Ayahuasca Scientific Papers

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J. C. Callaway et al. (1999). ... R. Harris & L. Gurel (2012). A Study ... (2013). LC/MS/MS Analysis of the Endogenous Dimethyltryptamine ...... can hallucinogenic drink, an ethnobotanical and chemical ...... actions of ayahuasca by means of quantitative-electro- ...... of Therapeutics, Ninth Edition, eds Hardman JG, Limbird LE.

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

Ayahuasca Scientific Papers -2013-

2013

Asociación 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: Genís Oña

Content _____________________________________ 1. What is ayahuasca? 2. Scientific papers about ayahuasca arranged chronologically (1969-2013) -

G. R. Dolmatoff (1969). El contexto cultural de un alucinógeno aborigen: Banisteriopsis Caapi

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C. Grob et al. (1996). Human Psychopharmacology of Hoasca, a Plant Hallucinogen Used in Ritual Context in Brazil

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J. C. Callaway et al. (1999). Pharmacokinetics of Hoasca Alkaloids in Healthy Humans

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B. Shanon (2000). Ayahuasca and Creativity

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J. Riba et al. (2001). Subjective Effects and Tolerability of the South American Psychoactive Beverage Ayahuasca in Healthy Volunteers

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J. Riba et al. (2002). Effects of Ayahuasca on Sensory and Sensorimotor Gating in Humans as Measured by P50 Supression and Prepulse Inhibition of the Startle Reflex, Respectively

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J. Riba et al. (2002). Topographic Pharmaco-EEG Mapping of the Effects of the South American Psychoactive Beverage Ayahuasca in Healthy Volunteers

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E. Frecksa et al. (2003). Effects of Ayahuasca on Binocular Rivalry with Dichoptic Stimulus Alternation

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J. Riba (2003). Human Pharmacology of Ayahuasca

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D. McKenna (2004). Clinical Investigations of the Therapeutic Potential of Ayahuasca: Rationale and Regulatory Changes

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J. Riba et al. (2004). Effects of the South American Psychoactive Beverage Ayahuasca on Regional Brain Electrical Activity in Humans: A Functional Neuroimaging Study Using Low-Resolution Electromagnetic Tomography

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P. R. Barbosa et al. (2005). Altered States of Consciousness and Short-Term Psychological AfterEffects Induced by the First Time Ritual Use of Ayahuasca in an Urban Context in Brazil

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D. X. da Silveira et al. (2005). Ayahuasca in Adolescence: A Neuropsychological Assessment.

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D. X. da Silveira et al. (2005). Ayahuasca in Adolescence: A Preliminary Psychiatric Assessment.

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D. E. Stuckey et al. (2005). EEG Gamma Coherence and Other Correlates of Subjective Reports During Ayahuasca Experiences

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J. Riba & M. Barbanoj (2005). Bringing Ayahuasca to the Clinical Research Laboratory

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J. Riba et al. (2006). Increased Frontal and Paralimbic Activation Following Ayahuasca, the PanAmazonian Inebriant

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R. Gable (2006). Risk Assessment of Ritual Use of Oral Dimethyltryptamine (DMT) and Harmala Alkaloids

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E. Rodrigues & E. A. Carlini (2006). Use of South American Plants for the Treatment of Neuropsychiatric Disorders

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M. Barbanoj et al. (2007). Daytime Ayahuasca Administration Modulates REM and Slow-Wave Sleep in Healthy Volunteers

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R. G. Santos et al. (2007). Effects of Ayahuasca on Psychometric Measures of Anxiety, Panic-Like and Hopelessness in Santo Daime Members

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J. H. Halpern et al. (2008). Evidence of Health and Safety in American Members of a Religion Who Use a Hallucinogenic Sacrament

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P. R. Barbosa et al. (2009). A Six-Month Prospective Evaluation of Personality Traits, Psychiatric Symptoms and Quality of Life in Ayahuasca-Naïve Subjects

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J. M. Fabregas et al. (2010). Assessment of Addiction Severity Among Ritual Users of Ayahuasca

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J. C. Bouso & J. Riba (2011). An Overview of the Literature on the Pharmacology and Neuropsychiatric Long Term Effects of Ayahuasca

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D. Frost et al. (2011). B-Carboline Compounds, Including Harmine, Inhibit DYRK1A and Tau Phosphorylation at Multiple Alzheimer’s Disease-Related Sites

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R. G. dos Santos et al. (2011). Autonomic, Neuroendocrine, and Immunological Effects of Ayahuasca

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R. G. dos Santos et al. (2011). Pharmacology of Ayahuasca Administered in Two Repeated Doses

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D. B. de Araujo et al. (2011). Seeing With the Eyes Shut: Neural Basis of Enhanced Imagery Following Ayahuasca Ingestion

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B. C. Labate (2011). Consumption of Ayahuasca by Children and Pregnant Women: Medical Controversies and Religious Perspectives

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A. Gaujac et al. (2012). Analytical Techniques for the Determination of Tryptamines and Bcarbolines in Plant Matrices and in Psychoactive Beverages Consumed During Religious Ceremonies and Neo-shamanic Urban Practices

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E. de Frecksa et al. (2012). Enhancement of Creative Expression and Entoptic Phenomena as Aftereffects of Repeated Ayahuasca Ceremonies

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M. B. Liester & J. I. Prickett (2012). Hypotheses Regarding the Mechanisms of Ayahuasca in the Treatment of Addictions.

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P. C. Barbosa et al. (2012) Health Status of Ayahuasca Users

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R. Harris & L. Gurel (2012). A Study of Ayahuasca Use in North America

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E. H. McIlhenny (2012). Ayahuasca Characterization, Metabolism in Humans, and Relevance to Endogenous N,N-Dimethyltryptamines

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J. C. Bouso et al. (2012). Personality, Psychopatology, Life Attitudes and Neuropsychological Performance Among Ritual Users of Ayahuasca: A Longitudinal Study

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G. Oña (2012). Ayahuasca. Una medicina que cambia nuestra vida.

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R. G. dos Santos (2013). Safety and Side Effects of Ayahuasca in Humans – An Overview Focusing on Developmental Toxicology

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J. C. Bouso et al. (2013). Acute Effects of Ayahuasca on Neuropsychological Performance:

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Differences in Executive Function Between Experienced and Occasional Users -

E. E. Schenberg (2013). Ayahuasca and Cancer Treatment

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G. Thomas et al. (2013). Ayahuasca-Assisted Therapy for Addiction: Results from a Preliminary Observational Study in Canada

3. Scientific papers about DMT arranged chronologically (1964-2013) -

D. E. Rosenberg et al. (1964). The Effect of N,N-Dimethyltryptamine in Human Subjects Tolerant to Lysergic Acid Diethylamide

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D. M. Ruffing & E. F. Domino (1981). Effects of Selected Opioid Agonists and Antagonists on DMT- and LSD-25-Induced Disruption of Food-Rewarded Bar Pressing Behavior in the Rat

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D. M. Stoff et al. (1982). Interaction of Monoamine Blocking Agents With Behavioral Effects of N,N-Dimethyltryptamine

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R. J. Strassman et al. (1994). Dose-Response Study of N,N-Dimethyltryptamine in Humans

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R. J. Strassman et al. (1996). Differential Tolerance to Biological and Subjective Effects of Four Closely Spaced Doses of N,N-Dimethyltryptamine in Humans

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R. J. Strassman (1996). Human Psychopharmacology of N,N-Dimethyltryptamine

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J. C. Callaway et al. (1996). Quantitation of N,N-Dimethyltryptamine and Harmala Alkaloids in Human Plasma After Oral Dosing With Ayahuasca

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E. Gouzoulis-Mayfrank et al. (2005). Psychological Effects of (S)-Ketamine and N,NDimethyltryptamine (DMT): A Double-Blind, Cross-Over Study in Healthy Volunteers

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K. Heekeren et al. (2007). Prepulse Inhibition of the Startle Reflex and its Attentional Modulation in the Human S-Ketamine and N,N-Dimethyltryptamine (DMT) Models of Psychosis

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N. V. Cozzi et al. (2009). Dimethyltryptamine and Other Hallucinogenic Tryptamines Exhibit Substrate Behavior at the Serotonin Uptake Transporter and the Vesicle Monoamine Transporter

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D. Fontanilla et al. (2009). The Hallucinogen N,N-Dimethyltryptamine (DMT) is an Endogenous Sigma-1 Receptor Regulator

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V. Cakic et al. (2010). Dimethyltryptamine (DMT): Subjective Effects and Patterns of Use Among Australian Recreational Users

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TP Su et al. (2011). When the Endogenous Hallucinogenic Trace Amine N,N-Dimethyltryptamine Meets the Sigma-1 Receptor

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J. Riba et al. (2012). Metabolism and Disposition of N,N-Dimethyltryptamine and Harmala Alkaloids After Oral Administration of Ayahuasca

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S. A. Barker et al. (2012). A Critical Review of Reports of Endogenous Psychedelic N,NDimethyltryptamine in Humans: 1955-2010

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E. de Frecksa et al. (2013). A Possibly Sigma-1 Receptor Mediated Role of Dimethyltryptamine in Tissue Protection, Regeneration, and Immunity

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A. Gaujac et al. (2013). Determination of N,N-Dimethyltryptamine in Beverages Consumed in Religious Practices by Headspace Solid-Phase Microextraction Followed by Gas Chromatography Ion Trap Mass Spectrometry

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S. A. Barker et al. (2013). LC/MS/MS Analysis of the Endogenous Dimethyltryptamine

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Hallucinogens, Their Precursors, and Major Metabolites in Rat Pineal Gland Microdialysate

What is Ayahuasca? ______________________________________

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Ayahuasca it’s the most investigated amazon psychedelic. Depending the region in the jungle where you are, ayahuasca will called by different names (Caapi, Yagé, etc.). This beverage is the result of a decoction of two plants: Ayahuasca (Banisteriopsis Caapi) and Chakruna (Psychotria Viridis). The first plant contains B-carboline compounds (Harmine, tetrahidroharmine and harmaline), which act as powerful inhibitors of monoamine oxidase-A (MAOI-A). The second plant contains DMT (N,NDimethyltryptamine). Although DMT is a strong psychedelic, it’s not psychoactive orally, because our MAO degrades it in the liver, and partially in the stomach. However, with the MAOI of the first plant, DMT can work normally.

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Scientific Papers About Ayahuasca Arrenged Chronologically

The Journal of Nervous & Mental Disease (c) Williams & Wilkins 1996. All Rights Reserved. Volume 184(2) February 1996 pp 86-94

vol 184 No. 2

Human Psychopharmacology of Hoasca, A Plant Hallucinogen Used in Ritual Context in Brazil GROB, CHARLES S. M.D1.; McKENNA, DENNIS J. Ph.D.2; CALLAWAY, JAMES C. Ph.D.3; BRITO, GLACUS S. M.D.4; NEVES, EDISON S. M.D.4; OBERLAENDER, GUILHERME M.D.4; SAIDE, OSWALDO L. M.D.5; LABIGALINI, ELIZEU M.D.6; TACLA, CRISTIANE Ph.D.6; MIRANDA, CLAUDIO T. M.D.6; STRASSMAN, RICK J. M.D.7; BOONE, KYLE B. Ph.D.1 A multinational, collaborative, biomedical investigation of the effects of hoasca (ayahuasca), a potent concoction of plant hallucinogens, was conducted in the Brazilian Amazon during the summer of 1993. This report describes the psychological assessment of 15 long-term members of a syncretic church that utilizes hoasca as a legal, psychoactive sacrament as well as 15 matched controls with no prior history of hoasca ingestion. Measures administered to both groups included structured psychiatric diagnostic interviews, personality testing, and neuropsychological evaluation. Phenomenological assessment of the altered state experience as well as semistructured and open-ended life story interviews were conducted with the long-term use hoasca group, but not the hoasca-naive control group. Salient findings included the remission of psychopathology following the initiation of hoasca use along with no evidence of personality or cognitive deterioration. Overall assessment revealed high functional status. Implications of this unusual phenomenon and need for further investigation are discussed. J Nerv Ment Dis 184:86-94, 1996 Hoasca is a hallucinogenic concoction of potent psychoactive plants that are indigenous to the Amazon basin of South America. It has been known under a variety of names, including ayahuasca, caapi, yage, mihi, dapa, natema, pinde, daime, and vegetal. Hoasca is the Portuguese transliteration for ayahuasca and is the accepted term utilized throughout Brazil. Prior to the European conquest, domination, and acculturation of South America, beginning in the 16th century, hoasca was widely used by the native peoples for purposes of magic and religious ritual, divination, sorcery, and the treatment of disease (Dobkin de Rios, 1972). In spite of prolonged and savage attempts by the European conquerors to repress and eradicate native culture and belief systems (Taussig, 1986), sacramental and medicinal use of hoasca remained extant. 1

Department of Psychiatry, Harbor-UCLA Medical Center, Box 498, 1000 West Carson Street, Torrance, California, 90509. Send reprint requests to Dr. Grob 2 Botanical Dimensions, Occidental, California. 3 Department of Pharmacology and Toxicology, University of Kuopio, Finland 4 Centro De Estudos Medicos, Sao Paulo, Brazil. 5 Departmento de Psiquitria, Universidade Estadual do Rio De Janeiro, Brazil 6 Departamento de Psiquitria, Escola Paulista de Medicina, Sao Paulo, Brazil 7 Department of Psychiatry, University of New Mexico, Albuquerque, New Mexico. The authors acknowledge the support of the Heffter Research Institute, Botanical Dimensions, and Jeffrey Bronfman.

