogues 13−15 showed little to no agonist activity at the KOP, which is consistent with observed SAR trends, wherein replacing the furan ring of salvinorin A generally reduces affinity, efficacy, and potency for the KOP.26 To determine whether the salvinorin A-derived analogues exhibited antiproliferative activity, their effects on the growth of the MCF7 breast cancer cell line were tested (Table 2). The

GDA 1,4-BQ salvinorin A 13 14 15

± ± ± ± ±

% transport equals ratio of basolateral to apical concentration of test compound. Data are mean ± SEM; n = 3−5. bOne-way ANOVA: F(4,13) = 93.33, p < 0.0001; Bonferroni post-test ###p < 0.001 versus all other compounds after vehicle pretreatment. c**p < 0.01, ***p < 0.001 versus vehicle pretreatment, Student’s t test.

EC50 = concentration for 50% maximal response; mean ± SEM, n = 2. bEmax = % stimulation compared to (−)-U-69,593; mean ± SEM n = 2. cEC50 could not be calculated because no activity was observed. d ND = not determined.

IC50 (μM)a

85.9 6.7 21.2 5.0 9.7

verapamilc ###

a

a

cmpd

vehicle

b

±3 ± 0.3 ±8 ±3 ±4

a IC50 = concentration for 50% growth inhibition. Data are mean ± SEM, n = 2−5. b% growth inhibition compared to DMSO-treated cells. Data are mean ± SEM, n = 2−5. cIC50 could not be calculated because no inhibition occurred. dND = not determined.

positive controls geldanamycin (GDA) and 1,4-benzoquinone (1,4-BQ) inhibited over 80% of cell growth compared to DMSO-treated controls, with potencies of 0.04 and 2.57 μM, respectively. Similarly, salvinorin A-derived analogues, 13−15, show antiproliferative efficacy, inhibiting between 77% and 86% of cell growth, with potencies of 6.54, 2.70, and 5.84 μM, respectively. Salvinorin A did not show any detectable antiproliferative effects, suggesting that the activity of analogues 13−15 is due primarily to the quinone moiety. Similar results were obtained from an additional breast cancer cell line (SKBr3; data not shown). Additional information is needed 1820

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equiv, 0.21 mmol) in MeCN (7 mL) at ambient temperature. The reaction was stirred until completion (TLC monitoring), CH2Cl2 (20 mL) was added, and the resulting mixture was washed with H2O and brine, dried (Na2SO4), and concentrated under reduced pressure. The resulting residue was purified by flash chromatography on silica gel using mixtures of EtOAc/n-hexanes. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-(2,5dimethoxybenzoyl)-6a,10b-dimethyl-4,10-dioxododecahydro1H-benzo[f ]isochromene-7-carboxylate (4). Compound 4 was synthesized from compound 3 using general procedure A and commercially available 2,5-dimethoxyphenylboronic acid to afford 0.250 g (48% yield) of a white solid, mp = 97−100 °C. TLC system: 45% EtOAc/55% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 7.36 (d, J = 3.2 Hz, 1H), 7.10 (dd, J = 9.0, 3.2 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H), 5.95 (t, J = 8.1 Hz, 1H), 5.13−5.03 (m, 1H), 3.90 (s, 3H), 3.80 (s, 3H), 3.71 (s, 4H), 2.78−2.66 (m, 2H), 2.31−2.22 (m, 2H), 2.19−2.07 (m, 7H), 1.80−1.60 (m, 3H), 1.54 (td, J = 13.4, 3.8 Hz, 1H), 1.42 (s, 3H), 1.40−1.32 (m, 1H), 1.07 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 201.84, 196.68, 171.61, 169.78, 153.78, 153.42, 124.04, 122.28, 114.45, 113.45, 79.12, 74.86, 65.02, 56.20, 55.84, 53.31, 51.96, 49.45, 42.04, 38.04, 37.88, 35.58, 30.75, 20.64, 18.31, 16.68, 16.05; HRMS (m/z) [M + Na]+ calcd for C28H34NaO10 553.2050, found 553.2063. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-[(2,5dimethoxyphenyl)(hydroxy)methyl]-6a,10b-dimethyl-4,10dioxododecahydro-1H-benzo[f ]isochromene-7-carboxylate (7). Compound 7 was synthesized from compound 4 using general procedure B to afford 0.31 g (90% yield) of a white solid, mp = 125− 128 °C. 1H NMR (500 MHz, CDCl3) δ 6.96 (dd, J = 11.1, 2.5 Hz, 1H), 6.85−6.70 (m, 2H), 5.27 (dd, J = 5.2, 3.1 Hz, 1H), 5.12 (dd, J = 12.4, 7.5 Hz, 1H), 3.78−3.76 (m, 5H), 3.72 (d, J = 4.7 Hz, 3H), 2.80− 2.70 (m, 2H), 2.35−2.19 (m, 2H), 2.19−2.09 (m, 5H), 2.02 (dd, J = 11.5, 3.3 Hz, 1H), 1.87 (dd, J = 13.2, 6.1 Hz, 1H), 1.75 (dt, J = 10.1, 2.9 Hz, 1H), 1.60−1.52 (m, 3H), 1.30 (s, 3H), 1.06 (d, J = 6.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 202.16, 172.13, 171.67, 169.60, 153.82, 150.10, 127.29, 113.79, 113.21, 111.26, 78.80, 74.92, 70.54, 64.28, 55.92, 55.72, 53.44, 51.94, 50.56, 42.13, 38.10, 34.85, 34.81, 30.91, 20.60, 18.18, 16.15, 15.21; HRMS (m/z) [M + Na]+ calcd for C28H36NaO10 555.2206; found 555.2217. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-(2,5dimethoxybenzyl)-6a,10b-dimethyl-4,10-dioxododecahydro1H-benzo[f ]isochromene-7-carboxylate (10). Compound 10 was synthesized from compound 7 using general procedure C to afford 0.11 g (36% yield) of a white solid, mp = 96−98 °C. TLC system: 40% EtOAc/60% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 6.79−6.73 (m, 3H), 5.12 (dd, J = 11.6, 8.4 Hz, 1H), 4.76 (dq, J = 11.3, 5.6 Hz, 1H), 3.76 (s, 3H), 3.75 (s, 3H), 3.72 (s, 3H), 2.91 (dd, J = 5.8, 2.7 Hz, 2H), 2.73 (dd, J = 11.7, 5.2 Hz, 1H), 2.33−2.23 (m, 3H), 2.17 (s, 3H), 2.14−2.06 (m, 2H), 1.92−1.83 (m, 1H), 1.79−1.71 (m, 1H), 1.59− 1.49 (m, 2H), 1.33 (s, 3H), 1.08 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 202.11, 171.61, 171.58, 169.88, 153.31, 151.83, 125.67, 117.47, 112.57, 111.30, 75.05, 64.20, 55.79, 55.73, 53.58, 51.96, 51.23, 42.25, 42.15, 38.22, 37.22, 35.08, 30.81, 20.60, 18.16, 16.31, 15.18; HRMS (m/z) [M + Na]+ calcd for C28H36NaO9 539.2257; found 539.2269. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-[(3,6dioxocyclohexa-1,4-dien-1-yl)methyl]-6a,10b-dimethyl-4,10dioxododecahydro-1H-benzo[f ]isochromene-7-carboxylate (13). Compound 13 was synthesized from compound 10 using general procedure D to afford 0.04 g (39% yield) of a yellow solid, mp = dec at 212−214 °C. 1H NMR (500 MHz, CDCl3) δ 6.80−6.72 (m, 2H), 6.70 (dd, J = 2.3, 1.2 Hz, 1H), 5.14 (dd, J = 11.5, 8.5 Hz, 1H), 4.69 (dddd, J = 11.9, 9.6, 4.9, 3.2 Hz, 1H), 3.73 (s, 3H), 2.81−2.70 (m, 2H), 2.60− 2.50 (m, 1H), 2.39 (dd, J = 13.3, 4.8 Hz, 1H), 2.34−2.25 (m, 2H), 2.18 (s, 3H), 2.12 (d, J = 3.7 Hz, 1H), 1.97 (dd, J = 11.5, 3.1 Hz, 1H), 1.81−1.74 (m, 1H), 1.65−1.57 (m, 1H), 1.53 (s, 1H), 1.35 (s, 3H), 1.32−1.22 (m, 1H), 1.09 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 201.91, 187.17, 187.10, 171.50, 170.74, 170.00, 143.61, 136.61, 136.59, 135.20, 75.11, 75.03, 64.04, 53.60, 52.00, 51.41, 43.04, 42.09, 38.14, 36.78, 35.26, 30.75, 20.59, 18.12, 16.37, 15.17; HRMS (m/z) [M + K]+ calcd for C26H30KO9 525.1527, found 525.1528; HPLC tR = 6.424; purity = 99.2%.