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Scientific study of hoasca began with the renowned English botanist Richard Spruce, who from 1849 to 1864 traveled extensively throughout the Brazilian, Venezuelan, and Ecuadorian Amazon to compile an inventory of the varieties of plant life found there (Schultes and Raffauf, 1992). Spruce made a number of valuable discoveries, including Hevea, the genus of the rubber tree, and cinchona, from which quinine is derived. He also identified one of the primary sources of a powerful hallucinogenic brew used by the Mazan and Zaparo Indians, called ayahuasca (Quechua for "vine of the souls" or "vine of the dead"), and previously described by the Ecuadorian Manuel Villavicencio (1858), as a large woody vine that would later be given the formal botanical designation of Banisteriopsis caapi (Ott, 1994; Spruce, 1908). Subsequent laboratory analysis would reveal the presence of the psychoactive beta-carboline alkaloids harmine, harmaline, and tetrahydroharmine, although when first isolated during the early 20th century they would receive the rather exotic appellation of telepathine. As identified by early field observers of hoasca use, additional psychoactive admixtures were often added to the cooking B. caapi preparations, most notably highly potent and hallucinogenic tryptamine-containing plants, including, such vision-inducing plants as Psychotria viridis ( McKenna and Towers, 1984). Throughout the Amazon basin, the use of hoasca remained so deeply rooted in tribal mythology and philosophy that modern investigators have been able to confidently conclude that its use extended back to the earliest aboriginal inhabitants of the region (Schultes and Hofmann, 1992). They have recorded the tradition of hoasca use by the indigenous peoples of the region for the purpose of freeing the soul from corporeal confinement and facilitating access to realms of alternate reality, allowing for a variety of

magical experiences, including accessing communication with the spirits of the ancestors. Anthropologists who have conducted ethnographic studies of the native inhabitants of the Amazon Basin have described such common hoasca-induced phenomena as visions of jaguars, snakes and other predatory animals, visions of distant persons, "cities" and landscapes, the sensation of "seeing" the detailed enactment of recent mysterious events, and the sense of contact with the supernatural (Harner, 1973). Hoasca, as is the case with other plant hallucinogens, has a prehistoric tradition of use by native aboriginal peoples as shamanic sacraments or catalysts (Bravo and Grob, 1989; Furst, 1976). It is considered a "great medicine" and is used to both diagnose and treat illness (Schultes and Hofmann, 1992). Its use is fully sanctioned by societal customs and laws and, in fact, is the core experience upon which tribal and collective consciousness rests. Utilization of such potent plant hallucinogens as hoasca typically occurs within a ritualized context, including the traditional rites of initiation (Grob and Dobkin de Rios, 1992). The powerful hypersuggestible effects induced by the hallucinogenic plant drug reinforce collective belief systems, strengthen group cohesion, and facilitate culturally conditioned and syntonic visions which provide revelation, blessing, healing, and ontological security (Dobkin de Rios and Grob, 1994). Use of hoasca for purposes of healing and religious sustenance has, during the centuries of European acculturation of Amazonia, emerged from the exclusive tribal domains of the rain forest and been incorporated into the contemporary fabric of rural and urban society, particularly among the indigenous Mestizo populations of Peru, Colombia, and Ecuador. Identified as a valuable adjunct to folk healing practices, hoasca is ritually administered by "ayahuasqueros" to carefully selected groups 2

of patients (Dobkin de Rios, 1972). Scrupulously adhering to the shamanic models practiced by the aboriginal peoples, these folk healers similarly use the sacramental hoasca for purposes of medical diagnosis and healing, divination, and as a path of access to the realms of the supernatural. During the 20th century, the use of hoasca within the context of modern syncretic religious movements, particularly in Brazil, has arisen. One such church, and the object of the current study, is the Uniao do Vegetal (UDV), whose translation from the Portuguese means "union of the plants." The UDV originated in the early 1950s when its founder, Gabriel de Costa, a rubber tapper who had first experienced the effects of hoasca with the native Indians of Bolivia, returned to the rapidly expanding Brazilian Amazon settlement of Rio Branco with his visions of spiritual revelation and personal mission. Gathering a group of loyal followers, Maestre Gabriel, as he came to be known, elaborated a mythology and structure for his new religion. Spreading first through the Brazilian Amazon and then to the more densely populated and urbanized South, the UDV grew over the subsequent four decades to achieve an eventual size of approximately 7000 members nationwide, drawing adherents from across the socioeconomic and professional spectra. Organized along the lines of an early Christian parish, local "nucleos," or congregations, are centers where sacramental hoasca is consumed in large bimonthly ritual ceremonies which are presided over by local "maestres," leaders of the religious sect. Although not the only Brazilian syncretic church to use hoasca as a ritual sacrament, the Santo Daime sect being the largest and most widely known, the UDV does have the strongest organizational structure as well as the most highly disciplined membership. Of all the hoasca churches in Brazil, the UDV was also most pivotal in convincing the government

narcotics commission to remove hoasca from the list of banned drugs, which was accomplished in 1987 for use within religious ceremonial contexts. Although achieving some attention and even notoriety in North American literature and the popular press, most notably the work of William Burroughs and Allen Ginsberg (1963), the psychological pheno menon induced by hoasca has been subjected to virtually no rigorous study. Various travelers to the Amazon Basin have reported their own first-hand accounts of experiences with hoasca (Weil, 1980), while both formal and informal anthropological narratives have excited the public imagination (Lamb, 1971; Luna and Amaringo, 1991). Indeed, interest in the exotic Amazonian traditions and effects of hoasca have sparked a steady stream of North American tourists, often attracted by articles and advertisements in popular and New Age magazines (Krajick, 1992; Ott, 1993). Concern over possible adverse psychological health effects incurred by such naive travelers has also been raised by a noted anthropological authority of hoasca use in the Amazon (Dobkin de Rios, 1994). Contrasted with testimonials of improved psychological and moral functioning by adherents of the syncretic hoasca churches in Brazil, a formal study exploring the effects of long-term use of this unusual hallucinogenic beverage would appear to be indicated. During the summer of 1993 a multinational group of biomedical researchers from the United States, Finland, and Brazil met in Manaus, the capital city of the Brazilian state of Amazonia, to conduct an examination of the psychological and biochemical effects of hoasca. Prior to the actual performance of the study, an invitation had been extended by the Uniao do Vegetal to conduct an investigation of the toxicity of their hoasca "tea." Given the long history of repression of their religious movement and use of the hoasca sacrament prior to government sanction in 1987, the leaders of the UDV had surmised that the 3

conclusions of a fair and objective scientific study might be of some protective value in the future if the political winds in Brazil were to shift. Consequently, and upon consultation with the North American research group, a decision was made to utilize the oldest nucleo outside of Rio Branco, in Manaus, where a large percentage of the membership had been ritually consuming hoasca on a regular basis for more than 10 years. Given the

Methods Fifteen members of the syncretic church, Uniao do Vegetal, living in the Brazilian Amazon city of Manaus, were randomly selected from a larger group of volunteers. Criteria for inclusion into the study included membership in the UDV for at least 10 years. Members of the UDV participate in church rituals utilizing hoasca as a psychoactive sacrament a minimum of twice monthly, but often as frequently as several times per week, although always within ritual context. In addition to regular participation in ceremonial consumption of hoasca, the UDV requires of its membership complete abstinence from all other psychoactive substances, including alcohol, tobacco, marijuana, cocaine, and amphetamines. Fifteen control subjects who had never consumed hoasca were also recruited, with the objective of matching them on all demographic parameters. Because of the relatively small sample size, and the need to limit the number of variables, all experimental and control subjects were men. Controls were compatibly matched to experimental subjects along the parameters of age, ethnicity, marital status, and level of education. Although attempts were made to control for diet and current consumption of alcohol, complete compliance was not possible to achieve. Because of difficult field

complicated logistics and demands placed upon subjects in this study, the tightly organized structure of the UDV and its highly disciplined membership proved to be invaluable in the successful completion of the project's goals. Part 1 of this report will detail the results of our investigation of the effects of the hoasca tea on psychological function and Part 2 will discuss our examination of the effects of hoasca on human biochemistry. conditions as well as limitations of time, it was not feasible to completely analyze all demographic data until after initiation of the actual study. At that time it was also identified that control subjects had significantly higher yearly incomes than experimental subjects. In endeavoring to explain this discrepancy we noted that the method of control subject recruitment had called for each of the experimental subjects to provide for the study a close friend or associate who had never participated in UDV ceremonies nor had consumed hoasca under any other circumstances. It was noted in retrospect that several experimental subjects had asked their supervisors at their places of employment to volunteer for the study. A variety of parameters were utilized to assess past and current levels of psychological function. Both experimental and control subject groups were administered structured psychiatric diagnostic interviews (Composite International Diagnostic Interview [CIDI]), personality testing (Tridimensional Personality Questionnaire [TPQ]), and neuropsychological testing (WHO-UCLA Auditory Verbal Learning Test). Experimental subjects, but not control subjects, were asked to fill out an additional questionnaire (Hallucinogen Rating Scale [HRS]) following a hoasca session. Each of the experimental subjects was also interviewed in a semistructured format designed to ascertain their life stories. All subjects were monolingual speakers of Portuguese. Portuguese versions of the CIDI and the TPQ were readily 4

available for this study, having been translated previously and validated in Portuguese by the creators of these instruments. Portuguese versions of the WHO-UCLA Auditory Verbal Learning Test and the HRS were developed for this study by Brazilian collaborators, who translated the instruments first into Portuguese, then back into English, and finally back once again into Portuguese. The CIDI and the WHO-UCLA Auditory Verbal Learning Test sessions were conducted by collaborating Brazilian mental health professionals instructed in their administration. The TPQ and HRS are selfreport questionnaires. The semistructured life story interviews were conducted by an English-speaking psychiatrist assisted by an interpreter fluent in both English and Portuguese. All life story interviews were audiotaped. Composite International Diagnostic Interview The CIDI is a comprehensive, fully standardized diagnostic interview for the assessment of mental disorders according to the definitions and criteria of ICD-10 and DSM-III-R (Robbins et al., 1988). The CIDI was conceived for use in a variety of cultures and settings. Although its primary application has been for epidemiological studies of mental disorders, the CIDI has also been utilized for clinical and research purposes. In the course of its development, the CIDI was subjected to a variety of tests in different settings, countries, and cultures for feasibility, diagnostic coverage, test-retest reliability, and procedural reliability (Wittchen et al., 1991). Tridimensional Personality Questionnaire The TPQ is a 100-item, selfadministered, paper-and-pencil, true/false instrument which takes approximately 15 minutes to complete (Cloninger, 1987a). The questionnaire measures the three higher order personality dimensions of novelty seeking,

harm avoidance, and reward dependence, each of which measures four lower order dimensions (Cloninger, 1987b). The novelty seeking domain measures the spectrums of exploratory excitability versus stoic rigidity (9 items), impulsiveness versus reflection (8 items), extravagance versus reserve (7 items), and disorderliness versus regimentation (10 items). The harm avoidance domain measures the spectrums of anticipatory worry versus uninhibited optimism (10 items), fear of uncertainty versus confidence (7 items), shyness with strangers versus gregariousness (7 items), and fatigability and asthenia versus vigor (10 items). The reward dependence domain measures the spectrums of sentimentality versus insensitiveness (5 items), persistence versus irresoluteness (9 items), attachment versus detachment (11 items), and dependence versus independence (5 items). The TPQ is based on a unified biosocial model of personality integrating concepts focused on the neuroanatomical and neurophysiological basis of behavioral tendencies, styles of learning, and the adaptive interaction of the three personality dimensions (Cloninger et al., 1991). WHO-UCLA Auditory Verbal Learning Test The WHO-UCLA Auditory Verbal Learning Test is a simple list-learning task similar to the Rey Auditory Verbal Learning Test (Rey, 1964), but which also is suitable for use in cross-cultural contexts and is sensitive to mild degrees of cognitive dysfunction. To be familiar to a variety of cultures, the test comprises a list of items carefully selected from categories such as parts of the body, tools, household objects, and common transportation vehicles (Maj et al., 1993). Subjects are read a list of 15 items at the rate of approximately one word per second, following which they are asked to recite as many words as they can recall. The same list is read to subjects a total of five 5

successive times, and on each occasion subjects are asked to recite as many words as they can remember. This is followed by an interference test where subjects are read 15 words from a second list and asked to recite as many as they can from the second list, following which they are asked to again recall the words from the first list. For the final trial, subjects are read from a list of 30 words, half of which (in random order) are from the original list. Subjects then are asked to indicate after each word whether they recognize it as part of the original list of 15 words.

interviews were conducted, with the aid of a translator, in a semistructured and open-ended manner. Each subject was asked to "tell the story of your life from the time before you first drank the hoasca tea... to how you first became acquainted with the UDV and the effects of the hoasca... to how your life has developed since the time you became a part of the UDV.”

Results Psychiatric Diagnoses

The HRS is a 126-item questionnaire originally developed to assess the range of effects induced by intravenous administration of synthetic dimethyltryptamine (Strassman et al., 1994). A 0 to 4 scale is utilized for most questions, with 0 = not at all, 1 = slightly, 2 = moderately, 3 = quite a bit, and 4 = extremely. Responses to items are analyzed according to six conceptually coherent "clusters": somesthesia (interoceptive, visceral, and cutaneous/tactile effects), affect (emotional/affective responses), perception (visual, auditory, gustatory, and olfactory experiences), cognition (alterations in thought processes or content), volition (a change in capacity to willfully interact with themselves, the environment, or certain aspects of the experience), and intensity (strength of the various aspects of the experience).

A structured psychiatric interview was conducted with each of the 15 experimental subjects and each of the 15 normal control subjects. Administration of the CIDI identified that whereas none of the UDV experimental subjects had a current psychiatric diagnosis, active diagnoses of alcohol abuse disorder and hypochondriasis were present in two of the matched control subjects. However, assessment of past (although no longer active) psychiatric diagnoses indicated that, according to ICD-10 and DSM-III-R criteria, five of the UDV experimental subjects had prior formal alcohol abuse disorders, two had past major depressive disorders, and three had past phobic anxiety disorders. On the other hand, among the 15 control subjects, only one subject had a past psychiatric disorder that was no longer present-an alcohol abuse disorder that had remitted 2 years before the study.

Life Story Interview

Personality Testing

Each of the 15 experimental subjects agreed to submit to an approximate hour-long interview conducted by a psychiatrist (C. S. G.). The interview addressed various facets of their lives related to their experience as members of the Uniao do Vegetal and their frequent participation in rituals utilizing the psychoactive sacrament, hoasca. The

The TPQ, measuring the three domains of novelty seeking, harm avoidance, and reward dependence, was administered to the 15 experimental long-term hoascadrinking subjects and to the 15 hoasca-naive control subjects. Means and standard deviations and results of t-test comparisons

Hallucinogen Rating Scale

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are shown in Table 1. Significant findings on the novelty seeking domain included UDV subjects having greater stoic rigidity versus exploratory excitability (p /= 15%) of the slope for any of the compounds was observed (Table 3.2), either between urine samples or for urine versus water, illustrating that relative and absolute matrix effects were generally not of concern [3.38]. Matrix effects are further mitigated in this method by the use of the matrix itself to generate standard curves. DMK was affected to the greatest degree of all the compounds examined. While of interest, this observation should not be an issue since it was determined from the present study that DMK does not appear as a urinary metabolite of DMT following ayahuasca administration and need not be included in future studies. 3.3.3 Major Constituents and Metabolites of Ayahuasca in Urine The application of this method to actual samples has also been demonstrated and the results obtained offer new data concerning the metabolism and clearance of the major harmala alkaloids of ayahuasca and of DMT, in particular. Because of the limited number of subjects, calculations as to various parameters of clearance and overall metabolism were not deemed

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appropriate. A larger study will need to be conducted that includes more subjects and correlation of the data with urine creatinine as well as other values. The overall chromatography and the detection of the selected alkaloids and metabolites present in pre- and post-enzyme treated urine samples collected after ayahuasca administration, are presented in Figures 4 and 5, respectively. Tabulation of the concentrations of these compounds pre- and post-enzyme treatment of urine as a function of collection interval are shown in Table 3.3. As shown in Table 3.3, prior to enzyme treatment of urine, the overall major metabolite observed was DMT-NO, peaking at 4-8 hour after ayahuasca administration with concentrations of approximately 11 µg/ml (n = 3 individuals). THH was the major component in the 8-24 hours samples, with concentrations greater than 5 µg/ml. Free harmalol and harmol were also observed in significant µg/ml concentrations. Much smaller amounts of unchanged DMT, harmaline and harmine and of 5-OH-DMT, and 2-MTHBC were also detected. In urine samples which underwent enzyme hydrolysis with glucuronidase/sulfatase, a 4060 fold increase (Table 3.3) in the amount of harmol in the urine samples was observed, making harmol (or harmol-glucuronide/sulfate) the most abundant product exctreted. Harmalol concentration was increased by a factor of at least 4-fold. Little discernible change in concentration was observed for excreted THH, harmine, harmaline, or DMT as a consequence of enzyme hydrolysis. DMT-NO concentrations were observed to decrease with enzyme treatment of urine but this may be caused by direct chemical effects on the compound produced by heating and hydrolysis [3.31]. Enzyme treatment of urine resulted in release of NMT, which was not detected in unhydrolyzed samples, and of 2-MTHBC. The presence of 5-OH-DMT was also noted in unhydrolyzed urine samples and its concentration was increased following enzyme treatment of urine (Table 3.3). 61

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5-OH-DMT DMT

205.2 → 160.1

189.2 → 144.1

DMK 2-MTHBC

193.1 → 58.3

187.2→ 143.1

Harmalol 201.1 → 160.1

THH 217.1→ 188.1

NMT 175.2 → 144.1 DMT-NO 205.2 → 144.1 Harmol 199.1 → 171.1 Harmine 5-MeO-DMT

213.1 → 170.1

219.2 → 130.1

Harmaline D4-5-MeO-DMT

215.1 → 171.1

223.2 → 178.1

Figure 3.4. Representative chromatogram from a urine sample (4RH12) obtained in the 8-24h interval after ayahuasca administration. 62

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5-OH-DMT DMT

205.2 → 160.1

189.2 → 144.1

DMK 2-MTHBC

193.1 → 58.3

187.2→ 143.1 Harmalol 201.1 → 160.1

THH 217.1→ 188.1

NMT 175.2 → 144.1 DMT-NO 205.2 → 144.1 Harmol 199.1 → 171.1 Harmine 213.1 → 170.1

5-MeO-DMT 219.2 → 130.1

Harmaline D4-5-MeO-DMT 215.1 → 171.1 223.2 → 178.1

Figure 3.5. Representative chromatogram from an enzyme treated urine sample (4RH12) obtained in the 8-24h interval after ayahuasca administration. 63

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Table 3.3: Concentration of compounds (µg/ml) in urine as a function of collection interval (hours) and enzyme (glucuronidase/sulfatase) treatment. Concentration of Compounds in Urine

Collection Interval (hours)

0 0 to 4 4 to 8 8 to 24

DMT

DMT-NO

THH

HARMINE

0 0.45 0.6 0.03

0 8.66 11.06 1.27

0.01 6.03 6.27 5.4

0 0.16 0.12 0.01

HARMALINE HARMALOL

0.02 0.51 0.5 0.32

0 3.61 4.04 1.25

HARMOL

2MTHBC

5-OH-DMT

0.04 2.3 3.09 0.77

0 0.02 0.13 0.01

0 0.14 0.16 0

HARMOL

2MTHBC

5-OH-DMT

0.17 126.18 115.49 38.93

0 0.12 0.11 0

0 0.2 0.2 0.02

Concentration of Compounds in Urine Following Enzyme Treatment

Collection Interval (hours)

0 0 to 4 4 to 8 8 to 24

DMT

DMT-NO

THH

HARMINE

0 0.5 0.48 0.02

0 8.74 8.67 1.02

0 7.25 6.3 4.85

0 0.21 0.18 0.06

The metabolism of

HARMALINE HARMALOL

0 0.53 0.36 0.25

0 14.16 11.06 5.32

the two major harmala alkaloids in ayahuasca, harmine and

harmaline, have previously been examined as individual compounds, in separate studies, to undergo metabolism by O-demethylation to harmol and harmalol, respectively, followed by glucuronidation and/or sulfation [3.41-3.44]. The data from the present study are in agreement with these previous findings. The amounts of harmol and harmalol present in the ayahuasca consumed in the present study would be expected to provide a minor contribution to the total observed in urine, being in the 10’s of µg/ml range versus the 100’s of µg/ml to mg/ml range for harmine and harmaline [3.8, 3.20, 3.22, 3.23, 3.30]. Thus, there appears to be significant metabolism of these latter compounds. In the current study, higher levels of THH were initially detected compared to harmine and harmaline in urine (Table 3.3). However, following enzyme hydrolysis of the urine samples, harmol and harmalol were found to be at far higher concentrations compared to THH.

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It is of interest that the hallucinogenic component of ayahuasca, DMT, when consumed alone by the oral route elicits no discernible psychoactivity, due to rapid oxidative deamination and peripheral degradation to indoleacetic acid (IAA) mediated by the enzyme monoamine oxidase A (MAO-A) [3.20, 3.24, 3.25 3.31, 3.45-3.50]. However, the beta-carboline alkaloids, such as harmine and harmaline and, to a lesser extent the THH also found in ayahuasca, are reversible inhibitors of MAO-A [3.3, 3.50]. Thus, co-administration of MAO inhibitors (MAOI) such as harmine or harmaline allows DMT to become orally active [3.3, 3.46] and would be expected to also affect its overall metabolism and excretion profile. Several studies have examined the pharmacokinetics of parenterally-administered DMT and it is well established that intramuscular (i.m.) or intravenous DMT administered without MAO inhibition is rapidly cleared from blood, [3.51, 3.52] with less than 0.1% of the parent DMT being recoverable in human urine within a 24-hour collection period [3.51, 3.53]. In vivo animal studies have also demonstrated a very rapid clearance of DMT from various tissues such as plasma, brain, and liver [3.47, 3.48, 3.54, 3.55]. While oxidative deamination appears to be a major metabolic route, N-oxidation has also been identified as an important metabolic pathway of DMT both in vitro [3.31, 3.51, 3.56] and in vivo in rodents and rabbits [3.24, 3.47-3.49]. It is of interest to note that DMT-NO does not appear to be a substrate for MAO [3.49, 3.56]. NMT has also been identified as a minor metabolite of DMT [3.24, 3.31, 3.51, 3.57] as has 2-methyl-THBC (2-MTHBC) and traces of tryptamine and 1,2,3,4-tetrahydro-ß-carboline (THBC) [3.31]. NMT is also a substrate for MAO and is subject to be metabolized to IAA. The finding of NMT in the present study suggests that formation of NMT from DMT in ayahuasca may also be a minor pathway in vivo and that it may be conjugated before excretion. One must also consider the possibility that the 2-MTHBC 65

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detected arises from the ayahuasca itself and not necessarily from DMT metabolism. Nonetheless, the latter possibility cannot be excluded. It has been demonstrated that iproniazid pretreatment (MAO-A inhibition) increases the levels of DMT in vivo in rat brain, liver, kidney, blood, as well as DMT-NO in rat liver [3.48], and to increase the urinary excretion of DMT, DMT-NO and NMT in rodents [3.47]. These data suggest that MAO inhibition may shift metabolism to these other routes as part of a possible compensatory metabolic mechanism. Given the MAO-inhibition effects of ayahuasca, similar effects on DMT metabolism would be expected and may explain some of the results. The present results suggest that N-oxidation represents a major metabolic route for DMT clearance in humans, particularly if MAO becomes inhibited, such as occurs with ayahuasca administration, in agreement with the work of Sitaram et al. [3.24]. Sitaram et al. found an approximately 10 times higher concentration of DMT-NO compared to DMT following iproniazid pretreatment in rodents [3.47] and noted a 6 times higher concentration of DMT-NO compared to DMT in control animals when no MAOI was administered. This suggests that DMT-NO may represent a major in vivo metabolite of DMT and may, thus, serve as a better marker for endogenous DMT production and metabolism in mammals as well [3.31, 3.45, 3.47].The data from the present study represent the first report of DMT-NO as a metabolite of DMT in the urine of humans following ayahuasca administration or, for that matter, DMT alone. This finding has implications for the further study of DMT in general, particularly its occurrence as a naturally occurring trace amine in man. The necessity for such studies has gained greater impetus with the recent report that DMT may be the endogenous ligand for the sigma-1 receptor [3.58, 3.59].

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The origins of the 5-OH-DMT observed are somewhat obscure. It is possible that the ayahuasca used in this study contained small quantities of either 5-OH or 5-MeO-DMT, which were not originally detected. However, 5-OH-DMT may also be endogenous and may, with MAO inhibition afforded by ayahuasca consumption, reach high enough concentrations to become more detectable in urine. Further analyses will be required to answer this question. DMK has also been reported to be a metabolite of DMT [3.60]. However, this compound has only been reported in vitro in whole blood following spiking with DMT. No study has yet to examine or determine the presence of this compound as an in vivo metabolite of DMT in any species. The present study suggests that, if formed in blood or in the tissues of humans, it is not consistently excreted as DMK in the urine, even with MAO inhibition, and after treatment with sulfatase/glucuronidase no free DMK is released. However, it remains possible that this compound undergoes metabolism to another, as yet undetermined, compound prior to excretion. The data also suggest that future studies with clinical administration of ayahuasca would benefit from urine collection beyond 24 hours after administration in order to monitor the complete disappearance of DMT-NO, THH, harmine, and harmol.

3.4 References [3.1]

R.E. Schultes. The identity of the maipighiaceous narcotics of South America. Botanical Museum Leaflets, Harvard University 1957, 18, 1-56.

[3.2]

R.E. Schultes, A. Hofmann. The Botany and Chemistry of Hallucinogens. Charles C. Thomas, Springfield, Illinois, 1980.

[3.3]

D.J. McKenna, G.H.N. Towers, F. Abbott. Monoamine oxidase inhibitors in South American hallucinogenic plants: tryptamine and beta-carboline constituents of ayahuasca. Journal of Ethnopharmacology 1984, 10, 195-223. 67

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[3.4]

DJ. McKenna, G.H.N. Towers. Biochemistry and pharmacology of tryptamines and betacarbolines: A minireview. Journal of Psychoactive Drugs 1984, 16, 347-358.

[3.5]

D.J. McKenna, L.E. Luna, G.H.N. Towers, in von Reis S, Schultes RE (Eds.). Ethnobotany: Evolution of a Discipline, Dioscorides Press, Portland, OR, 1995, p. 349.

[3.6]

D.J. McKenna. Clinical investigations of the therapeutic potential of ayahuasca: rationale and regulatory challenges. Pharmacology and Therapeutics 2004, 102, 111-129.

[3.7]

R.S. Gable. Risk assessment of ritual use of oral dimethyltryptamine (DMT) and harmala alkaloids. Addiction 2007, 102, 24-34.

[3.8]

J. Riba, M.J. Barbanoj. Bringing ayahuasca to the clinical research laboratory. Journal of Psychoactive Drugs 2005, 37, 219-230.