biological probes in the study of certain brain cancers. This represents the first report of salvinorin A-derived compounds that exhibit antiproliferative activity.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Unless otherwise indicated, all reagents were purchased from commercial sources and were used without further purification. Melting points were determined on a Thomas-Hoover capillary melting apparatus. NMR spectra were recorded on a Bruker DRX-400 with a qnp probe or a Bruker AV500 with a cryoprobe using δ values in ppm (TMS as internal standard) and J (Hz) assignments of 1H resonance coupling. HRMS data were collected on either an LCT Premier (Waters Corp., Milford, MA, USA) TOF mass spectrometer or an Agilent 6890 N gas chromatograph in conjunction with a Quarto Micro GC mass spectrometer (Micromass Ltd., Manchester, UK). TLC was performed on 0.25 mm Analtech GHLF silica gel plates using EtOAc/n-hexanes, in 1:1 v/v ratio, as the solvent unless otherwise noted. Spots on TLC were visualized by UV (254 or 365 nm), if applicable, and phosphomolybdic acid in EtOH. Column chromatography was performed with silica gel (40−63 μm particle size) from Sorbent Technologies (Atlanta, GA, USA). Analytical HPLC was carried out on an Agilent 1100 Series capillary HPLC system with diode array detection at 254 nm on an Agilent Eclipse XDB-C18 column (250 × 10 mm, 5 μm) with isocratic elution in CH3CN/H2O (3:2, v/v) unless otherwise specified. General Procedure A: Liebskind−Srogl Coupling Reaction. Thioester 3 (1 equiv), appropriate boronic acid (3 equiv), bis(dibenzylideneacetone)palladium(0) (5 mol %), and copper(I) thiophene carboxylate (1.5 equiv) were placed in a 100 mL roundbottom flask and flushed twice with argon. Anhydrous THF was added immediately followed by triethylphosphite (20 mol %, color change from red to green with a brown tint). The reaction was allowed to stir at ambient temperature and upon completion (TLC monitoring) was diluted with Et2O (30 mL). The organic portion was washed with saturated NaHCO3 and brine, dried (Na2SO4), and concentrated under reduced pressure. The resulting residue was purified by flash chromatography on silica gel using mixtures of EtOAc/n-hexanes. General Procedure B: CBS Reduction. The corresponding aromatic ketone (1 equiv) was placed in a round-bottom flask and flushed three times with argon. A solution of (R)-(+)-2-methyl-CBSoxazaborolidine (1 M in toluene, 1 equiv) was added, the temperature was cooled to −78 °C, and the solution was allowed to stir for 5 min. A solution of borane-dimethyl sulfide complex (2 M in diethyl ether, 1 equiv) was added dropwise, and the mixture was warmed to −30 °C and allowed to stir until completion (TLC monitoring). Upon completion, MeOH (1 mL), H2O (0.5 mL), and Et2O (5 mL) were added, and the mixture was warmed to room temperature over a period of 1 h. H2O (20 mL) and Et2O (20 mL) were added, and the two layers separated. The aqueous layer was washed with two additional portions of Et2O, the combined organic layers were washed with brine and dried over Na2SO4, and the solvent was removed under reduced pressure. The resulting residue was purified by flash chromatography on silica gel using mixtures of EtOAc/n-hexanes. General Procedure C: Deoxygenation Procedure. The corresponding alcohol (1 equiv, 0.21 mmol) was placed in a roundbottom flask and flushed with argon. CH2Cl2 (10 mL) was added, and the mixture was cooled to 0 °C. Triethylsilane (2 equiv) was added, and the mixture was stirred for 2 min. Boron trifluoride diethyl etherate (2 equiv) was added dropwise, and the resulting mixture was allowed to stir at 0 °C until completion (TLC monitoring). The reaction was quenched with H2O, and the mixture was warmed to ambient temperature. The mixture was extracted with EtOAc (3 × 20 mL), dried (Na2SO4), and concentrated to dryness under reduced pressure. The resulting residue was purified by flash chromatography on silica gel using mixtures of EtOAc/n-hexanes. General Procedure D: Quinone Formation. An aqueous solution of ceric(IV) ammonium nitrate (2−3 equiv, 0.42 mmol, 1.4 mL) was added dropwise to a solution of salvinorin A derivative (1 1821

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(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-6a,10b-dimethyl-4,10-dioxo-2-(2,3,4,5-tetramethoxybenzoyl)dodecahydro-1H-benzo[f ]isochromene-7-carboxylate (5). Compound 5 was synthesized from compound 3 using general procedure A and 2,3,4,5-tetramethoxyphenylboronic acid24 to afford 0.57 g (79% yield) of an amorphous solid. TLC system: 45% EtOAc/ 55% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 7.06 (s, 1H), 5.88 (t, J = 8.2 Hz, 1H), 5.13−5.04 (m, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.90 (s, 3H), 3.86 (s, 3H), 3.72 (s, 3H), 2.71 (ddd, J = 12.1, 8.3, 2.5 Hz, 2H), 2.28 (td, J = 9.5, 2.2 Hz, 2H), 2.18−2.17 (m, 3H), 2.14−2.13 (m, 3H), 2.12 (s, 1H), 1.76 (dt, J = 13.4, 3.2 Hz, 1H), 1.72−1.58 (m, 2H), 1.43 (s, 3H). 1.07 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 201.81, 196.30, 171.58, 171.37, 169.80, 149.46, 148.77, 148.54, 146.62, 122.46, 107.23, 78.79, 74.89, 64.92, 62.02, 61.25, 56.16, 53.38, 51.97, 49.51, 42.07, 38.38, 37.91, 35.55, 30.95, 30.71, 20.60, 18.29, 16.51, 16.05; HRMS (m/z) [M + Na]+ calcd for C30H38NaO12 613.2261, found 613.2289. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2(hydroxy(2,3,4,5-tetramethoxyphenyl)methyl)-6a,10b-dimethyl-4,10-dioxododecahydro-1H-benzo[f ]isochromene-7-carboxylate (8). Compound 8 was synthesized from compound 5 using general procedure B to afford 0.78 g (89% yield) of a white solid, mp = 104−107 °C. TLC system: 55% EtOAc/44% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 6.65 (s, 1H), 5.26 (dd, J = 4.7, 2.9 Hz, 1H), 5.11 (dd, J = 12.2, 7.7 Hz, 1H), 4.73 (ddd, J = 11.5, 5.8, 3.0 Hz, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.83 (d, J = 5.1 Hz, 6H), 3.72 (s, 3H), 2.96 (s, 1H), 2.88 (s, 1H), 2.74 (dd, J = 12.7, 4.1 Hz, 1H), 2.65 (d, J = 4.9 Hz, 1H), 2.31−2.22 (m, 2H), 2.17 (s, 2H), 2.12 (s, 3H), 1.97 (td, J = 13.2, 12.6, 7.0 Hz, 2H), 1.81−1.74 (m, 1H), 1.31 (s, 3H), 1.06 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 202.18, 171.89, 171.55, 169.53, 149.68, 146.51, 144.04, 142.65, 125.54, 104.67, 79.77, 74.90, 70.07, 64.23, 61.07, 61.05, 60.99, 56.33, 53.51, 51.91, 50.64, 42.19, 38.14, 34.81, 34.62, 30.82, 20.50, 18.12, 16.15, 15.14; HRMS (m/z) [M + Na]+ calcd for C30H40NaO12 615.2418, found 615.2435. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-6a,10b-dimet hy l - 4, 10 - d i o x o - 2 - ( 2 , 3 , 4 , 5 - te t r a m e t ho xy b e n zy l)dodecahydro-1H-benzo[f ]isochromene-7-carboxylate (11). Compound 11 was synthesized from compound 8 using general procedure C to afford 0.42 g (55% yield) of a white solid, mp = 91−94 °C. TLC system: 45% EtOAc/55% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 6.45 (s, 1H), 5.12 (dd, J = 11.5, 8.4 Hz, 1H), 4.71 (dq, J = 11.3, 5.5 Hz, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.80 (d, J = 9.0 Hz, 6H), 3.72 (s, 3H), 2.88 (qd, J = 13.8, 5.9 Hz, 2H), 2.77−2.70 (m, 1H), 2.33−2.22 (m, 3H), 2.16 (s, 3H), 2.10 (d, J = 3.9 Hz, 2H), 1.87 (dd, J = 11.6, 3.1 Hz, 1H), 1.80−1.72 (m, 1H), 1.59−1.49 (m, 2H), 1.34 (s, 3H), 1.30−1.24 (m, 1H), 1.08 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 202.14, 171.58, 171.49, 169.85, 149.22, 146.94, 145.69, 141.92, 123.97, 108.53, 77.69, 75.05, 64.12, 61.15, 61.06, 61.04, 56.23, 53.57, 51.97, 51.28, 42.28, 42.14, 38.20, 36.72, 35.07, 30.78, 20.58, 18.17, 16.33, 15.18; HRMS (m/z) [M + Na]+ calcd for C30H40NaO11 599.2468, found 599.2459. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-[(4,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)methyl]-6a,10b-dimethyl-4,10-dioxododecahydro-1H-benzo[f ]isochromene-7carboxylate (14). Compound 14 was synthesized from compound 11 using general procedure D to afford 0.11 g (28% yield) of an orange solid, mp = 102−104 °C. TLC system: 55% EtOAc/45% nhexanes. 1H NMR (500 MHz, CDCl3) δ 6.55−6.48 (m, 1H), 5.14 (dd, J = 11.7, 8.3 Hz, 1H), 4.68 (dddd, J = 11.7, 9.5, 4.7, 3.2 Hz, 1H), 4.02 (s, 3H), 4.00 (s, 3H), 3.73 (s, 3H), 2.80−2.70 (m, 2H), 2.54 (ddd, J = 14.6, 9.3, 1.1 Hz, 1H), 2.38 (dd, J = 13.3, 4.9 Hz, 1H), 2.34−2.26 (m, 2H), 2.18 (s, 3H), 2.14−2.09 (m, 2H), 2.00−1.93 (m, 1H), 1.81−1.75 (m, 1H), 1.60−1.51 (m, 2H), 1.34 (s, 3H), 1.30−1.21 (m, 1H), 1.09 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 201.92, 183.85, 183.80, 171.51, 170.80, 169.98, 145.04, 144.87, 141.74, 133.24, 75.19, 75.02, 64.01, 61.31, 61.24, 53.58, 52.00, 51.38, 42.97, 42.08, 38.12, 36.43, 35.25, 30.74, 20.59, 18.11, 16.36, 15.17; HRMS (m/z) [M + Na]+ calcd for C28H34NaO11 569.1999, found 569.1960; HPLC tR = 6.959; purity = 98.1%. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-6a,10b-dimethyl-4,10-dioxo-2-(2,4,5-trimethoxybenzoyl)dodecahydro1H-benzo[f ]isochromene-7-carboxylate (6). Compound 6 was