[3.9]

D.J. Moura, M.F. Richter, J.B. Boeira, J.A.P. Henriques, J. Saffi. Antioxidant properties of β-carboline alkaloids are related to their antimutagenic and antigenotoxic activities. Mutagenesis 2007, 22, 293-302.

[3.10] W. Andritzky. Sociopsychotherapeutic functions of ayahuasca healing in Amazonia. Journal of Psychoactive Drugs 1989, 21, 77-89. [3.11] J. Mabit, R. Giove, J. Vega. in M. Winkelman and W. Andritzky (Eds.), Yearbook of Cross-Cultural Medicine and Psychotherapy 1996; VMB Press, Berlin 257. [3.12] J.C. Callaway, M.M. Airaksinen, D.J. McKenna, G.S. Brito, C.S. Grob. Platelet serotonin uptake sites increased in drinkers of ayahuasca. Psychopharmacology 1994, 116, 385-387. [3.13] J. Tiihonen, J.T. Kiukka, K.A. Bergstorm, J. Karhu, H. Viinamaki, J. Lehtonen, T. Hallikainen, J. Yang, P. Hakola. Single-photon emission tomography imaging of monoamine transporters in impulsive violent behavior. European Journal of Nuclear Medicine 1997, 24, 1253-1260. [3.14] T. Hallikainen, H.M. Saito, J. Lachman, T. Volavka, O.P. Pohjalainen, J. Ryynanen, J. Kauhanenm, E. Syvalahti, J. Hietala, J. Tiihonen. Association between low activity serotonin transporter promoter genotype and early onset alcoholism with habitual impulsive violent behavior. Molecular Psychiatry 1999, 4, 385-388. [3.15] T. Mantere, E. Tupala, H. Hall, T. Sarkioja, P. Rasanen, K. Bergstorm, J.C. Callaway, J. Tiihonen. Serotonin transporter distribution and density in the cerebral cortex of alcoholic and nonalcoholic comparison subjects: a whole-hemisphere autoradiography study. American Journal of Psychiatry 2002, 159, 599-606. [3.16] L. Rivier, J.E. Lindgren. “Ayahuasca,” the South American hallucinogenic drink: an ethnobotanical and chemical investigation. Economic Botany 1972, 26, 101-129. [3.17] Y. Hashimoto, K. Kawanishi. New organic bases from Amazonian Banisteriopsis caapi. Phytochemistry 1975, 14, 1633-1635.

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[3.18] Y. Hashimoto, K. Kawanishi. New alkaloids from Banisteriopsis caapi. Phytochemistry 1976, 15, 1559-1560. [3.19] J.C. Callaway, D.J. McKenna, C.S. Grob, G.S. Brito, L.P. Raymon, R.E. Poland, E.N. Andrade, E.O. Andrade, D.C. Mash. Pharmacokinetics of Hoasca alkaloids in healthy humans. Journal of Ethnopharmacology 1999, 65, 243-256. [3.20] J. Riba. Human Pharmacology of Ayahuasca. Doctoral Thesis, Universitat Autonoma de Barcelona. 2003, http://www.tdx.cesca.es/TDX-0701104-165104/ [3.21] J. Ott. Pharmahuasca: Human pharmacology of oral DMT plus harmine. Journal of Psychoactive Drugs 1999, 31, 171-177. [3.22] J. Riba, M. Valle, G. Urbano, M. Yritia, A. Morte, M.J. Barbanoj. Human pharmacology of ayahuasca: Subjective and cardiovascular effects, monoamine metabolite excretion, and pharmacokinetics. Journal of Pharmacology and Experimental Therapeutics 2003, 306, 73-83. [3.23] E.H. McIlhenny, K.E. Pipkin, L.J. Standish, H.A. Wechkin, R.J. Strassman, S.A. Barker. Direct analysis of psychoactive tryptamine and harmala alkaloids in the Amazonian botanical medicine ayahuasca by liquid chromatography-electrospray ionization-tandem mass spectrometry. Journal of Chromatography A 2009, 1216, 8960-8968. [3.24] B.R. Sitaram, W.R. McLeod. Observations on the metabolism of the psychotomimetic indolealkylamines: Implications for future clinical studies. Biological Psychiatry 1990, 28, 841-848. [3.25] J.C. Callawa, L.P. Raymon, W.L. Hearn, D.J. McKenna, C.S. Grob, G.S. Brito, D.C. Mash. Quantitation of N,N-dimethyltryptamine and harmala alkaloids in human plasma after oral dosing with ayahuasca. Journal of Analytical Toxicology 1996, 20, 492-497. [3.26] M. Yritia, J. Riba, J. Ortuno, A. Ramirez, A. Castillo, Y. Alfaro, R. De La Torre, M.J. Barbano. Determination of N,N-dimethyltryptamine and beta carboline alkaloids in human plasma following oral administration of Ayahuasca. Journal of Chromatography B 2002, 779, 271-281. [3.27] J.C. Callaway, G.S. Brito, E.S. Neves. Phytochemical analyses of Banisteriopsis caapi and Psychotria viridis. Journal of Psychoactive Drugs 2005, 37, 145-150. [3.28] G. Frison, D. Favretto, F. Zancanaro, G. Fazzin, S.D. Ferrara. A case of beta-carboline alkaloid intoxication following ingestion of Peganum harmala seed extract. Forensic Science International 2008, 179, e37-e43. [3.29] K. Bjornstad, O. Beck, A. Helander. A multi-component LC-MS/MS method for the detection of ten plant-derived psychoactive substances in urine. Journal of Chromatography B 2009, 877, 1162-1168. [3.30] J. Riba, A. Rodríguez-FornellS, G. Urbano, A. Morte, R. Antonijoan, M. Montero, J.C. Callaway, M.J. Barbanoj. Subjective effects and tolerability of the South American 69

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psychoactive beverage Ayahuasca in healthy volunteers. Psychopharmacology(Berlin) 2001, 154, 85-95. [3.31] S.A. Barker, J.A. Monti, S.T. Christian. Metabolism of the hallucinogen N,Ndimethyltryptamine in rat brain homogenates. Biochemical Pharmacology 1980, 29, 1049-1057. [3.32] K. Kawanishi, K. Saiki, H. Tomita, Y. Tachibana, N.R. Farnsworth, M. Bohlke. Chemical components of the Brazilian shamanistic drink “Ayahuasca.” Advances in Mass Spectrometry 1998, 14, D053560/1. [3.33] R.G. Santo, J. Landeira-Fernandez, R.J. Strassman, V. Motta, A.P.M. Cruz. Effects of ayhuasca on psychometric measures of anxiety, panic-like and hopelessness in Santo Daime members. Journal of Ethnopharmacology 2007, 112, 507-513. [3.34] G. Gambelungh, K. Aroni, R. Rossi, L. Moretti, M. Bacci. Identification of N,Ndimethyltryptamine and beta-carbolines in psychotropic ayahuasca beverage. Biomedical Chromatography 2008, 22, 1056-1059. [3.35] A.P.S. Pire, C.D.R. De Oliveir, S. Moura, F.A. Dorr, W.A.E. Silva, M. Yonamine. Gas chromatographic analysis of dimethyltryptamine and beta-carboline alkaloids in ayahuasca, an Amazonian psychoactive plant beverage. Phytochemical Analysis 2009, 20, 149-153. [3.36] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Analytical Chemistry 2003, 75, 3019-3030. [3.37] B.K. Matuszewski. Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis. Journal of Chromatography B 2006, 830, 293-300. [3.38] Food and Drug Administration, Guidance for Industry on Bioanalytical Method Validation, Federal Register 23 May 2001, 66, 28526. [3.39] P.J. Taylor. Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometery. Clinical Biochemistry 2005, 38, 328-334. [3.40] E. Chambers, D.M. Wagrowski-Diehl, Z. Lu, J.R. Mazzeo. Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses. Journal of Chromatography B 2007, 852, 22-34. [3.41] T.A. Slotki, V. DiStefano, W.Y.W. Au. Blood levels and urinary excretion of harmine and its metabolites in man and rats. Journal of Pharmacology and Experimental Therapeutics 1970, 173, 26-30. [3.42] D.J. Tweedie, M.D. Burke. Metabolism of the beta-carbolines, harmine and harmol, by liver microsomes from phenobarbitone- or 3-methylcholanthrene-treated mice. Identification and quantitation of two novel harmine metabolites. Drug Metabolism and Disposition 1987, 15, 74-81. 70

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[3.43] A. Yu, J.R. Idle, K.W. Krausz, A. Küpfer, F.J. Gonzalez. Contribution of individual cytochrome P450 isozymes to the O-demethylation of the psychotropic beta-carboline alkaloids harmaline and harmine. Journal of Pharmacology and Experimental Therapeutics 2003, 305, 315-322. [3.44] A. Yu. Indolealkylamines: biotransformations and potential drug-drug interactions. American Association of Pharmaceutical Scientists Journal 2008, 10, 242-253. [3.45] S.A. Barker, J.A. Monti, S.T. Christian. N,N-Dimethyltryptamine: an endogenous hallucinogen. International Review of Neurobiology 1981, 22, 83-110. [3.46] O. Suzuki, Y. Katsumata, M. Oya. Characterization of eight biogenic indoleamines as substrates for type A and type B monoamine oxidase. Biochemical Pharmacology 1981, 30, 1353-1358. [3.47] B.R. Sitaram, L. Lockett, G.L. Blackman, W.R. McLeod. Urinary excretion of 5methoxy-N,N-dimethyltryptamine, N,N-dimethyltryptamine and their N-oxides in the rat. Biochemical Pharmacology 1987a, 36, 2235-237. [3.48] B.R. Sitaram, L. Lockett, R. Talomsin, G.L. Blackman, W.R. McLeod. In vivo metabolism of 5-methoxy-N,N-dimethyltryptamine and N,N-dimethyltryptamine in the rat. Biochemical Pharmacology 1987b, 36, 1509-1512. [3.49] B.R. Sitaram, R. Talomsin, G.L. Blackman, W.R. McLeod. Study of metabolism of psychotomimetic indolealkylamines by rat tissue extracts using liquid chromatography. Biochemical Pharmacology 1987c, 36, 1503-1508. [3.50] H. Kim, S.O. Sablin, R.R. Ramsay. Inhibition of monoamine oxidase A by beta-carboline derivatives. Archives of Biochemistry and Biophysics 1997, 337, 137-142. [3.51] J. Kaplan, L.R. Mandel, R. Stillman, R.W. Walker, W.J.A. VandenHeuvel, J.C. Gillin, R.J. Wyatt. Blood and urine levels of N,N-dimethyltryptamine following administration of psychoactive doses to human subjects. Psychopharmacologia 1974, 38, 239-245. [3.52] J.C. Gillin, J. Kaplan, R. Stillman, R.J. Wyatt. The psychedelic model of schizophrenia: the case of N,N-dimethyltryptamine. American Journal of Psychiatry 1976, 133, 203208. [3.53] R.J. Strassman, C.R. Qualls, E.H. Uhlenhuth, R. Kellner. Dose-response study of N,Ndimethyltryptamine in humans. II. Subjective effects and preliminary results. Archives of General Psychiatry 1994, 51, 98-108. [3.54] I. Cohen, W.H. Vogel. Determination and physiological disposition of dimethyltryptamine and diethyltryptamine in rat brain, liver and plasma. Biochemical Pharmacology 1972, 21, 1214-1216. [3.55] L.R. Mande, R. Prasad, B. Lopez-Ramos, R.W. Walker. The biosynthesis of dimethyltryptamine in vivo. Research Communications in Chemical Pathology and Pharmacology 1977, 16, 47-58. 71

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[3.56] M.S. Fish, N.M. Johnson, E.P. Lawrence, E.C. Horning. Oxidative N-dealkylation. Biochemical and Biophysical Acta 1955, 18, 564-565. [3.57] S. Szara, J. Axelrod. Hydroxylation and N-demethylation of N,N-dimethyltryptamine. Experientia 1959, 15, 216-217. [3.58] D. Fontanilla, M. Johannessen, A.R. Hajipour, N.V. Cozzi, M.B. Jackson, A.E. Ruoho. The hallucinogen N,N-dimethyltryptamine is an endogenous Sigma-1 receptor regulator. Science 2009, 323, 934-937. [3.59] T-P. Su, T. Hayashi, D.P. Vaupel. When the endogenous hallucinogenic trace amine N,N-dimethyltryptamine meets the Sigma-1 receptor. Science Signaling 2009, 2, 1-4. [3.60] L.M. Hryhorczuk, J.M. Rainey, C. Frohman, E. Novak. A new metabolic pathway for N,N-dimethyltryptamine. Biological Psychiatry 1986, 21, 84-93.