synthesized from compound 3 using general procedure A and 2,4,5trimethoxyphenylboronic acid25 to afford 1.01 g (90% yield) of a white solid, mp = 103−106 °C. TLC system: 60% EtOAc/40% n-hexanes. 1 H NMR (500 MHz, CDCl3) δ 7.47 (s, 1H), 6.47 (s, 1H), 5.94 (dd, J = 8.8, 7.3 Hz, 1H), 5.07 (ddd, J = 10.8, 9.3, 1.0 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.87 (s, 3H), 3.70 (s, 3H), 2.78 (dd, J = 13.7, 8.8 Hz, 1H), 2.72−2.65 (m, 1H), 2.30−2.22 (m, 2H), 2.21−2.07 (m, 6H), 1.74 (dt, J = 12.9, 3.1 Hz, 1H), 1.65 (ddd, J = 14.8, 11.6, 3.3 Hz, 1H), 1.57−1.49 (m, 1H), 1.42 (s, 3H), 1.35−1.27 (m, 1H), 1.06 (s, 3H); 13 C NMR (126 MHz, CDCl3) δ 201.89, 171.94, 171.63, 169.81, 155.64, 155.37, 143.68, 118.18, 114.77, 112.83, 96.12, 78.93, 74.89, 65.22, 56.30, 56.26, 56.20, 53.30, 51.95, 49.16, 42.07, 38.37, 37.88, 35.62, 30.76, 20.66, 18.35, 16.95, 16.00; HRMS (m/z) [M + K]+ calcd for C29H36KO11 599.1895, found 599.1903. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2(hydroxy(2,4,5-trimethoxyphenyl)methyl)-6a,10b-dimethyl4,10-dioxododecahydro-1H-benzo[f ]isochromene-7-carboxylate (9). Compound 9 was synthesized from compound 6 using general procedure B to afford 0.78 g (64% yield) of a white solid, mp = 133−136 °C. TLC system: 60% EtOAc/40% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 6.92 (s, 1H), 6.49 (s, 1H), 5.28 (dd, J = 5.0, 2.8 Hz, 1H), 5.15−5.05 (m, 1H), 4.75 (ddd, J = 11.6, 5.8, 3.0 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.71 (s, 3H), 2.74 (dd, J = 12.7, 4.2 Hz, 1H), 2.63 (d, J = 5.2 Hz, 1H), 2.32−2.19 (m, 2H), 2.14 (d, J = 19.1 Hz, 5H), 2.02−1.94 (m, 1H), 1.91 (dd, J = 13.2, 5.8 Hz, 1H), 1.80−1.72 (m, 1H), 1.58−1.50 (m, 2H), 1.30 (s, 3H), 1.05 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 202.19, 172.04, 171.63, 169.55, 150.16, 149.20, 143.24, 117.56, 111.01, 96.89, 79.36, 74.93, 69.90, 64.31, 56.73, 56.12, 56.02, 53.56, 51.94, 50.67, 42.23, 38.20, 34.85, 34.74, 30.89, 20.55, 18.18, 16.18, 15.19; HRMS (m/z) [M + K]+ calcd for C29H38KO11 601.2051, found 601.2029. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-6a,10b-dimethyl-4,10-dioxo-2-(2,4,5-trimethoxybenzyl)dodecahydro1H-benzo[f ]isochromene-7-carboxylate (12). Compound 12 was synthesized from compound 9 using general procedure C to afford 0.42 g (56% yield) of a white solid, mp = 108−111 °C. TLC system: 55% EtOAc/45% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 6.70 (s, 1H), 6.51 (s, 1H), 5.15−5.01 (m, 1H), 4.71 (dd, J = 11.4, 5.5 Hz, 1H), 3.89 (s, 3H), 3.81 (s, 3H), 3.78 (s, 3H), 3.71 (s, 3H), 2.96−2.80 (m, 2H), 2.76−2.67 (m, 1H), 2.32−2.21 (m, 3H), 2.17 (s, 3H), 2.07 (d, J = 11.7 Hz, 2H), 1.85−1.71 (m, 2H), 1.57−1.46 (m, 2H), 1.32 (s, 3H), 1.20 (dd, J = 13.5, 11.6 Hz, 1H), 1.07 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 202.13, 171.65, 171.60, 169.89, 151.77, 148.48, 142.77, 115.84, 115.16, 97.43, 77.47, 75.07, 64.24, 56.62, 56.18, 56.15, 53.60, 51.95, 51.20, 42.18, 41.92, 38.23, 36.07, 35.02, 30.80, 20.60, 18.15, 16.32, 15.20; HRMS (m/z) [M + K]+ calcd for C29H38KO10 585.2102, found 585.2084. (2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-[(4-methoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)methyl]-6a,10b-dimethyl-4,10-dioxododecahydro-1H-benzo[f ]isochromene-7carboxylate (15). Compound 15 was synthesized from compound 12 using general procedure D to afford 0.50 g (72% yield) of a yellow solid, mp = 127−130 °C. TLC system: 60% EtOAc/40% n-hexanes. 1 H NMR (500 MHz, CDCl3) δ 6.63 (t, J = 1.1 Hz, 1H), 5.93 (s, 1H), 5.14 (dd, J = 11.6, 8.4 Hz, 1H), 4.70 (dddd, J = 14.6, 8.6, 4.8, 3.3 Hz, 1H), 3.83 (s, 3H), 3.73 (s, 3H), 2.83−2.71 (m, 2H), 2.56 (ddd, J = 14.4, 9.2, 1.0 Hz, 1H), 2.38 (dd, J = 13.3, 4.9 Hz, 1H), 2.34−2.28 (m, 2H), 2.18 (s, 4H), 2.11 (d, J = 4.1 Hz, 2H), 2.00−1.94 (m, 1H), 1.82− 1.75 (m, 1H), 1.54 (d, J = 12.9 Hz, 1H), 1.35 (s, 3H), 1.27 (t, J = 12.5 Hz, 1H), 1.09 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 201.90, 187.00, 181.77, 171.52, 170.77, 169.98, 158.75, 144.44, 133.15, 107.66, 75.35, 75.02, 64.04, 56.31, 53.59, 52.00, 51.40, 43.01, 42.08, 38.14, 36.65, 35.26, 30.76, 20.60, 18.11, 16.37, 15.17; HRMS (m/z) [M + K]+ calcd for C27H32KO10 555.1633, found 555.1678; HPLC tR = 6.049; purity = 95.4%. Calcium Mobilization Assay. The assay was performed as described previously.23,32 Briefly, CHO cells stably expressing KOP RGαq16 were maintained in F-12 media with 10% FBS, 1% penicillin and streptomycin, and 0.2% normocin (Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO2. Cells were plated at 30 000 cells/well 1822

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not shown).30,37 Transepithelial electrical resistance was randomly monitored in monolayers prior to the assay to ensure development of resistance and monolayer integrity using chopstick-style electrodes (EVOM, World Precision Instruments). Each study was conducted with one or two independent samples of each compound and controls. Studies were repeated two or three times, and data were averaged to produce mean and SEM; therefore, each value represents data from n = 3−5 independent measurements. LC/MS Methods. Sample preparation: 20 μL of media was mixed with 180 μL of 0.1% HCO2H in MeCN. Samples were centrifuged, and 100 μL of supernatant was mixed with 300 μL of 50:50 MeOH/ H2O in 96-well plates. Sample analysis was conducted in positive electrospray mode using an Applied Biosystems API 5000 triple quadrupole mass spectrometer (Framingham, MA, USA) interfaced to a Waters Acquity UPLC system (Waters Corporation, Milford, MA, USA). Chromatography was accomplished using a Phenomenex Luna C18 column (50 mm × 2 mm i.d., 5 μm particle size) fitted with a guard cartridge. Injection volumes were 20 μL. Two mobile phase solutions were used: (A) 0.1% HCO2H with 10 mM ammonium formate in H2O; (B) 0.1% HCO2H with 10 mM ammonium formate in MeOH. The flow rate was 0.5 mL/min. Initial chromatographic conditions were 90% A held for 1 min, decreasing to 5% A over 2.5 min, and held for 1 min before returning to initial conditions. Compound 14 was monitored with a multiple reaction monitoring (MRM) transition of 547.18 → 487.00 with DP = 186, CE = 17, and CXP = 20. Compound 15 was monitored with an MRM transition of 517.35 → 457.20 with DP = 201, CE = 17, and CXP = 18. Salvinorin A was monitored with an MRM transition of 433.19 → 373.30 with DP = 141, CE = 15, and CXP = 30. Prazosin was monitored with an MRM transition of 384.19 → 247.00 with DP = 186, CE = 39, and CXP = 18. Caffeine was monitored with an MRM transition of 195.32 → 138.00 with DP = 86, CE = 25, and CXP = 22. Mass spectrometer parameters were as follows: CUR = 10, GS1 = 40, GS2 = 60, IS = 2000, TEM = 650 °C, CAD = 5.