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Chapter 4. Methodology for Determining Major Constituents of Ayahuasca and Their Metabolites in Blood*

Author Names and Affiliations: Ethan H. McIlhennya, Jordi Ribab, Manel J. Barbanojb, Rick Strassmanc, and Steven A. Barkera* a

Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70806 USA [email protected]

b

Centre d'Investigació de Medicaments, Institut de Recerca, Servei de Farmacologia Clínica, Hospital de Sant Pau, Barcelona. Departament de Farmacologia i Terapèutica, Universitat Autònoma de Barcelona. Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM. [email protected] [email protected]

c

Department of Psychiatry, School of Medicine, University of New Mexico, Albuquerque, New Mexico 87131; Cottonwood Research Foundation, Taos, New Mexico 87571 USA [email protected]

________________________________________________________________________ *Reprinted with the permission of John Wiley and Sons and the Journal of Biomeidcal Chromatography 73

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4.1 Introduction Ayahuasca, also known as hoasca, yage, daime, or vegetal in the pharmacopeia of various South American groups, is a psychotropic plant tea that has a long cultural history of indigenous medical and religious use. The major components of ayahuasca are the hallucinogen N,Ndimethyltryptamine (DMT) and members of the harmala alkaloid family [4.1-4.6]. There is an increasing interest in potential medical applications of ayahuasca [4.7-4.8], including its antioxidant, antimutagenic and antigenotoxic activity [4.9]. In addition, there are suggestions of its putative psychotherapeutic and rehabilitative effects for conditions such as alcoholism, violence, suicidal behaviors, and severe depression, as well as other disorders [4.104.17]. Thus, a detailed examination and understanding of the biochemical parameters affected by ayahuasca is needed and will no doubt add to our understanding of this Amazonian medicine. Clinical research assessing the potential medicinal uses for ayahuasca will require information regarding the pharmacokinetics, metabolism, and clearance of ayahuasca’s major components. Thus, specific methods for the characterization and quantitation of the major constituents of ayahuasca and their metabolites in blood and urine are needed. In this regard, the authors have recently reported such a method for urine [4.18]. The present research provides a protocol for conducting such analyses in blood. Historically, a combination of two analytical techniques, one based on high performance liquid chromatography (HPLC) with ultraviolet (UV) and/or fluorescence detection [4.19-4.20] and another, using gas chromatography with nitrogen-phosphorus detection (GC-NPD) [4.193.20], have been used for the blood analysis of many of the constituents of ayahuasca following oral administration. The major components analyzed in these studies were N,Ndimethyltryptamine (DMT, 4.1..8 in Table 4.1; GC-NPD) and the harmala alkaloids: harmine (1, 74

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Table 4.1), harmaline (4.1.3), and tetrahydroharmine (THH, 4.1.5; HPLC-UV or fluoresence) [4.19-4.20], as well as harmol (4.1.2) and harmalol (4.1.4; HPLC-fluoresence; [4.20]). These methods have been applied to blood samples to examine the pharmacokinetics of these compounds [4.11, 4.19-4.21]. Such analyses have also been used to correlate blood levels of the major components with ayahuasca’s effects on subjective and cardiovascular variables as well on monoamine metabolite excretion [4.21]. Other than the obvious complexity created by this approach in using two different methods to obtain data from the same sample, samples analyzed by these methods also required separate isolation of the DMT from blood by liquid-liquid extraction [4.19-4.20], as well as preparation of the samples for HPLC analysis by solid phase extraction [4.20] or protein precipitation/dilution [4.19]. However, recent data [4.6] show that a larger number of compounds in ayahuasca, as well as several previously known and potential metabolites of both DMT and the harmala alkaloids [4.18], also need to be monitored in blood in such studies. Given the increasing interest in ayahuasca and the potential for future clinical research, it was determined that a need existed for newer methods of analysis, an expanded list of compounds of interest, and simpler analytical and analyte isolation procedures. We report here a single methodology for the direct analysis of the known alkaloid components of ayahuasca as well as several known and potential metabolites of DMT and the harmala alkaloids in blood. The method developed is based on a 96-well plate/protein precipitation/filtration of plasma samples and analysis by HPLC-ion trap-ion trap-mass spectrometry using heated electro-spray ionization to reduce matrix effects. In the present work the list of compounds examined in previous studies has been expanded to include indoleacetic acid (IAA, 4.1.15), DMT-N-oxide (DMT-NO, 4.1.10), 5-hydroxy- and 5-methoxy-DMT

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(OHDMT, 4.1.11, MeODMT, 4.1.12, potential components of ayahuasca itself), N,Ndimethylkynuramine (DMK, 4.1.14), N-methyltryptamine (NMT, 4.1.9), 2-methyl-tetrahydrobeta-carboline (2-MTHBC, 4.1.6) as well as a recently identified O-desmethyl-metabolite of THH, the major harmala component of ayahuasca, 7-hydroxy-THH (THHOH, 4.1.7; [4.22]). As a demonstration of its performance, the method described has also been applied to a small number of blood samples collected from individuals administered ayahuasca. Further, selected blood samples were treated with glucuronidase/sulfatase and analyzed by the same method to determine the presence of glucuronic acid or sulfate conjugates of ayahuasca’s major constituents and metabolites. 4.2 Experimental 4.2.1 Standards and reagents HPLC-grade methanol was purchased from Honeywell Burdick and Jackson (Morristown, New Jersey, USA). HPLC-grade water, high purity formic acid, and acetonitrile were purchased from J. T. Baker (Phillipsburg, NJ, USA). N-methyltryptamine (NMT), DMT, 5-hydroxy-DMT (5OH-DMT), 5-methoxy-DMT (5-MeO-DMT), harmine, harmaline hydrochloride dihydrate, harmol hydrochloride dihydrate, harmalol hydrochloride dihydrate, indoleacetic acid (IAA) and N,N-diethyltryptamine (DET, 4.1.13, Table 4.1, used as an internal standard), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetrahydroharmine was purchased from THC Pharm (Frankfurt am Main, Germany). DMT-NO and 2-MTHBC were prepared as previously described [4.23]. N,N-Dimethylkynuramine (DMK) was a gift from Dr. Laurent Micouin, Laboratoire de Chimie Thérapeutique, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France. An authentic reference standard of 7-hydroxy-tetrahydroharmine (THHOH) was a gift from THC Pharm (Frankfurt am Main, Germany). 76

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Harmine: R1 = CH3O, 4.1.1 Harmol: R1 = OH, 4.1.2

Harmaline: R1 = CH3O, 4.1.3 Harmalol: R1 = OH, 4.1.4

THH: R1 = H; R2 = CH3; R3 = CH3O, 4.1.5 2-MTHBC: R1 = CH3; R2 = R3 = H, 4.1.6 THHOH: R1 = H; R2 = CH3; R3 = OH 4.1.7

DMT: R1 = R3 = CH3; R2 = Ø; R4 = H, 4.1.8 NMT: R1 = CH3; R2 = Ø; R3 = H; R4 = H, 4.1.9 DMT-NO: R1 = R3 = CH3; R2 = O+; R4 = H, 4.1.10 5-OH-DMT:

R1 = R3 = CH3; R2 = Ø; R4 = OH, 4.1.11

5-MeO-DMT: R1 = R3 = CH3; R2 = Ø; R4 = CH3O, 4.1.12 DET: R1 = R3 = C2H5; R2 = Ø; R4 = H, 4.1.13

DMK: 4.1.14

IAA: 4.1.15

Figure 4.1. Structures of the compounds examined in blood samples. 77

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Stock standard solutions (1 mg/ml) of the 14 selected compounds and of the internal standard (DET) were prepared individually in methanol in 10 ml amber glass vials with Teflonlined screw-cap closures and stored in a freezer at -20o C. Working mixed standards were prepared in methanol at selected concentrations (0, 1, 2.5, 5, 10, 25, 50, 100, 250 500 ng/ml) by serial dilution and pipetted into 5 ml conical tubes. The methanol was removed by gentle evaporation over dry nitrogen and the residues were dissolved in amounts of 97% H2O/0.1% formic acid: 3% acetonitrile/0.1% formic acid mobile-phase (MP) solution corresponding to the appropriate sample concentrations desired. 4.2.2 Freeze Dried Ayahuasca Administration and Plasma Collection Ayahuasca was prepared as an encapsulated lyophilizate obtained from a previously analyzed, freeze-dried and homogenized 10 L batch. One gram of freeze-dried material contained 8.33 mg DMT, 14.13 mg harmine, 0.96 mg harmaline, and 11.36 mg THH. A dose of 1.0 mg DMT/kg body weight was chosen based on previous ayahuasca dosing studies [4.8, 4.21, 4.24, 4.25]. Subjects were 3 healthy male volunteers with previous experience with psychedelic drugs. The study was approved by the local ethics committee (Hospital de Sant Pau, Barcelona) and the Spanish Ministry of Health. Signed informed consent was obtained from all participants. After oral administration of 1mg/kg DMT blood was collected in10 ml EDTA tubes and centrifuged at 2000 rpm for 10 min at 4 ºC and the resulting plasma immediately frozen at -20 ºC. The frozen plasma samples were stored at -80 ºC until analysis. The following time points relative to ayahuasca administration were collected for pilot analyses: Basal (pre-dose), 1.5 h, 4.5

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h and 10 h. Samples were shipped and received on dry ice and underwent a single freeze-thaw cycle prior to analysis. 4.2.3 Sample preparation Protein precipitation 96-well plates (Thermo Scientific, Waltham, MA, USA) were used to prepare the samples. Basal plasma was used for negative controls and for spiking to generate standard curves. Standard curve data points at 7 selected concentrations (0, 1, 2.5, 5, 10, 25, 50 ng/ml; IAA was examined at 0, 10, 25, 50 100, 250 and 500 ng/ml) specific to the expected concentration range for each compound (determined from preliminary analyses) were prepared in blank plasma and used to examine linearity of response, to collect data for the quantification of samples, and to determine method performance. Spiked MP which did not go through the protein precipitation process was also prepared and was used to examine matrix effects and to determine relative analyte recovery. Two sets of 200 μl mixed standards in MeOH were dried completely under nitrogen and brought up with 200 μl of MP or 200 μl of blank plasma. To each well of a 96-well protein precipitation plate were added 580 μl ACN, 20 μl of 1.0 μg/ml DET (20 ng; IS) in MeOH and either 200 μl spiked MP (MP curve), 200 μl MP (Blank with IS), 200 μl spiked plasma (recovery curve), 200 μl blank plasma (matrix effects curve), or 200 μl of ayahuasca administration plasma sample. The plate was shaken for 3 minutes using a microplate genie (Scientific Industries, Bohemia, NY, USA) and then placed in a -40 oC freezer for 30 minutes. The protein precipitation plate was then placed on a Porvair Sciences (Leatherhead, UK) vacuum manifold with an Agilent Technologies (Santa Clara, CA, USA) 96 deep-well receiver plate placed underneath to collect the filtrate. Minimum vacuum was applied for approximately 3 minutes.

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The filtrate was shaken for 3 minutes and transferred to a new 96-well protein precipitation plate which was again placed in a -40o C freezer for another 30 minutes. The protein precipitation plate was then placed back onto the vacuum manifold with minimum suction for 3 minutes and filtrate was collected in a new Agilent 96 deep well plate which was shaken for 3 minutes and then dried completely using a SPE Dry 96 Dual plate dyer (Argonaut, Mid Glamorgan, UK). All wells were brought up to 200 μl with MP. Thus, there was no relative dilution of the original sample. 4.2.4 Determination of Matrix Effects Matrix effects were examined using four sets of blank plasma that were processed through the protein precipitation protocol and then fortified with 0, 10, 25, 50, 100, 250, and 500 ng/ml of the 15 analytes, and samples of similarly spiked MP (n = 3) which were not processed through the protocol. The resulting samples were analyzed as described with 20 μl being injected for analysis. The data were examined to determine the slopes of the curves so generated. The slopes were compared (plasma samples to one another and plasma samples to MP samples) in order to determine the percent relative standard deviation (%RSD) of the slopes of extracted/fortified plasma samples with the slopes for the MP spiked samples for each compound. 4.2.5 Enzyme Hydrolysis A 1.0 M sodium acetate buffer at pH 5.0 was prepared by dissolving 82 g of anhydrous sodium acetate with 967 mL HPLC water and 33 ml of glacial acetic acid. Glucuronidase/ sulfatase buffer was prepared by dissolving 1 bottle (2 million units) of βglucuronidase/sulfatase from limpets (Patella vulgata) Type L-II (Sigma-Aldrich, St. Louis, MO, USA) in 400 ml of distilled water. Acetate buffer (1200 ml) was then mixed with 400 ml glucuronidase/sulfatase 80

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buffer. Glucuronidase/sulfatase /acetate buffer mix (120 μl) was then added to each tube containing (100 μl) of individual plasma as well as 10 μl of 1.0 μg/ml DET in MP. Standard curves were similarly treated and prepared using 90 μl of spiked blank plasma. The samples, blanks, and standards were vortexed and incubated for 1 hour in a 65 o C shaking water bath, then allowed to cool to room temperature. Samples were then treated as described above for protein precipitation. The dilution caused by the hydrolysis process was accounted for by using 680 μl ACN plus 320 μl of hydrolysis mixture and bringing the final volume up to only 100 μl MP (no dilution of original sample). 4.2.6 LC-HESI-MS/MS analysis The LC/MS/MS analyses were based, in part, on direct methods of analysis we have previously described for ayahuasca itself and for ayahuasca’s major constituents [4.16], as well as for their metabolites in urine [4.18], with modifications. In the present study the compounds IAA and THHOH have been added to the analyses (as compared to [4.18]) and the internal standard used in the previous studies was changed from a deuterated 5-methoxy-DMT to DET. Furthermore, the LC mobile phase/separation system and the mass spectrometer were changed as was the sample preparation step. Thus, analyses were conducted using a Thermo Open Autosampler and a Thermo Accela pumping system interfaced to a Thermo Velos linear ion trap-ion trap system equipped with a heated electrospray ionization (HESI) probe and operated in the positive ion mode. Chromatographic separation was achieved on a 1.8 μm 4.6 x 50 mm (i.d.) 600 bar Agilent ZORBAX Eclipse Plus C18 rapid resolution HT threaded column with an Alltech Direct-Connect Column 2 μm pre-filter (Deerfield, IL, USA) using gradient elution. The following gradient

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system was used as the mobile phase: A (0.1% formic acid in H20) and a mobile phase B (0.1% formic acid in acetonitrile) delivered at a constant flow rate of 0.3 ml/min throughout the analysis; A:B 97:3 (0 min)- 50:50 (14 min)- 2:98 (16 min)- 2:98 (18 min)- 97:3 (21 min)- 97:3 (25 min), allowing for re-equilibration. The MS/MS analysis was performed using selected reaction monitoring (SRM) of the protonated molecular ions for the analytes and scanning the target ions in seven (7) different segments (Table 4.1). The heated ESI source temperature was 300o C, sheath gas pressure was 25 psi, the auxillary gas pressure was 7 psi, the spray voltage (kV) was 5, the capillary temperature was 275o C and the S-lens RF level was 35%. The collision pressure was 1.5 psi of high purity argon. Data for the molecular ions and energies utilized to generate diagnostic fragment ions are shown in Table 4.1. Detection data were collected and integration of chromatographic peaks was performed by Xcalibur 2.0.7 Thermo Fisher Scientific (Waltham, MA, USA) LCquan 2.5.6 QF 30115 software. Identification of the compounds was based on the presence of the molecular ion at the correct retention time (+/- 1%), the presence of at least two transition ions, and the correct ratio of these ions to one another (+/- 25% relative).

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Table 4.1: Mass spectrometric parameters for the analytes and internal standard (IS) (CE = collision energy in volts, v; MW= molecular weight; Product ion 1 was used for quantitation in every case). (Segment): Runtime in minutes (1): 5.35 (2):0.50 (3): 0.90 (4): 1.60 (5): 0.90 (6): 1.80 (7): 13.95 Analytes

Scan Segme nt

MW

[M + H]+

5-OH-DMT

1

204

205

THHOH

2

202

203

DMK

3

192

193

HARMALOL

4

200

201

NMT

4

174

175

DMT

4

188

189

5-MeO-DMT

4&5

218

219

2-MTHBC

4&5

186

187

DMT-NO

5&6

204

205

5

216

217

DET (IS)

5&6

216

217

HARMOL

4&6

198

199

HARMALINE

6

214

215

HARMINE

6

212

213

IAA

7

175

176

THH

Product Ion 1 (Scan Range) 160.0 (159-161) 174.0 (173-175) 58.0 (57-59) 160.0 (159-161) 144.0 (143-145) 58.0 (57-59) 174.0 (173-175) 144.0 (143-145) 144.0 (143-145)

CE 1 (v)

188.0 (187-189) 86.0 (85-87) 171.0 (170-172) 174.0 (173-175) 198.0 (197-199) 130 (129-131)

25

83

30 30 30 35 25 38 25 25 42

25 40 35 35 34

Product Ion 2 (Scan Range) 134.0 (133-135) 186.0 (185-187) 148.0 (147-149) 184.0 (183-185) 132 (131-133) 144.0 (143-145) 148.0 (147-149) 158.0 (157-159) 160.0 (159-161)

CE 2 (v)

200.0 (199-201) 144.0 (143-145) 157.0 (156-158) 200.0 (199-201) 213.0 (212-215) 158.0 (157-159)

25

Product Ion 3 (Scan Range)

CE 3 (v)

160.0 (159-161)

30

68.0 (67-69)

35

174.0 (173-175) 74.0 (73-75) 181.0 (180-182) 215.0 (214-216) 185.0 (184-186)

25

30 30 30 35 25 38 25 25 42

25 40 35 35 34

25 40 35 35

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4.2.7 Calculations The concentration of compounds in blood samples was determined from the ratio of the peak area of the target analyte to that of the internal standard (DET), by reference to calibration curves prepared by spiking blank plasma samples with each of the 14 substances plus a consistent amount of internal standard. The transition ions used for quantitation are shown in Table 4.1. If the concentration observed for the actual administration samples tested was outside the measured range, the samples were re-analyzed after further dilution with mobile phase and the original concentration calculated by extrapolation. Values determined from repeated analyses of n aliquots of samples or standards were expressed as their arithmetic mean. For the standards the percent relative standard deviation (%RSD) was calculated, as was the method bias, as a function of concentration. Standard deviations were calculated for n determinations of samples as noted. Inter- and intra-assay %RSDs were also determined. Limits of detection (LOD; concentration response greater than 3 times baseline noise) and limits of quantification (LOQ: the lowest concentration having a proven %RSD of less than 15%) were also determined for each compound. To examine matrix effects, slopes were calculated for standard curves generated from fortifying 4 pre-extracted plasma samples and 4 water samples as described. The slope of individual plasma samples were compared to each other and the %RSD was determined. The mean of these slopes was also compared to the mean of spiked water curves and the %RSD was calculated for each compound.

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4.3 Results and Discussion 4.3.1 Method Performance Each of the 15 compounds gave high yields of molecular ions (M+H)+ using the mobile phase and heated electrospray ionization parameters described. However, for many of the compounds the yield of a third product ion was less than 5% of the base peak and was not sufficient to be included in the identification criteria (Table 4.1). However, the criteria for identification were sufficiently rigorous [correct retention time, molecular ion plus two transitions, and an acceptable ratio of ions (+/- 25% absolute)] to provide high confidence in the data. Using the LC/HESI/MS/MS multi-component method described, almost all of the 15 compounds examined were separated temporally (Figure 4.2A). All of the compounds were, nonetheless, completely separated by mass. A thorough examination of ion contribution between each of the compounds and the masses monitored indicated that, even at the highest concentrations, no measurable interference (“cross-talk”) occurred. The LC method gave excellent peak shapes and consistency of retention time for the 15 compounds despite their rather diverse chemical character. Furthermore, no potentially interfering naturally occurring substances from plasma with the same masses and retention times were observed, in either the method blanks or basal samples collected from subjects. Similarly, enzyme treatment did not produce any new compounds that interfered with the analysis. Although this is a limited examination, it appears that the necessary specificity for the assay can be met, especially when additional parameters of ion fragmentation and ratios are also applied. A representative chromatogram for 50 ng/ml standards spiked in blank plasma is shown in Figure 4.2A, and a

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representative chromatogram for a blank plasma sample containing only the internal standard (DET) is shown in Figure 4.2B. A representative chromatogram from the analysis of a volunteer’s sample is shown in Figure 4.2C. Data for the retention time of each compound and their reproducibility are presented in Table 4.2. The calibration curves for each compound were linear over the range of concentrations examined (1-50 ng/ml for all compounds except IAA which was 10-500 ng/ml). The linear regression equations, coefficients of correlation, and the performance parameters for the method (% bias, %RSD, LOD, LOQ, intra- and inter-assay variation) for the 14 compounds using DET as the internal standard are also presented in Table 4.2. Calculations of method bias and %RSDs show that performing the minimal sample manipulations required in conducting protein precipitation/filtration result in acceptable accuracy and precision for quantitation, as well as the appropriateness of DET as the internal standard for all of the compounds tested. The modest inter- and intra-assay variations observed also reflect the method’s reliability and reproducibility for analysis of human blood samples after ayahuasca administration. Similarly, the limits of detection observed are more than adequate. Proven limits of quantitation (% bias less than 15%) at 1.0 ng/ml were met for all of the 14 analytes.

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Figure 4.2A. Representative chromatogram of reference standards spiked into blank plasma illustrating retention times and the molecular ion and product ion monitored (50 ng/ml of each; RT= retention time in minutes). 87

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Figure 4.2B. Representative chromatogram of a blank plasma sample (Basal) obtained preayahuasca administration. 88

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Figure 4.2C. Representative chromatogram from a plasma sample obtained 1.5 hours after ayahuasca administration. 89

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Table 4.2 Method Perform Parameters

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Table 4.2 Continued

Absolute recovery of the individual analytes was also examined (Table 4.2) and ranged from a mean of 59-102% (n = 8) for these 15 chemically diverse compounds compared to responses of equal concentrations spiked into MP and analyzed directly. Much of the loss of analytes appears to occur during the protein precipitation/filtration steps since the remaining procedures involve little more than pipetting, diluting, or drying without transfers. Because of the natural occurrence of IAA in all plasma samples, an absolute recovery could not be accurately 91

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calculated but its response was observed to be linear with concentration (Table 4.2). Correction for endogenous IAA suggests that its recovery is greater than 90%. The method described has distinct advantages over previous methods used for determination of ayahuasca’s major components in blood in that a greater number of relevant compounds can be monitored, only a single analysis is required, and sample manipulation is minimal. 4.3.2 Matrix Effects Matrix effects were assessed by comparing the linear regression line slopes of standards spiked into MP over a range of concentrations versus standards similarly spiked into 4 blank plasma samples after being processed through the protein precipitation protocol (n = 4 for each) and by comparing the slopes for the compounds spiked into extracted plasmas from individuals to each other [4.26, 4.27]. Table 4.3 shows the %RSD for the slopes of standards spiked into preextracted plasma and water. Comparing the slopes of the 4 plasmas to each other, a %RSD of 15% or less was observed for all compounds. This suggests that little variability will be observed in conducting patient-to-patient plasma analyses due to matrix effects and that standard curves derived from spiking blank plasma will likewise provide appropriate results. Comparing the slopes for the compounds spiked into extracted plasma to the slopes for the same compounds spiked into water, a %RSD of less than 15% was also obtained for all compounds except for DMK. This suggests that some matrix effects occur for this compound but, in terms of attaining accurate quantitation, they may be overcome by fortification into blank plasma. There was little variability or deviation from linearity for standards spiked in water or in different plasma matrices. Reference standards spiked into extracted plasma showed no deviation from a linear

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response, and the accuracy of the results was readily reproduced over a broad range of concentrations (Table 4.3). Similarly, shifts in retention times were not evident, regardless of the relative concentrations of the standards (Table 4.1). Thus, the use of a HESI probe, which has been shown to greatly diminish matrix effects [4.26-4.29], as well as the overall methodology, provides an accurate, sensitive and specific analysis, apparently unaffected by co-eluting or other interferences. Table 4.3. Matrix effects. The slopes of the individual plasma samples, pre-extracted and fortified with analytes, were compared to each other and to fortified mobile phase (MP).

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Table 4.4. Mean (n =6) concentrations (ng/ml) of ayahuasca components and metabolites detected in plasma from three (3) volunteers.

4.3.3 Analysis of Administration Samples Representative chromatograms for the detection and quantitation of the ayahuasca alkaloids and metabolites present in plasma samples collected before and after administration are presented in Figures 4.2(B and C), respectively. Tabulation of the concentrations of these compounds in samples from three individuals is shown in Table 4.4. Enzyme treatment of a selected volunteer’s samples did not show any significant increase in any of the major alkaloids or their metabolites (data not shown). Considering that conjugates have been observed in urine [4.22], their absence in plasma suggests that they are rapidly excreted. In the three individuals’ samples examined, DMT concentrations were observed to be lower at all time points than the major DMT metabolite recently identified in human urine [4.18], DMT-NO. This metabolite was observed to be present in the plasma at 3-to-4 times the concentration of DMT itself, peaking at 1.5 hrs after ayahuasca administration at approximately 45 ng/ml. The formation of the N-oxide appears to occur rapidly after ayahuasca administration. Enzymes responsible for this conversion may be N-oxidases in blood as well as liver or kidney CYPs (cytochrome P450 superfamily) [4.23, 4.30-4.34]. Others have reported DMT-NO to be a major metabolite of DMT in rat plasma and urine [4.31-4.34]. The present study is the first report of its presence in human blood following ayahuasca or DMT administration. In addition, 94

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inhibition of MAO-A in rats, such as occurs with the harmala alkaloids in ayahuasca [4.35], shifts the normal metabolism of DMT to IAA to the formation of the N-oxide in vitro and in vivo [4.31-4.34]. It may be assumed that a similar mechanism is at work following ayahuasca administration to humans. Nonetheless, rather modest levels of DMT continue to be observed and are consistent with previous studies of ayahuasca pharmacokinetics [4.11, 4.19-4.21]. For the major harmala components, THH was observed to peak in the 4.5 hr samples with concentrations greater than 55 ng/ml. THH is the major harmala component of ayahuasca and it is also the major harmala excretion product in urine in man [4.6, 4.18]. Harmalol and harmol as well as THHOH, harmaline, harmine, and 2-MTHBC were also detected in most samples. 5-OHDMT, a potential component of some ayahuasca preparations, and NMT, a known demethylation metabolite of DMT, were randomly detected and no 5-MeO-DMT, another potential component of ayahuasca, was observed. The compound DMK, a DMT metabolite reported to be formed in human blood in vitro [4.36], was not detected. This was also the case in our examination of urine samples collected from humans administered ayahuasca [4.18], and suggests that, while this compound was identified in in vitro studies, apparently it is not a metabolite of DMT in vivo in man. Despite the MAO inhibiting effect of the harmala alkaloids [4.25, 4.35, 4.37], significant increases in the plasma levels of IAA were observed following ayahuasca administration. The reason for this observation is, at present, unclear. However, the data suggest that, to the degree that MAO inhibition and a resultant shift to N-oxide formation occur, a significant quantity of the administered DMT may still be converted to IAA, apparently via incomplete inhibition of MAOA.

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4.4 Conclusions The present method expands the list of compounds capable of being monitored in blood following ayahuasca administration in humans while providing a simplified approach to their analysis. The characteristics of the method suggest that its sensitivity, specificity, and reproducibility are adequate for future clinical research with ayahuasca. The results also show for the first time that the major DMT urinary metabolite, DMT-N-oxide, is also a major circulating product in the blood following ayahuasca administration. Thus, the method and data provide the most complete profile of DMT, harmala alkaloids, and their respective metabolite concentrations in the blood following ayahuasca administration to date.

4.5 References [4.1]

L. Rivier and J.E. Lindgren. “Ayahuasca,” the South American hallucinogenic drink: an ethnobotanical and chemical investigation. Economic Botany 1972, 26, 101-129.