in the same media in a black Costar 96-well optical bottom plate and incubated at 37 °C overnight. The media was replaced with fluorescent calcium dye (Calcium 5 dye, Molecular Devices, Sunnyvale, CA, USA) in 225 μL of assay buffer (HBSS buffer containing 20 mM HEPES, 0.25% BSA, 1% DSMO, and 10 μM probenecid (Sigma, St. Louis, MO, USA)), and cells were incubated for 1 h at 37 °C. Stock solutions (10 mM) were prepared for all compounds in DMSO. Stocks were used to make fresh serial dilutions in DMSO for each experiment and diluted to 10× in assay buffer. Cells were stimulated with 25 μL of 10× compounds using the Flexstation 3 plate-reader, and the change in fluorescence over baseline was recorded for 60 s. Maximum change over baseline was determined from two independent experiments performed on different days (n = 2). For each experiment, 10-point dose−response curves spanning at least seven log units were generated in duplicate (two wells per dose per compound) for each compound. The dose range for salvinorin A was chosen based on literature precedent26 and to provide clear lower and upper asymptotes for the semilog plots of the dose−response curves. The dose range for test compounds began at the highest concentration allowed by solubility (10 μM). Data from each experiment were analyzed separately using nonlinear regression (GraphPad Prism 5.0, San Diego, CA, USA) to determine the potency (EC50) and efficacy (Emax) values. Antiproliferation Assay.33 MCF7 and SKBr3 cells were maintained in Advanced DMEM/F12 (1:1; Life Technologies) supplemented with L-glutamine (2 mM), streptomycin (500 μg/ mL), penicillin (100 units/mL), and 10% FBS at 37 °C and 5% CO2. Cells were grown to confluence, seeded (2000 cells/well, 100 μL total media) in clear, flat-bottom 96-well plates, and allowed to attach overnight. Stock solutions (10 mM) were prepared for all compounds in DMSO. Stocks were used to make fresh dilutions in DMSO for each experiment. Six-point dose−response curves were generated in duplicate (two wells per dose per compound) using the following dose ranges: GDA, 1 nM to 1 μM; all test compounds, 100 nM to 100 μM. Compounds were added (1% DMSO final concentration), and cells were incubated at 37 °C for 72 h. After 72 h, cell growth was determined using an MTS/PMS cell proliferation kit (Promega) per the manufacturer’s instructions. Cells incubated in 1% DMSO were used as controls (i.e., 0% inhibition), and the relative growth of cells incubated with each compound concentration was normalized to DMSO-treated controls. Data from at least two independent experiments performed on different days (n ≥ 2) were analyzed by nonlinear regression using GraphPad Prism 5.0 to generate IC50 and % inhibition values. In Vitro Predictive Model of BBB Penetration. MDCK-MDR1 cells (Madin-Darby canine kidney cells stably transfected with MDR1, which codes for P-gp) obtained from The Netherlands Cancer Institute were maintained in DMEM/F12 (Life Technologies) media with 10% FBS and antibiotics at 37 °C and 5% CO2. MDCK-MDR1 cells were plated (40 000 cells/well) on semipermeable membranes in a 24-well Transwell system with a 0.4 μm membrane pore size, and media was changed the next day. Cells were grown to confluence (generally 3 to 4 days after plating), and growth media was replaced with transport media (Hanks’ balanced salt solution supplemented with 25 mM glucose and 25 mM HEPES) containing vehicle or verapamil (100 μM) for 15 min at 37 °C. Transport media was replaced with fresh transport media containing 10 μM test compound (and vehicle or verapamil) on the apical chamber and incubated at 37 °C for 60 min. The concentration of DMSO was maintained at 1% throughout the experiment to facilitate solubility and minimize aggregation. Samples (20 μL) from the apical and basolateral chambers were collected and analyzed by LC/MS for the presence of test compound (LC/MS details below). The difference in peak areas between the apical and basolateral sides with account of volume differences in each side was used to determine transport across a monolayer.34−36 Caffeine, which penetrates the BBB efficiently, was used as a positive control for membrane permeability. Prazosin was used as a control for P-gp-mediated transport, and 5-(4-chlorophenyl)1-(2,4-dichlorophenyl)-N-{[4-(methanesulfonamidomethyl)cyclohexyl]methyl}-4-methylpyrazole-3-carboxamide was used as a negative control for transport across MDCK-MDR1 monolayers (data

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ASSOCIATED CONTENT

S Supporting Information *

Supporting data including HPLC, 1H NMR, and 13C NMR spectra of compounds 13−15. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 785-864-3267. Fax: 785-864-5326. E-mail: prisinza@ ku.edu. Present Address ‡

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank J. Hall and Dr. B. S. J. Blagg for their technical assistance. This work was supported in part by a grant from the National Institute on Drug Abuse (DA018151).

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REFERENCES

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HYPOTHESIS AND THEORY ARTICLE published: 26 February 2014 doi: 10.3389/fnint.2014.00020

The claustrum’s proposed role in consciousness is supported by the effect and target localization of Salvia divinorum Klaus M. Stiefel 1 *, Alistair Merrifield 2 and Alex O. Holcombe 3 1

The MARCS Institute, University of Western Sydney, Sydney, NSW, Australia NPS Medicinewise, Sydney, NSW, Australia 3 School of Psychology, University of Sydney, Sydney, NSW, Australia 2

Edited by: John J. Foxe, Albert Einstein College of Medicine, USA Reviewed by: Lawrence Edelstein, Medimark Corporation, USA John Smythies, University of California at San Diego, USA Peter Addy, Yale University School of Medicine, USA *Correspondence: Klaus M. Stiefel, The MARCS Institute, University of Western Sydney, Penrith/Kingswood Campus, Room no XB.1.09F, Building XB, Sydney, NSW 2751, Australia e-mail: [email protected]

This article brings together three findings and ideas relevant for the understanding of human consciousness: (I) Crick’s and Koch’s theory that the claustrum is a “conductor of consciousness” crucial for subjective conscious experience. (II) Subjective reports of the consciousness-altering effects the plant Salvia divinorum, whose primary active ingredient is salvinorin A, a κ-opioid receptor agonist. (III) The high density of κ-opioid receptors in the claustrum. Fact III suggests that the consciousness-altering effects of S. divinorum/salvinorin A (II) are due to a κ-opioid receptor mediated inhibition of primarily the claustrum and, additionally, the deep layers of the cortex, mainly in prefrontal areas. Consistent with Crick and Koch’s theory that the claustrum plays a key role in consciousness (I), the subjective effects of S. divinorum indicate that salvia disrupts certain facets of consciousness much more than the largely serotonergic hallucinogen lysergic acid diethylamide (LSD). Based on this data and on the relevant literature, we suggest that the claustrum does indeed serve as a conductor for certain aspects of higher-order integration of brain activity, while integration of auditory and visual signals relies more on coordination by other areas including parietal cortex and the pulvinar. Keywords: claustrum, consciousness, Salvia divinorum, salvinorin A, κ-opioid receptor

CRICK AND KOCH’S IDEAS ON THE ROLE OF THE CLAUSTRUM The late Francis Crick proposed that at any one moment, human subjective consciousness of perceptual contents1 is brought about by the activity of a limited number (∼105 ) of neurons (Crick, 1995; Crick and Koch, 2003). According to Crick’s analysis, these neurons must: (1) Be central in the connection scheme of the human brain, not too close to primary sensory or motor areas. (2) Involve a number of sensory areas, since consciousness integrates several sensory modalities. (3) Have activity correlated with conscious experience, even in situations where it is dissociated from direct sensory input (for instance during the perception of visual illusions). Importantly, the identity of these neural populations will likely change as the contents of conscious experience change. Crick and other authors have suggested that some brain region must act as a “conductor” of this dynamic “conscious field” (Tononi and Edelman, 1998a)2 , “dynamical core” (Tononi and Edelman, 1998b; Dehaene and Changeux, 2004) or “neuronal workspace” (Crick and Koch, 2005). In the last paper Crick authored before his death, he and Koch argued that the claustrum is an ideal candidate for this role (Crick and Koch, 2005). The claustrum is a brain region located

1 Crick’s

definition of consciousness (Crick, 1995) is used here. nomenclature for this concept is used, without hyphens, in the remainder of the paper. 2 Searle’s

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between the insular cortex, piriform cortex and the caudateputamen (Franklin and Paxinos, 2007), see Figure 1. It is highly connected to a number of cortical areas in a mostly reciprocal manner (Carman et al., 1964; Shameem et al., 1984; Neal et al., 1986; Sadowski et al., 1997). This strong and complex interconnectivity with the cortex makes it a prime candidate for the role of the director of the conscious field. Crick and Koch’s hypothesis about the central role of the claustrum in consciousness has received some attention (Smythies et al., 2012, 2013, 2014) but unfortunately the evidence has been limited to anatomy and behavioral effects on animals other than primates. Here we argue that because the psychoactive plant Salvia divinorum has its effect primarily on neuromodulator receptors concentrated in the claustrum, analysis of the subjective effects caused by S. divinorum’s active compound provide a novel source of evidence regarding the role of the claustrum in humans. This new evidence essentially supports Crick and Koch’s arguments from anatomy. A detailed look at the subjective effects together with a review of some of the literature leads us to extend Crick and Koch’s theory of the claustrum’s role in human consciousness. Specifically, we suggest that the claustrum is one of several brain-wide integrators, together with the parietal cortex and the pulvinar (the role of the pulvinar is already discussed in Smythies et al., 2013). We discuss these theoretical proposals, and suggest specific tests using neuroimaging and neural recordings in conjunction with administration of Salvia.

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FIGURE 1 | Connectivity and κ-opioid receptor density of the claustrum in the human brain. (A) The claustrum is strongly connected to diverse cortical areas, with the sections of the claustrum receiving cortical inputs overlapping. This anatomical connection pattern, amongst other things, leads Crick and Koch to propose that the claustrum acts as a director of consciousness. The connectivity was established by injecting horseradish

THE PSYCHOACTIVE COMPOUND OF S. divinorum AND THE κ-OPIOID RECEPTORS OF THE BRAIN The S. divinorum plant, of the Lamiaceae mint family, is native to the Oaxaca region in southern Mexico and is traditionally orally ingested by Mazatecs as an inebriate in religious and spiritual contexts (Siebert, 1994). Its main active compound, salvinorin A, has a threshold effective dose of 250 μg to evoke mind-altering effects in an average-sized adult (humans: Siebert, 1994; rodents: Ansonoff et al., 2006). Salvinorin A is a κ-opioid receptor agonist (Cooper et al., 2002; Roth et al., 2002; Chavkin et al., 2004). Opioid receptors are a class of neuromodulators-receptor proteins embedded in the neural membrane. They convey a signal from the outside of the neuron, the binding of an opioid molecule, to the inside of the neuron, by releasing signaling molecules (2nd messengers) into the cytoplasm. Four classes (with several subtypes) of such receptors, δ, κ, μ, and nociception receptors exist, each with different agonist specificities and signal transduction mechanisms. In the human brain, a number of endogenous agonists activate these receptors. Smoking or orally ingesting S. divinorum is also believed to activate them. An intracellular IP3 and cAMP3 based 2nd messenger cascade (Law et al., 2000) elicited by the κ-opioid receptors yields downstream cellular effects (Henry et al., 1995). Cellular excitability decreases via an increase of the inward rectifier potassium currents (Tallent et al., 1994). Additionally, κ-opioid receptors down-regulate N-type calcium currents, which, via the reduction of presynaptic Ca2+ influx, likely leads to a reduction of excitatory and inhibitory neurotransmitter release. The effects of κ-opioid receptors are thus inhibitory, both by reducing the amount of input a neuron is receiving and by reducing the response to that input. The distributions of neurotransmitter κ-opioid receptors in brains have been measured both by detecting the density of receptor mRNAs and by detecting the receptor-mediated metabolism of 3 Inositol

triphosphate and cyclic adenosine monophosphate.

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peroxidase into the cortex, and subsequently tracing the marked cells in the claustrum. Figure from Crick and Koch (2005), as modified from the original study (Phelps and LeDoux, 2005). (B) In situ hybridization signal of κ-opioid receptor mRNA. The darker the staining of the tissue, the higher the density of κ-opioid receptor mRNA, and subsequently of κ-opioid receptors. Cl: claustrum. Scale bar = 5 mm. Figure from Peckys and Landwehrmeyer (1999).

radioactively labeled 2nd messenger precursors. In human brains, κ-opioid receptor expression was measured by mRNA in situ hybridization (Peckys and Landwehrmeyer, 1999). High densities were found in the striatum, hippocampal dentate gyrus, deep cortical layers (V and VI, with more expression in the prefrontal than in the occipital cortex) and, especially, in the claustrum. The claustrum showed the strongest signal, in fact it was the only brain region in which nearly all cells were labeled with dense to very dense labeling density (Table 1). In the macaque monkey brain, κ-opioid receptor activity was measured by monitoring the agonist-induced binding of a radioactively labeled GTP-analog ([35 S]GTPγS)4 . Strong activity was found in the limbic and association cortex, ventral striatum, caudate, putamen, globus pallidus, claustrum, amygdala, hypothalamus, and substantia nigra (Sim-Selley et al., 1999). The authors report that “A very high level of κ1 -stimulated [35 S]GTPγS binding was observed in the claustrum, with an area of especially high stimulation in the ventral claustrum, adjacent to the amygdala”. While there was evidence for κ-opioid receptor activity in other brain regions as well, the densities were markedly higher in the aforementioned regions. The unusually high κ-opioid receptor density in the claustrum makes it a particularly good candidate area for the consciousness altering effects of salvinorin A. Compared to other brain regions, this high receptor density will likely lead to an onset of inhibition of activity in the claustrum at lower concentrations, and to a stronger inhibition at equal concentrations of salvinorin A. While the receptor density already strongly suggests that the consciousness-altering effects of Salvia are related to claustrum disruption, we should also consider the other parts of the brain with significant κ-opioid receptor densities. The striatum, caudate, putamen, substantia nigra, and globus pallidus are commonly 4 Guanosine triphosphate, another molecule involved in signaling by κ-opioid receptors. [35 S]GTPγS is a radioactively labeled molecule similar in structure to GTP. κ-opioid receptor activity leads to binding of the radioactive label in [35 S]GTPγS, which then can be measured.

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Table 1 | κ-opiod receptor densities in the human brain. Brain region/layer

Density of

Grain density

labeled

per labeled

neurons

neuron

Layer I

0

0

Layer II

++

+ to ++

Layer III

+ to ++

+ to ++

Layer IV

0

0

Layer V

+++

++ to +++

Layer VI

+++

++

Layer I

0

0

Layer II

+

+ to ++

Prefrontal cortex

Primary visual cortex

Layer III

+

+

Layer IV

0

0

Layer V

++ to +++

+ to ++

Layer VI

++

+

Dentate gyrus

+++

++

CA1

+

++

CA2

+

++

CA3

++

+++

CA4

+

++

Accumbens nucleus

+++

++

Putamen anterior part

+++

+++

Putamen posterior part

++

+ to ++

Caudate nucleus anterior

+++

+ to ++

Caudate nucleus posterior

++

+ to ++

Ventral pallidum

++

+

Globus pallidus external

0

0

Globus pallidus internal

0

0

Claustrum

++++

+++ to

Hippocampus

Striatal region

++++ Adapted from Peckys and Landwehrmeyer (1999). 0, no signal; + to +++, increasing receptor densities.

considered to be part of an integrated system involved in action selection, reinforcement learning, and motor control, and are not likely neural correlates of consciousness (Wilson, 2004). The hypothalamus is considered to be responsible for the regulation of metabolic processes as part of the autonomous nervous system. It is an unlikely candidate for a role in consciousness other than creating certain states of arousal necessary for consciousness. The amygdala is a brain region thought to be involved in emotional processing, such as fear and fear conditioning (Phelps and LeDoux, 2005). Some subjects report a component of fear in their S. divinorum evoked experiences. However, this effect

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is distinct from the consciousness-altering effects we are discussing here. This leaves us with the deep layers of the frontal and prefrontal cortex and the claustrum as relevant S. divinorum/salvinorin A target areas. Arguments both from receptor densities, as well as from exclusion of brain regions due to known functions point in their direction. Most likely, these are the brain areas which, when inhibited by salvinorin A, give rise to the intense consciousness-altering experiences reported by users of S. divinorum. The frontal and prefrontal cortex are known as areas involved in planning, higher-order executive and social functions (Cicerone and Tanenbaum, 1997; Beer et al., 2006). Two recent studies have also found dopaminergic activity of salvinorin A (Grundmann et al., 2007; Listos et al., 2011), which could have additional influence on the frontal cortex. A disruption of this area could explain some effects of S. divinorum (see below); however the proposed roles of the claustrum are sufficiently different from the proposed roles of the frontal and prefrontal cortex to allow some distinction in the analysis of the subjective effects.

CONSCIOUSNESS-ALTERING EFFECTS OF SALVINORIN A Unfortunately for scientists, human consciousness is not accessible to outside observers. Observers’ reports about their consciousness can be unreliable, but such subjective reports are the only source of information about a conscious experience, and are therefore valuable for understanding consciousness. For S. divinorum, subjective reports indicate marked differences between the experiences associated with it versus those of other psychoactive drugs. Baggott et al. (2010) conducted an online survey of Salvia users, asking them to compare it to other methods of altering consciousness. The most frequent (38%) response was that it is unique. Experiences with Salvia are sometimes likened to lucid dreams and are usually described as highly interesting but frequently also as terrifying and unpleasant. In the discussion below, the effects of Salvia described are based on anecdotal reports together with a quantitative analysis of subjective reports obtained from Erowid.org (http://www.erowid.org/). Erowid.org is a curated website with tens of thousands of informational documents about drugs. Members of the public post accounts of their experiences after ingesting various substances, and the administrators of the site sometimes work with researchers (Baggott et al., 2010). We (independently of the administrators of the site) initially chose 63 Salvia experiences and 63 lysergic acid diethylamide (LSD) experiences randomly from the site. We are well aware of the limitations of these reports, including a lack of control over the dosage and no prior screening of participants. However, there is on average no reason to doubt the sincerity of these reports, and they constitute a large dataset of human experiments with psychoactive substances that are controlled in many countries. Because the effect of S. divinorum may result from a somewhat specific disruption of the activity of the claustrum, the reports are valuable subjective descriptions of the effects of such a disruption. Our exploratory analysis of the trip reports, the methodology and results of which is described in detail at http://dx.doi.org/10.6084/m9.figshare.902215, used questions from inventories developed to assess the effects of psychoactive