[4.2]

J.C. Callaway. Various alkaloid profiles in decoctions of Banisteriopsis caapi. Journal of Psychoactive Drugs 2005, 37, 1-5.

[4.3]

J.C. Callaway, G.S. Brito and E.S Neves. Phytochemical analyses of Banisteriopsis caapi and Psychotria viridis. Journal of Psychoactive Drugs 2005, 37, 145-150.

[4.4]

D.J. McKenna and G.H.N. Towers. Biochemistry and pharmacology of tryptamines and beta-carbolines: A minireview. Journal of Psychoactive Drugs 1984, 16, 347-358.

[4.5]

A.P.S. Pires, C.D.R. De Oliveira, S. Moura , F.A. Dorr, W.A.E. Silva and M. Yonamine. Gas chromatographic analysis of dimethyltryptamine and beta-carboline alkaloids in ayahuasca, an Amazonian psychoactive plant beverage. Phytochemical Analysis 2009, 20, 149-153.

[4.6]

E.H. McIlhenny, K.E. Pipkin, L.J. Standish, H.A. Wechkin, R.J. Strassman and S.A. Barker. Direct analysis of psychoactive tryptamine and harmala alkaloids in the Amazonian botanical medicine ayahuasca by liquid chromatography-electrospray ionization-tandem mass spectrometry. Journal of Chromatography A 2009, 1216, 89608968.

[4.7]

D.J. McKenna. Clinical investigations of the therapeutic potential of ayahuasca: rationale and regulatory challenges. Pharmacology and Therapeutics 2004, 102, 111-129. 96

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[4.8]

J. Riba and M.J. Barbanoj. Bringing ayahuasca to the clinical research laboratory. Journal of Psychoactive Drugs 2005, 37, 219-230.

[4.9]

D.J. Moura, M.F Richter, J.B. Boeira, J.A.P. Henriques and J. Saffi. Antioxidant properties of β-carboline alkaloids are related to their antimutagenic and antigenotoxic activities. Mutagenesis 2007, 22, 293-302.

[4.10] W. Andritzky. Sociopsychotherapeutic functions of ayahuasca healing in Amazonia. Journal of Psychoactive Drugs 1989, 21, 77-89. [4.11] J.C. Callaway, D.J. McKenna, C.S. Grob, G.S. Brito, L.P. Raymon, R.E. Poland, E.N. Andrade, E.O. Andrade, D.C. Mash. Pharmacokinetics of Hoasca alkaloids in healthy humans. Journal of Ethnopharmacology 1999, 65, 243-256. [4.12] J. Mabit, R. Giove, J. Vega. In M. Winkelman and W. Andritzky (Eds.), Yearbook of Cross-Cultural Medicine and Psychotherapy 1996, VMB Press, Berlin 257. [4.13] J. Tiihonen, J.T. Kiukka, K.A. Bergstorm, J. Karhu, H. Viinamaki, J. Lehtonen, T. Hallikainen, J. Yang, P. Hakola. Single-photon emission tomography imaging of monoamine transporters in impulsive violent behavior. European Journal of Nuclear Medicine 1997, 24, 1253-1260. [4.14] T. Hallikainen, H.M. Saito, J. Lachman, T. Volavka, O.P. Pohjalainen, J. Ryynanen, J. Kauhanenm, E. Syvalahti, J. Hietala, J. Tiihonen. Association between low activity serotonin transporter promoter genotype and early onset alcoholism with habitual impulsive violent behavior. Molecular Psychiatry 1999, 4, 385-388. [4.15] T. Mantere, E. Tupala, H. Hall, T. Sarkioja, P. Rasanen, K. Bergstorm, J.C. Callaway, J. Tiihonen. Serotonin transporter distribution and density in the cerebral cortex of alcoholic and nonalcoholic comparison subjects: a whole-hemisphere autoradiography study. American Journal of Psychiatry 2002, 159, 599-606. [4.16] R.G. Santos, J. Landeira-Fernandez, R.J. Strassman, V. Motta, A.P.M Cruz. Effects of ayhuasca on psychometric measures of anxiety, panic-like and hopelessness in Santo Daime members. Journal of Ethnopharmacology 2007, 112, 507-513. [4.17] J.M. Fábregas, D. González, S. Fondevila, M. Cutchet, X. Fernández, P.C. Barbosa, M.Á. Alcázar-Córcoles, M.J. Barbanoj, J. Riba, J.C. Bouso. Assessment of addiction severity among ritual users of ayahuasca. Drug and Alcohol Dependence 2010, 111, 257-261. [4.18] E.H. McIlhenny, J. Riba, M.J. Barbanoj, R. Strassman, S.A. Barker. Methodology for and the determination of the major constituents and metabolites of the Amazonian botanical medicine ayahuasca in human urine. Biomedical Chromatography 2010, (wileyonlinelibrary.com) DOI 10.1002/bmc.1551. [4.19] J.C. Callaway, L.P. Raymon, W.L. Hearn, D.J. McKenna, C.S. Grob, G.S. Brito, D.C. Mash. Quantitation of N,N-dimethyltryptamine and harmala alkaloids in human plasma after oral dosing with ayahuasca. Journal of Analytical Toxicology 1996, 20, 492-497.

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[4.20] M. Yritia, J. Riba, J. Ortuno, A. Ramirez, A. Castillo, Y. Alfaro, R. De La Torre, M.J. Barbanoj. Determination of N,N-dimethyltryptamine and beta carboline alkaloids in human plasma following oral administration of Ayahuasca. Journal of Chromatography B 2002, 779, 271-281. [4.21] J. Riba, M. Valle, G. Urbano, M. Yritia, A. Morte, M.J. Barbanoj. Human pharmacology of ayahuasca: Subjective and cardiovascular effects, monoamine metabolite excretion, and pharmacokinetics. Journal of Pharmacology and Experimental Therapeutics 2003, 306, 73-83. [4.22] J. Riba, E.H. McIlhenny, M. Valle, M. Barbanoj, S.A.Barker. Metabolism and disposition of N,N-dimethyltryptamine and harmala alkaloids after oral administration of ayahuasca. Drug Testing and Analysis 2012, DOI 10.1002/dta.1344 [4.23] S.A Barker, J.A. Monti, S.T Christian. Metabolism of the hallucinogen N,Ndimethyltryptamine in rat brain homogenates. Biochemical Pharmacology 1980, 29, 1049-1057. [4.24] J. Riba, A. Rodríguez-Fornells, G. Urbano, A. Morte, R. Antonijoan, M. Montero, J.C. Callaway, M.J. Barbanoj. Subjective effects and tolerability of the South American psychoactive beverage Ayahuasca in healthy volunteers. Psychopharmacology(Berlin) 2001, 154, 85-95. [4.25] J. Riba. Human Pharmacology of Ayahuasca. Doctoral Thesis, Universitat Autonoma de Barcelona. 2003, http://www.tdx.cesca.es/TDX-0701104-165104/ [4.26] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Analytical Chemistry 2003, 75, 3019-3030. [4.27] B.K. Matuszewski. Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis. Journal of Chromatography B 2006, 830, 293-300. [4.28] P.J. Taylor. Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometery. Clinical Biochemistry 2005, 38, 328-334. [4.29] E. Chambers, D.M Wagrowski-Diehl, Z. Lu and J.R. Mazzeo. Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses. Journal of Chromatography B 2007, 852, 22-34. [4.30] S.A. Barker, J.A. Monti, S.T. Christian. N,N-Dimethyltryptamine: an endogenous hallucinogen. International Review of Neurobiology 1981, 22, 83-110. [4.31] B.R. Sitaram, W.R. McLeod. Observations on the metabolism of the psychotomimetic indolealkylamines: Implications for future clinical studies. Biological Psychiatry 1990, 28, 841-848.

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[4.32] B.R. Sitaram, L. Lockett, G.L. Blackman, W.R. McLeod. Urinary excretion of 5methoxy-N,N-dimethyltryptamine, N,N-dimethyltryptamine and their N-oxides in the rat. Biochemical Pharmacology 1987a, 36, 2235-237. [4.33] B.R. Sitaram, L. Lockett, R. Talomsin, G.L. Blackman, W.R. McLeod. In vivo metabolism of 5-methoxy-N,N-dimethyltryptamine and N,N-dimethyltryptamine in the rat. Biochemical Pharmacology 1987b, 36, 1509-1512. [4.34] B.R. Sitaram, R. Talomsin, G.L. Blackman, W.R McLeod. Study of metabolism of psychotomimetic indolealkylamines by rat tissue extracts using liquid chromatography. Biochemical Pharmacology 1987c, 36, 1503-1508. [4.35] D.J. McKenna, G.H.N. Towers, F. Abbott. Monoamine oxidase inhibitors in South American hallucinogenic plants: tryptamine and beta-carboline constituents of ayahuasca. Journal of Ethnopharmacology 1984, 10, 195-223. [4.36] L.M. Hryhorczuk, J.M. Rainey, C. Frohman, E. Novak. A new metabolic pathway for N,N-dimethyltryptamine. Biological Psychiatry 1986, 21, 84-93. [4.37] H. Kim, S.O. Sablin, R.R Ramsay. Inhibition of monoamine oxidase A by beta-carboline derivatives. Archives of Biochemistry and Biophysics 1997, 337, 137-142.

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Chapter 5. Summary and Conclusions 5.1 Summary and Conclusions In the research presented in this dissertation we have established, expanded, and modified an analytical method which we applied to study various ayahuasca samples and urine or blood samples before and following administration of ayahuasca to humans. This method has proved useful in the study of ayahuasca in human and ethnobotanical research, and examinations of ayahuasca preparations or human samples. The characteristics of the methods suggest their sensitivity, specificity and reproducibility are adequate for use in further toxicological and clinical research on ayahuasca as well as functioning as a potential assay to screen biological samples for endogenous hallucinogens. In chapter 2 we presented a manuscript describing the development of a liquid chromatography–electrospray ionization-tandem mass spectrometry procedure capable of the simultaneous quantitation of 11 of the major alkaloid components of ayahuasca, including several known and potential metabolites. This method affords rapid detection of alkaloids by a simple dilution assay requiring no extraction procedures while demonstrating an extremely high degree of specificity for the compounds in question, as well as lower limits of detection and quantitation than reported by previous methods while also eliminating potential matrix effects. The major components present in ayahuasca were identified as THH and harmine, followed by DMT and harmaline with quantities appearing very similar to those reported by others. The samples were also examined for the presence of DMT-NO which, to our knowledge, represented the first such effort to assay for this compound in ayahuasca preparations. Although a major DMT metabolite in mammals, it appears to be absent from the preparations of ayahuasca examined. 100

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After establishing the method we applied the assay to 11 ayahuasca samples collected by Dr. Leanna Standish in either North or South America as presented in the manuscript contained in Appendix A. DMT, harmine, harmol and tetrahydroharmine concentrations among ayahuasca teas made and used in healing ceremonies in South or North America were remarkably similar. Concentrations of two of the three minor beta-carbolines (harmaline and harmalol) were statistically different. North American tea samples were higher in harmaline and harmolol suggesting that the tea formula made and used in North America may include a higher ratio of B. caapi vine relative to P. viridis leaves or possibly a different strain of B. caapi. Seasonal, growth and or methodological practices could also account for some differences. We were able expand the method to include a number of potential metabolites and modify the preparation protocol in order to make the method suitable to analyze urine samples from participants before and after they had consumed a freeze dried form of ayahuasca thanks to the help of our collaborator Dr. Jordi Riba whose lab collected the samples. This manuscript was presented as Chapter 4: the overall major metabolite observed was DMT-NO, peaking at 4-8 h after ayahuasca administration with concentrations of approximately 11 µg/ml representing the first report of DMT-NO as a metabolite of DMT in the urine of humans and suggesting that Noxidation may represent a major metabolic route for DMT clearance in humans, particularly if MAO becomes inhibited, such as occurs with ayahuasca administration. DMT-NO could also represent a major in vivo metabolite of DMT and thus, may serve as a better marker for endogenous DMT production and metabolism in mammals. THH represented the major component in the 8-24 h samples, with concentrations greater than 5 µg/ml. However, following enzyme hydrolysis harmol and harmalol were found to be at far higher concentrations compared to THH because enzyme hydrolysis with glucuronidase / sulfatase appeared to produce a 40-60 101

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fold increase in the amount of harmol, making harmol the most abundant product excreted in urine. DMK did not appear to be consistently excreted in urine, even with MAO inhibition. However; it remains possible that this compound undergoes metabolism to another, as yet undetermined, compound prior to excretion. Next we modified and expanded the method to make it appropriate for the analysis of blood plasma samples following freeze-dried ayahuasca administration as presented in Chapter 4. The method expanded the list of compounds capable of being monitored in blood following ayahuasca administration in humans while providing a simplified approach to their analysis. The results also demonstrated for the first time that the major DMT urinary metabolite, DMT-NO, was also a major circulating product in the blood following ayahuasca administration. DMT-NO was observed to be present in the plasma at 3-to-4 times the concentration of DMT itself, peaking at 1.5 h after ayahuasca administration at approximately 45 ng/ml. THH was observed to peak in the 4.5 h samples with concentrations greater than 55 ng/ml. THH thus represented the major harmala component of ayahuasca and also the major harmala excretion product in urine and plasma in man. Despite the MAO inhibiting effect of the harmala alkaloids, significant increases in the plasma levels of IAA were observed following ayahuasca administration. The reason for this observation remains unclear. However, the data suggest that a significant quantity of the administered DMT may still be converted to IAA. The method applied and the resulting data provide the most complete profile of DMT, harmala alkaloids, and their respective metabolite concentrations in the blood following ayahuasca administration to date. In a recent study we applied our established blood method and collaborated with Rafael Santos and Dr. Jordi Riba’s lab on a study entitled ‘Autonomic, Neuroendocrine, and Immunological Effects of Ayahuasca: A Comparative Study With D-Amphetamine’ which was 102