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drugs (Dittrich, 1985; Strassman et al., 1994; Studerus et al., 2010), together with some novel questions relevant to Crick and Koch’s conductor of consciousness theory. One student and one postdoctoral scholar of psychopharmacology discussed with us the questions before reading all the trip reports and scoring them with the questions. We were interested in whether certain aspects of experience were disrupted, and grouped the questions into several categories. Inferential statistics suggest that Salvia experiences differed from LSD experiences on at least four categories, which are depicted in Figure 2. The four categories of questions that yielded significantly higher scores with Salvia are “ego dissolution” (a concept used by Dittrich, 1985 for what became the ASC questionnaire), “another environment,” “beings,” and “nonvisual sensory.” Regarding Salvia’s particularly strong effect on the “another environment” category, the high ratings reflect frequent loss of awareness of the subjects’ current surroundings, replaced by the experience of being in a completely different location. The different location sometimes is a real place, possibly visited decades earlier, and in other cases completely imaginary. One subject reported that he “all of a sudden was in my childhood bedroom as it looked twenty years ago”. Another subject reported being in a scene that “almost alluded to an African or Haitian village” (Arthur, 2008). Disturbances of consciousness of that kind seems consistent with Crick and Koch’s theory of the claustrum as a conductor of consciousness, as experience of presence in a certain locality can reasonably be interpreted as a synthesis of a number of qualitatively different contents of consciousness. But while Crick and Koch wrote of disturbances to the synthesis specifically of sensations associated with a perceived object, these other-location experiences suggest wholesale substitution of current sensory input with other sensations, sometimes constructed from memories, often forming a new coherent whole entirely distinct from the current sensory environment. A second prominent feature of S. divinorum experiences is its stronger effects on the “beings” questions. These correspond to

FIGURE 2 | Comparison of the average rating (top row) and number of trip questions that received ratings (bottom row) for S. divinorum and LSD trip reports. See trip report analysis deposited at http://dx.doi.org/ 10.6084/m9.figshare.902215 for associated statistics. Error bars are 95% confidence intervals. For the counts, the confidence intervals were calculated with Agresti and Caffo’s (2000) add-4 method and for the mean ratings, they were bootstrapped.

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the subjective presence of non-existent people or sentient beings. One subject reported, for example “The Salvia spirits (so I believed at the time) begun pulling me with fuzzy green arms covered in eyes to take me into their dimension. Their vague form was a green loop functioning as both head and arms, with a translucent body in between. They were playful, unthreatening yet determined. . .The most powerful part of the hallucination was my belief that the spirits were real” (erowid.org/exp/72338) While there are also encounters with beings in hallucinations induced by serotonergic psychedelics, they appear to be less common and our data supports this for the case of LSD. Of the trip reports analyzed, 31 of the Salvia reports mentioned interactions with other beings, against 18 of the LSD reports. Also, experiencing actual conversations with such beings seems to be rare with other drugs, for which beings appear as visual hallucinations, but are rarely perceived to utter speech. One possibility is that these effects are caused by disruption of self image, as discussed below. Another possibility is that the aforementioned physiological effects of salvinorin A on the social regions in the frontal and prefrontal cortical areas are responsible for the experiences of beings. A third feature of the influence of S. divinorum is severely altered body image. Subjects sometimes report perceiving their bodies as geometric objects, losing any feeling for the existence of their bodies whatsoever, and sometimes existing entirely non-spatially or outside of time. One subject stated: “It’s as if I have no body, but I can still feel that I’m connected by the roof of my mouth to this slab thing. It’s completely absurd. Then I start to feel like the slab is being held up vertically by somebody. I can’t see him, but it’s as if he has been carrying the slab around for eternity, and that I have always been here attached to it.” (erowid.org/exp/78727). Several component questions contributed to the significantly higher ratings for the associated “ego dissolution” category for the Salvia trip reports. These included “I was not able to complete a thought, my thought repeatedly became disconnected,” “It seemed to me as though I did not have a body anymore,” “Thoughts of present or recent past personal life,” and “Feel removed, detached, separated from body.” While alterations of representation of the self and body also occur under the influence of serotonergic psychedelics (including, in our dataset, LSD), those changes may be less extreme. It seems that under serotonergic psychedelics, the proportions and sizes of body parts are altered, but the modified percepts still originate from the subject’s current sensory input. In contrast, during S. divinorum experiences, the origin of body experiences is completely altered. Substantial progress has been made recently in understanding the neural basis of body image and perspective. Imaging of functioning human brains, in conjunction with visuo-tactile stimulation situations that elicit out-of-body experiences and lesion studies point to the temporo-parietal cortical junction as an area that encodes location of the self (Ionta et al., 2011). It has been suggested that the same function of encoding self location may, when disrupted, result in the experience of other beings. In a woman undergoing surgery for epilepsy, stimulation of the left temporo-parietal junction yielded the feeling of a shadowy person immediately behind her that mimicked her posture and actions (Arzy et al., 2006). While the patient interpreted the figment as being not herself, the researchers suggest that it was a duplicated

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and displaced self body image, interpreted as another being. With Salvia however typically it does not appear that the other being mimics the subject’s movements (or even intended movements). While the temporoparietal cortex did not show a strong signal in the κ-opiate receptor localization studies discussed above, connections between the claustrum and the parietal cortex do exist (Baizer et al., 1993). We suggest that this indirect effect onto the parietal cortical areas is most likely responsible for the effects of Salvia on body image. A fourth aspect of experience for which Salvia scored significantly higher was the “non-visual sensory” category, with the difference mainly driven by the following questionnaire items: “Gravity was in the wrong direction, or wrong force field,”“Feel as moving/falling/flying through space,” as well as the non-specific “Body feels different”. We suspect that the response to the former two questions, and possibly the last question, is related to the ego dissolution and body image disruption that occurs with Salvia. Recent work on neurological patients with impaired perception of the direction of gravity has emphasized that somatosensory information is very important, rather than solely vestibular signals (Barra et al., 2010). Blanke and Metzinger (2009) have linked disruption of somatosensory, vestibular, and gravity percepts to abnormal experiences of phenomenal selfhood. Somatosensory processing is also integral to body image, which as discussed above is highly disrupted in Salvia experiences. An alternative interpretation for the experiences in the “non-visual sensory” category is more in accord with Crick and Koch’s conductor hypothesis – a lack of a proper focus of consciousness aimed at the sensory inputs coming from the present surroundings. This interpretation would implicate the claustrum in a different way than body image distortion to bring about the non-visual sensory effects. In summary, the non-visual sensory effect (which statistically was weaker than the other categories) might reflect the same processes that disrupt body image, and/or substitution of current sensory inputs and representations with others unrelated to the immediate environment. A recently published study reports that S. divinorum evokes synesthesia (Luke and Terhune, 2013). In our sample, both LSD and S. divinorum users reported synesthesia. In sum, certain effects of Salvia consistent with claustral function disruption and Crick and Koch’s theory of the claustrum as a conductor of consciousness (effects on “another environment” and possibly “non-visual sensory”). Another class of Salvia effects was found that are at most indirect effects of claustral inactivation (“body image”), and effects likely brought about by an influence of Salvia on other brain areas (“beings”).

SUMMARY AND DISCUSSION Crick and Koch (2005) highlighted the mystery of the claustrum, and provided a stimulating theory of the claustrum’s possible role in consciousness. Here, consideration of the high density of κ-opioid receptors in the claustrum sparked the realization that insights into the claustrum’s role in consciousness might be gained by assessing the effects of S. divinorum/salvinorin A on humans. Using Salvia profoundly disturbs subjective experience, supporting the idea of Crick and Koch that the claustrum is important for consciousness. The analysis of the subjective reports

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presented here is in agreement with several studies which report significant hallucinogenic effects of S. divinorum, and effects different from those of serotroninergic hallucinogens (Johnson et al., 2011; Addy, 2012; Ranganathan et al., 2012; MacLean et al., 2013). The fact that besides the differences a considerable overlap exists between the effects of different hallucinogens is, in our opinion, evidence of the complexity and unified nature of the human psyche. In principle, the analysis of the Salvia-mediated effects (likely affecting the claustrum) agrees with the role of the claustrum as a large-scale integrator or “conductor” of numerous far-flung cortical regions. However, our analysis of subjective reports of the effect of Salvia suggests that the claustrum is not the sole brain area concerned with across-modality or within-modality binding (see also Smythies et al., 2013). Relative to LSD, Salvia was more likely to give users the impression of being in hallucinated locations with hallucinated beings, while severely distorting or disrupting their representation of their own body, sometimes with the experience of a more abstract, less physical existence. This is also characteristic of rapideye-movement sleep, during dreams. In both cases, conscious experience is decoupled from the signals being provided by the senses, with the brain given free rein to concoct novel scenarios, presumably based on recombination of previous experiences. Recent neuroimaging work has revealed brain areas whose activity appears to both correlated with and causally involved in the experience of scenes (the occipital place area and parahippocampal place area, Epstein and Kanwisher, 1998; Dilks et al., 2013), and future investigation should examine the involvement of these areas. A possible interpretation of these effects would be that the constructivist nature of perception is even more pronounced than in a sober state, with Salvia disrupting the proper coordination of sensory input and memories. Salvia also disrupted the user’s representation of his body and self. Veridical representation of the self, like audiovisual binding, requires the integration of distinct cortical areas. Specifically, the representation of body image appears to involve somatosensory, vestibular, proprioceptive, and visual signals (Barra et al., 2010). The maintenance of body image may therefore be particularly dependent on simultaneous integration of multiple cortical areas. This is consistent with Crick and Koch’s likening of the claustrum to a symphony conductor. The analysis of the subjective experiences caused by Salvia also suggests that the claustrum is involved in coordinating some brain areas, but not critical for (though possibly involved in) the binding of auditory and visual signals or color and motion (Stiefel and Holcombe, 2014) . If so, then what areas of the brain do mainly serve these functions? Neuropsychological evidence published after Crick and Koch’s paper have implicated another subcortical area, the pulvinar, in perceptual binding (Ward and Arend, 2007; Arend et al., 2008) along with the already-known importance of parietal cortex (Cohen and Rafal, 1991; Friedman-Hill et al., 1995). Functional brain imaging of humans under the influence of S. divinorum should improve our understanding of the role of the claustrum. The fast pharmacokinetics of salvinorin A (onset in seconds, duration of minutes) are advantageous for

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electroencephalography (EEG) and functional magnetic resonance (fMRI) imaging studies in humans, as well as more invasive techniques in other animals. If salvinorin A indeed inhibits the claustrum and disrupts large-scale cortical coordination, these proposed studies should yield a massive reorganization of cortical activity. It is impossible to predict the exact nature of the reorganization, since a specific theory of cortico-claustral interaction does not yet exist, but we speculate that one result will be decorrelation of the activity in somatosensory, vestibular, and other cortical areas.

AUTHOR CONTRIBUTIONS Klaus M. Stiefel, conceived the approach and researched the literature. Klaus M. Stiefel and Alex O. Holcombe, wrote the manuscript, designed, and directed the coding of the trip reports. Alex O. Holcombe and Alistair Merrifield performed preliminary analysis. Alex O. Holcombe, conducted the final data analysis and created the plots.. ACKNOWLEDGMENTS We thank Drs. Charles F. Stevens, Gordon W. Arbuthnott, G. Bard Ermentrout, Danko Nikolic, John Jacobson, Alex Marshall, Craig Motbey, Matt Baggott and the reviewers of a previous version of this manuscript for helpful discussion and critical reading.

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The effect of Salvia divinorum and Mitragyna speciosa extracts, fraction and major constituents on place aversion and place preference in rats Kenneth J. Sufka a,b,d,n, Melissa J. Loria a, Kevin Lewellyn c, Jordan K. Zjawiony c,d, Zulfiqar Ali e, Naohito Abe e, Ikhlas A. Khan c,d,e a

Department of Psychology, University of Mississippi, University, MS 38677, USA Department of Pharmacology, University of Mississippi, University, MS 38677, USA c Department of Pharmacognosy, University of Mississippi, University, MS 38677, USA d Research Institute of Pharmaceutical Science, University of Mississippi, University, MS 38677, USA e National Center for Natural Product Research, University of Mississippi, University, MS 38677, USA b

art ic l e i nf o

a b s t r a c t

Article history: Received 12 August 2013 Received in revised form 22 October 2013 Accepted 23 October 2013 Available online 7 November 2013

Ethnopharmacological relevance: Consumer use of botanicals has increased despite, in many instances, the paucity of research demonstrating efficacy or identifying liabilities. This research employed the place preference/aversion paradigm to characterize the psychoactive properties of Salvia divinorum extract (10, 30, 100 mg/kg), salvinorin A (0.1, 0.3, 1.0 mg/kg), Mitragyna speciosa MeOH extract (50, 100, 300 mg/kg), Mitragyna speciosa alkaloid-enriched fraction (12.5, 25, 75 mg/kg) and mitragynine (5, 10, 30 mg/kg) in rats. Material and methods: Following apparatus habituation and baseline preference scores, male SpragueDawley rats were given eight counter-balanced drug versus vehicle conditioning trials followed by a preference test conducted under drug-free states. S( þ)-amphetamine (1 mg/kg) served as the positive control (in Exp. 2) and haloperidol (0.8, 1.0 mg/kg) served as the negative control in both studies. Results: Rats displayed place aversion to both Salvia divinorum and salvinorin A that exceeded that of haloperidol. Rats showed place preference to mitragynine that was similar to that of S( þ)-amphetamine. This CPP effect was much less pronounced with the Mitragyna speciosa extract and its fraction. Conclusions: These findings suggest that both botanicals possess liabilities, albeit somewhat different, that warrant caution in their use. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Salvia divinorum Salvinorin A Mitragyna speciosa Mitragynine Place preference/aversion

1. Introduction Numerous botanical products are widely available to consumers and used not only to treat various medical conditions but also, in some instances, for their pleasurable/euphoric properties (Dennehy et al., 2005). Botanical products give consumers the impression that since they are “all natural” they are safe and pose no physical or psychological health risks (Marcus and Grollman, 2002. Whereas botanicals used therapeutically often have some supportive evidence of their efficacy, those used recreationally are typically under-researched. Such recreational botanicals contain a wide array of constituents whose properties, such as toxicity and abuse potential, may make them potentially dangerous. One recent example of this is the hepatotoxic effect of the purported anxiolytic kava–kava (Humberston et al., 2003; Teschke et al.,

n Corresponding author at: Peabody Building, University of Mississippi, University, MS 38677, USA. Tel.: þ1 662 915 7728. E-mail address: [email protected] (K.J. Sufka).

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.10.059

2008). Research to characterize the putative liabilities of botanicals and their constituents are as necessary as research that establishes claims of therapeutic efficacy. One procedure to evaluate a compound's abuse potential is the Conditioned Place Preference (CPP) procedure. This associative learning paradigm is based on the notion that animals prefer environments previously paired with positively reinforcing drugs (Bardo and Bevins, 2000). It should be noted that compounds that possess unpleasant properties produce conditioned place aversion (CPA) in the paradigm. CPP/CPA, in its traditional use of studying single-entity compounds, may not lend itself well to studying complex botanical products. Botanicals possess a wide range of constituents that may have antagonistic or synergistic effects that mask or exacerbate liabilities, respectively. Thus, studying only a major constituent or the entire extract alone may fail to identify potential liabilities. One approach is to concomitantly evaluate the full extract, one or more of its fractions and its major constituent(s) in the paradigm. This strategy would reveal antagonistic or synergistic effects within the extract and fraction and more fully characterize constituent liabilities.

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For the present research, we used this extract-fractionconstituent strategy to study the liabilities of the widely available Salvia divinorum (Salvia divinorum) and Mitragyna speciosa (Mitragyna speciosa) both of which are used recreationally. Salvia divinorum and its major active metabolite salvinorin A possess hallucinogenic properties. Salvinorin A is a κ-opioid receptor (KOR) agonist (Roth et al., 2002; Chavkin et al., 2004) and KOR agonists, including salvinorin A, are reported to produce unpleasant effects in humans and, not surprisingly, cause CPA in rodent models (Mucha and Herz, 1985; Pfeiffer et al., 1986; Zhang et al., 2005). Mitragyna speciosa and its major active alkaloid mitragynine possess stimulant like effects at low doses and opiate-like effects at higher doses (US-DOJ). Mitragynine is a high affinity m-opioid receptor (MOR) agonist (Watanabe et al., 1997; Yamamoto et al., 1999) and the role MOR in addiction is well-documented (for review see Koob et al., 1998). In rodents, m-opioid receptor agonists produce CPP (Tzschentke, 2007). We expect salvinorin A and mitragynine to produce CPA and CPP, respectively. Whether Salvia divinorum and Mitragyna speciosa contain other psychoactive constituents that mask or exacerbate the effects of their major metabolites is unknown.

2. Materials and methods 2.1. Subjects The research protocols detailed below were approved on 12 June 2012 by the university's IACUC (protocol # 12–020). Male Sprague Dawley rats (175–200 g, 6–7 weeks old; Harlan, Indianapolis, IN) were housed in pairs and maintained under a 12-h light/dark cycle in a temperature and humidity controlled vivarium. Food and water were available ad libitum. Animals were handled daily (3 d) prior to experimental manipulations to reduce experimenter-related stress. 2.2. Apparatus and procedure Five place preference chambers (Model MED CPP RS; Med Associates, St. Albans, VT) were used for these experiments. Each chamber has two stimulus-distinct (black versus white colored walls and wire mesh or metal rod flooring) drug-conditioning chambers and a third central start chamber (colored gray with smooth solid surface floor). Guillotine doors provide confinement/ access to the conditioning chambers. The CPP/CPA procedure involves four phases: (1) a 15 m apparatus habituation trial, (2) a 15 m baseline preference trial, (3) eight 30 m drug conditioning trials, and (4) a final 15 m place preference trial. Animals had access to the entire place preference apparatus during the drugfree habituation, baseline preference and final preference trials. The conditioning phase involved alternate day, counterbalanced (for drug order) pairings of test compound in one compartment (Sþ ) and vehicle in the other (S ). Conditioning trials were counter-balanced (drug/vehicle) within treatments conditions. Assignment of test compound to a given compartment (Sþ ) was based on baseline preference scores where compounds expected to produce CPP and CPA were assigned to the non-preferred and preferred compartments, respectively. Test apparatus was thoroughly cleaned after each trial.

University of Mississipp (Oxford, MS USA). Salvia divinorum voucher no. 13458 and Mitragyna speciosa voucher no. 12433. Salvia divinorum extract (10, 30, 100 mg/kg) was prepared by exhaustive extraction of dry plant material with ethanol. The extract was filtered and then concentrated. Because it is wellestablished that the psychoactive properties of Salvia divinorum are mediated by salvinorin A, we opted to not include analysis of a Salvia divinorum fraction. Salvinorin A (0.1, 0.3, 1.0 mg/kg) was isolated from Salvia divinorum leaves as previously described (Munro and Rizzacasa, 2003). Briefly, Salvia divinorum leaves were extracted with acetone and subsequently recrystallized from 95% ethanol to yield 99% (HPLC) pure salvinorin A. The salvinorin A doses selected were based on previously published studies in rodent models (McCurdy et al., 2006) and, for Salvia divinorum, dosing equivalence based on concentrations of salvinorin A in the extract. The HPLC fingerprinting analysis of Salvia divinorum extract showed that salvinorin A existed as one of the major constituents of Salvia divinorum (3.1%; see Supplemental materials. In this study, haloperidol (1 mg/kg, Sigma-Aldrich Inc., 498% purity) served as the negative control for CPA. Two vehicles were employed in this study (n ¼5). For Salvia divinorum and salvinorin A the vehicle was 10% DMSO and 10% Tween80 in saline. For haloperidol the vehicle was 50% DMSO in saline. ANOVA in vehicle groups did not reveal significant differences on preferences scores and these groups were combined for subsequent analyses. The leaves of Mitragyna speciosa (550 g) were extracted with methanol (3 L) for 24 h at room temperature for four times. The solvent was removed under reduced pressure to yield a dried extract. An aliquot was suspended in 5% HCl in water and extracted with ethyl acetate. The water-soluble part was basified (pH 9–10) with liquid ammonia and extracted with ethyl acetate. The ethyl acetate-soluble part, separated from basic media, was dried under reduced pressure to get an alkaloid-enriched fraction. Mitragynine (97% pure) was isolated from the fraction by repeated column chromatogaraphy over silica gel using chloroform/methanol (9:1) and hexanes/acetone/liq. ammonia (210:90:1) solvent systems (see Supplemental materials). From these processes, Mitragyna speciosa extract (50, 100, 300 mg/kg), an alkaloidenriched fraction (12.5, 25, 75 mg/kg) and mitragynine (5, 10 and 30 mg/kg) were used in this study. The mitragynine doses selected were based on previously published studies in rodent models (Sabetghadam et al., 2013) and for Mitragyna speciosa, the dosing was based to maximize concentrations of mitragynine in the extract and fraction (approximately 0.5, 1.0, 3.0 mg/kg of mitragynine, respectively) but to avoid its preparation as a suspension. Mitragynine was found to be the major compound in the crude extract (3.5%) and alkaloidal enrich fraction (4.3%) during HPLC fingerprinting analysis (see Supplemental materials). In this study, S( þ)-amphetamine (1 mg/kg in saline Sigma-Aldrich Inc., 4 99% purity) served as the positive control for CPP and haloperidol (0.8 mg/kg) served as the negative control for CPA. As before, two vehicles (n¼ 5) were employed in this study. Mitragyna speciosa extracts, fractions and constituents were dissolved 20% Tween80 in saline. The haloperidol vehicle was 50% DMSO in saline. ANOVA in vehicle groups did not reveal significant differences on preferences scores and these groups were combined for all subsequent analyses. All test compounds or vehicles were administered via intraperitoneal (IP) injection in a volume of 1 mL/kg immediately before each conditioning trial. Sample sizes were n¼ 10.

2.3. Test compounds 2.4. Statistics The leaves of Salvia divinorum and Mitragyna speciosa were purchased from the Salvia divinorum Research Center (Malibu, CA USA) and Bouncing Bear Botanicals (Lawrence, KS USA), respectively. The plant material was identified by Dr. Vijayasankar Raman at The National Center for Natural Prooducts Research at the

Data acquisition was handled by infrared photo-beam detection via MED-PCs IV software. Data analyses were conducted using SPSSs software. Group differences (CPP or CPA) were analyzed using one-way ANOVAs. CPP/CPA scores were defined as time in

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Fig. 1. The effects of Salvia divinorum and salvinorin A on place preference. Values represent mean change in time (seconds) spent in S þ (drug-paired) chamber between baseline and preference trials. Solid horizontal line reflects the mean preference score for the vehicle group and is provided for comparative purposes. Scores significantly above and below baseline reflect CPP and CPA, respectively. n indicates a significant difference from the vehicle group. Sample sizes were 9–10.

Sþpost-conditioning—time in Sþ baseline preference. Post-hoc analyses were performed using Fisher's LSD. Rewarding (CPP) or aversive (CPA) properties of test compounds were determined by a statistically significant (po0.05) increase or decrease in preference scores relative to vehicle treated rats, respectively.

3. Results Preference scores from the Salvia divinorum and salvinorin A study are summarized in Fig. 1. In the vehicle group, preference scores changed little over their initial baseline scores (þ23 s, see solid line). Haloperidol animals tended to have lower preference scores (CPA) as compared to the vehicle group, respectively. In general, both Salvia divinorum and salvinorin A showed CPA. Consistent with these findings, a one-way ANOVA revealed a significant main effect for treatment condition, F(8, 73) ¼3.986, p ¼0.001. Post-hoc analyses revealed that the preference score for haloperidol was lower than the vehicle group and this effect approached significance (p ¼0.083). Mean preference scores for 10 and 100 mg/kg Salvia divinorum were significantly lower than the vehicle (ps o0.05, respectively; 30 mg/kg Salvia divinorum at p ¼0.073). The mean preference scores for 0.3 and 1.0 mg/kg salvinorin A groups were significantly lower than the vehicle group, (ps o0.005; 0.1 mg/kg salvinorin A at p ¼0.051). Preference scores for Mitragyna speciosa and mitragynine study are summarized in Fig. 2. As before, vehicle group preference scores changed little over their initial baseline scores (þ30 s, see solid line). The S(þ)-amphetamine group displayed higher (CPP) and haloperidol animals displayed lower (CPA) preference scores compared to the vehicle group. In general, the Mitragyna speciosa extract, its fraction and mitragynine showed higher preference scores than the vehicle group. Consistent with these findings, a one-way ANOVA was performed on these data and revealed a significant main effect for treatment condition, F(11, 101) ¼2.97, p ¼0.002. Post-hoc analyses revealed that preference scores for S(þ)-amphetamine were significantly higher than vehicle (p¼0.016). As before, the preference score for haloperidol was lower than vehicle but this effect only approached significance (p¼0.09). The preference scores for the 5 and 30 mg/kg mitragynine groups were significantly higher than the vehicle (ps¼ 0.027 and 0.026; 10 mg/kg mitragynine at p¼ 0.076). While patterns of CPP appeared in the Mitragyna speciosa

extract and fraction groups, these were not statistically different from vehicle.

4. Discussion The goal of these experiments was to utilize the CPP/CPA paradigm to characterize the potential liabilities of two botanical products (salvinorin A and mitragynine) widely available in the consumer marketplace. The methodological approach employed entailed not only screening these individual isolated constituents, but also their parent botanical extracts, and in the case of Mitragyna speciosa, an extract fraction. This strategy enabled one to more fully characterize extracts that may have additional psychoactive constituents that could potentially mask or exacerbate psychoactive effects of the major constituent. In both experiments, the CPP/CPA procedure yielded a balanced designed where baseline preferences did not show a strong compartment bias; furthermore, vehicle-treated group postconditioning preference scores did not significantly change from these baseline measures. The positive and negative reference compounds in these experiments produced both CPP and CPA. For example, 1 mg/kg S(þ)-amphetamine produced a robust increase in preference score and this finding is consistent with numerous reports that S( þ)-amphetamine supports CPP in rodent models (Yamamoto et al., 1999). Haloperidol was selected as the negative control and in both studies decreased preference scores to a degree. This CPA was not as robust as expected and, once again, may reflect too high a dose to support CPA associative learning (0.8 and 1.0 mg/kg). As expected, salvinorin A produced robust CPA. This is not surprising as salvinorin A is a KOR agonist and compounds with this mechanism of action, such as U50,488H, produce place aversion (Skoubis et al., 2001; Tzschentke, 2007). In addition, this work is consistent with research showing salvinorin A produces CPA in mice (Zhang et al., 2005). One additional noteworthy finding is that Salvia divinorum also produced CPA at a level similar to its major constituent. These observations illustrate (1) the main constituent responsible for place aversion is salvinorin A and (2) that there appears to be no other constituent in the extract that antagonizes salvinorin A's psychoactive properties. In the second experiment, mitragynine produced a robust increase in preference scores indicative of CPP. This finding was also expected given mitragynine is a MOR agonist and such compounds produce CPP in rodent models (for review see Koob et al., 1998). In general,

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Fig. 2. The effects of Mitragyna speciosa, its fraction, and mitragynine on place preference. Values represent mean change in time (seconds) spent in S þ (drug-paired) chamber between baseline and preference trials. Solid horizontal line reflects the mean preference score for the vehicle group and is provided for comparative purposes. Scores significantly above and below baseline reflect CPP and CPA, respectively. n indicates a significant difference from the vehicle group. Sample sizes were 9–10.

Mitragyna speciosa extract and its fraction increased preference scores but did so to a lesser degree than mitragynine. We interpret this to suggest (a) this may represent a diminished effect due to lower concentration of mitragynine in the extract and fraction or (b) there may be other psychoactive constituents in the extraction and or fraction that have partial antagonistic properties on mitragynine's rewarding effects. It is interesting to note that the pattern of CPP in rodents in the present study are consistent with anecdotal evidence of the abuse liability of mitragynine in that consumers often have difficulty stopping use of this botanical product (Ahmad and Aziz, 2012). 5. Conclusion The evaluation of botanicals products remains a challenge due to the complexity in which constituents may show antagonistic or synergistic interactions. A multi-tiered approach that incorporates screening the botanical extract, one or more of its fractions and its major constituent(s) appears to be a useful strategy for identifying potential liabilities of botanicals reaching the consumer marketplace as well as potential leads that may attenuate harmful effects of major constituents. For example, further studies exploring psychoactive effects of constituents in the Mitragyna speciosa fraction may reveal one with properties that lessen the liability of drugs of abuse. Current research in this laboratory is addressing this possibility. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2013.10.059. References Ahmad, K., Aziz, Z., 2012. Mitragyna speciosa use in the northern states of Malaysia: a cross-sectional study. J. Ethnopharmacol. 141, 446–450. Bardo, M.T., Bevins, R.A., 2000. Conditioned place preference: what does it add to our preclinical understanding of drug reward? Psychopharmacology 153, 31–43.

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