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published in December 2011 in the Journal of Clinical Psychopharmacology. This study was not included here as it constituted part of the dissertation of one of Dr. Riba’s students (Santos). Nonetheless, we found that ayahuasca led to measurable DMT plasma levels and distinct subjective and neurophysiological effects that were absent after amphetamine. Prolactin levels were significantly increased by ayahuasca but not by amphetamine, and cortisol was increased by both, with ayahuasca leading to the higher peak values. Natural killer cells were also increased by both. These findings indicate that ayahuasca has a moderate impact on the nervous system with a modulatory capacity on cell-mediated immunity [B.25]. If present, endogenous DMT and -carbolines could also potentially produce these effects. In a follow up to our preliminary urine study where we established the urine method in Appendix B, we presented a manuscript further characterizing the metabolism and disposition of DMT and harmala alkaloids after oral administration of ayahuasca. O-demethylation plus conjugation seems to represent an important but probably not the only degradation route for the harmala alkaloids in humans. Harmol and harmalol concentrations were 10-fold and 5-fold the amounts ingested with ayahuasca demonstrating, that the vast majority of harmol and harmalol recovered in urine after ayahuasca ingestion must necessarily be formed through the metabolic breakdown of harmine and harmaline. The recoveries of each harmala alkaloid plus its Odemethylated metabolite varied greatly between 9 and 65%. N-oxidation appears to represents a major degradation pathway of DMT in humans when administered together with -carbolines in ayahuasca suggesting the existence of an alternative metabolic route to biotransformation by MAO. Less than 1% of the administered DMT dose was recovered as the unmetabolized parent compound. The main DMT metabolite found in the urine was IAA. The second highest concentration metabolite detected was DMT-NO, with recoveries 103

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around 10%. DMT and metabolite excretion was maximal during the first third of the 24 h collection period. DMT-NO made up around 20% of the compounds measured. Recovery of DMT plus metabolites reached 68%. These findings support the notion that MAO plays a prominent role in the degradation of DMT. However, MAO-inhibition after ayahuasca appeared to be either incomplete or short-lived, as large amounts of IAA were already detected in the first 4 h collection interval. Partial inhibition of MAO by the harmalas in ayahuasca appeared to be sufficient to allow psychoactive effects. DMT-NO does not seem to function as an intermediate during the formation of IAA by MAO-A, but it does appear to represent the major metabolite of DMT in the absence of or after inhibition of mitochondrial MAO. MAO inhibition could consequently shift metabolism from oxidative deamination to N-oxidation as a compensatory metabolic mechanism. Future investigations could address the metabolism of oral DMT in humans without the presence of -carbolines, in order to assess the contribution of the different metabolic pathways for its degradation under physiological conditions. This would allow us to estimate the degree of metabolic compensation induced by the harmala alkaloids in ayahuasca. We have already collected and analyzed preliminary data for this study which compared urine samples from participants following orally administered or smoked DMT. Alternatively no studies have yet pretreated humans with MAO inhibitors alone and measured the parent compounds and their corresponding N-oxides. The advantage of such a study would be that the N-oxide, as opposed to the indoleacetic acid, retains the original structure of the parent molecule, permitting a cumulative association. Therefore, monitoring the N-oxide metabolites rather than the parent compounds alone in MAO-inhibited humans could provide a substantial advantage in detecting and quantitating endogenous psychedelic compounds. We suggest that MAO inhibition in

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humans could substantially enhance detection and quantitation of endogenous hallucinogenic compounds in the periphery, especially if the N-oxide metabolites are monitored. It would appear necessary that in order to sufficiently detect parent compounds sensitivity of assays must be improved to 1.0 pg/ml or less and include more frequent sampling and longer collection times given the possible intermittent presence of these compounds in the periphery, blood and urine. In this regard, much of our interest in ayahuasca pharmacology and metabolism was based on the need to further understand the possible role and needed methods of analysis for endogenous psychedelics and to provide a more definitive answer to the question, “Are ‘hallucinogenic’ tryptamines naturally present in human metabolism?” In Appendix C this question was addressed, and we concluded that the quantity of mass spectral evidence in the literature demonstrated that DMT and 5-OH-DMT do indeed appear endogenously and may be sufficiently measured in human body fluids although the tissue sources of these compounds in human remains unknown. The highest levels of INMT mRNA have been reported in adrenal gland and lung, although no large amount of INMT mRNA was detectable in brain [C.96, C.97]. The active transport of DMT across the blood-brain barrier [C.98] suggests that peripheral synthesis may still affect central function. A fluorescent antibody to INMT and confocal microscopy [C.99], have identified INMT in spinal cord, brain, retina, and pineal gland, and suggest the future possibility for mapping and characterizing the regulation of the endogenous psychedelic pathway. Future molecular biological approaches and advances in assay methodology could potentially help characterize the biochemistry and physiology of these compounds in humans. Data regarding psychodynamics, concentrations, circadian variation, metabolism and clearance as assessed by validated analytical methods applied to biological samples represent an accessible approach to more clearly determining potential roles in human

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psychophysiology. We propose that future studies assay CSF, blood and urine and monitor for DMT, 5-OH-DMT, 5-MeO-DMT and their corresponding N-oxides using validated mass spectrometric methodology. Pretreatment of study subjects with an MAO inhibitor could optimize results. The search for endogenous psychedelic tryptamines could also turn toward solid organs such as adrenal, brain, lung, pineal, retina, and other tissues in which INMT activity has been demonstrated. Mapping of INMT within certain cell types and locations could reveal its intracellular distribution and possible associations with various receptors. The creation of an INMT knockout mouse and characterization of the phenotype could also greatly aid in understanding the role of this enzyme and endogenous psychedelics. Why might organisms produce endogenous psychointegrative substances such as DMT and 5-MeO-DMT? The current research conducted leaves this question open to speculation and hypotheses, which abound. Could DMT represent a molecular interface to the experience of consciousness? Indeed, there are many hypotheses that have attempted to link our more mundane experiences of the world to the more extraordinary psychic states through quantum mechanical mechanisms and the possibilities of a multidimensional reality beyond normal perception. These quantum mechanical-to-cosmological hypotheses appear to be untestable at present. However, it may not necessarily remain this way. Perhaps further study of these molecules which have been claimed to afford the capability of perceived transport to “other worlds” might help answer some of these questions and expand our knowledge of the origins of religious and mystical states and other extraordinary states of consciousness. Perhaps these molecules function as dynamic regulators or integrated “tuners” of conscious, unconscious and preconscious perception,

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sensation, emotions, cognition, intuition, inspiration and intention. Perhaps they afford an awareness of different resolutions of perceived ontological existence.

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Appendix A. Cross-cultural variations in psychoactive alkaloid content in ayahuasca teas used in spiritual ceremonies

Leanna J. Standish, ND PhDa, Dawn E. Reardona, Bu Huang PhDa, Ethan McIlhenny, PhD,b and Steve A. Barker, PhDb a. Bastyr University Research Institute, 14500 Juanita Drive NE, Kenmore WA 98028 Leanna J. Standish ([email protected]); Dawn E. Reardon ([email protected]) Bu Huang ([email protected]). b. Louisiana State University, School of Veterinary Medicine, Baton Rouge LA 70803 Ethan Mclhenny [email protected]; Steven A . Barker ([email protected]).

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A.1 Introduction The purpose of this study was to compare the concentrations of psychoactive alkaloids present in ayahuasca teas used in healing ceremonies conducted in South and North America. Ayahuasca, an ethnobotanical medicine used by indigenous peoples of South America for thousands of years, is primarily derived from the synergistic interactions of two Amazonian plants, Psychotria viridis and Banisteriopsis caapi. The leaves of P. viridis contain the alkaloid N, N-dimethyltryptamine (DMT). The woody vine of B. caapi contains three main active indole alkaloids, beta-carboline alkaloids, harmine, tetrahydroharmine (THH) and harmaline [A.1]. Ayahuasca tea is a unique serotonin agonist with both short and long-term action that, with repeated use, modifies 5-HT receptor sites to increase serotonin availability. DMT contained in ayahuasca tea is a potent serotonin agonist at the 5-HT1A presynaptic and 5-HT2A post-synaptic membrane receptor sites [A.1, A.2] and possibly increases neocortical GABA levels [A.4]. DMT is believed to be primarily responsible for the psychoactive effects of ayahuasca. Orally administered DMT is metabolized by an enzyme in the gut and liver, monoamine oxidase A (MAO-A) before it can cross the blood-brain barrier. Inhibition of MAO by beta-carbolines present in ayahuasca tea protects DMT from oxidative deamination in gut and liver , thus enabling DMT to be active when administered orally [A.2, A.5]. Beta-carbolines are tricyclic indole alkaloids that are structurally related to tryptamines and have been identified in both plant and mammalian tissue [A.6]. The primary mechanism of action of two of the three principal beta carbolines found in ayahuasca, harmine and harmaline, is the reversible inhibition of MAO-A, which is responsible for breaking down serotonin, dopamine and norepinephrine [A.3]. The third beta carboline, tetrahydroharmine weakly inhibits the uptake of serotonin via serotonin transporter binding.

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We sought to measure the concentration of nine known active alkaloid constituents present in ayahuasca tea:

N,N-dimethyltryptamine (DMT), 5-OH-DMT (bufotenine)

and N-

methyltryptamine (precursor of DMT) from the P. viridis plant and six beta-carbolines (harmine, harmol, tetrahydroharmine, harmalol, harmaline and tetrahydroharmol (THHO)) from the B. Caapi vine [A.3, A.7]. A.2 Materials and methods A.2.1 Materials Eleven ayahuasca tea samples were collected from two regions, South America (N=6) and North America (N=5). Five ayahuasca ceremony leaders were approached to provide research samples of the tea used in a ceremony that had been conducted within the previous 24 hours. Each of the ayahuasqueros approached agreed and 10 ml of the tea was collected into sterile brown glass vials then immediately frozen at -80 degrees C until batch analysis. Eleven samples were provided to the researchers. Six of the tea samples were brewed by traditional ethnomedicine methods in South America (N=6) and five samples from non-denominational ayahuasca ceremonies brewed and conducted on the Hawaiian Islands or in the mainland U.S. South American samples differed from the North American samples by level of freshness of the tea decoction. South American samples were shipped from indigenous groups in the Amazon basin to the ayahuasquero ceremony leader and thus were not as fresh as those made for ceremonies conducted in Hawaii and on the U.S. mainland. A.2.2 Methods The concentration of nine known - constituents of each sample of ayahuasca tea were measured (mg/ml) using high performance liquid chromatography-electrospray ionization111

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tandem mass spectroscopy methods developed by our research team and performed at the Louisiana State University in a single-blind manner; see [A.7] for detailed methods. In brief, standard solutions of the 9 compounds analyzed were prepared using a 10 ppm stock standard in MeOH stored in -80oC. A 1.0 ml aliquot of this solution was dried under nitrogen and brought up with 1.0 ml 97/3 (water + 0.1% formic acid: acetonitrile +0.1% formic acid) mobile phase (MP). This 10 ppm stock solution in 97/3 MP was then serially diluted to 5ppm, 2.5ppm, 1ppm, 500ppb, 250ppb, 100ppb, 50ppb, 25ppb, 10ppb, 5ppb, 2.5ppb, and 1 ppb with MP. Extracts were prepared and analyzed in duplicates. For all the ayahuasca extracts 50 μl of each were diluted with 950 μl MP (=20x dilution) in a 96 well Thermo protein precipitation plate (PPT), then shaken for 3 minutes on an orbital shaker. The samples were then transferred to a second PPT using a vacuum manifold, shaken again for 3 minutes and then placed again on a vacuum manifold. Vacuum was applied for 5 minutes with the filtrate being collected in a 96 well deep-well plate from which the samples were injected for analysis. To 180 μl aliquots of the 9 compound mix-serial dilutions (5ppm-1ppb) were added 20ul of 1 ppm DET internal standard in a separate Thermo PPT plate. Similarly, 100 ul aliquots of the 20X diluted samples were mixed with 100 ul of 1 ppm DET and 800 ul MP in a Thermo PPT plate, shaken for 3 minutes, placed on the vacuum manifold and again suctioned with minimal vacuum for 5 minutes. Standards were treated in the same manner. The filtrate was collected into a new 96 well deep-well plate and 20 ul were injected for analysis. Analyses were conducted in duplicate and as described in [A.7] with two modifications; the mobile phase conditions for LC separation were slightly altered to afford improved separation of the target compound, as described in [A.8] and the instrument used was a Thermo Velos ion trap [A.8] as opposed to a Thermo TSQ [A.7].

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We evaluated whether concentration of the nine alkaloids present in the 11 ayahuasca tea samples was related to the concentration of any of the other eight active constituents measured in this study in two ways, first as a simple correlation then in a regression model. T-tests were used to detect statistically significant difference in alkaloid concentrations between ayahuasca made in South versus North America. Correlations and regression models were used to investigate the relationship among the nine alkaloids. Principle components analysis was used to detect the effect of geographic location (South versus North American) tea samples. A.3 Results A.3.1 DMT and 5-OH-DMT concentrations in ayahuasca teas DMT concentrations vary widely but there were no differences in mean DMT concentration between South and North American ayahuasca tea samples. Nine alkaloid constituents were measured in each of 11 tea samples. See Table A.1 for raw data, mean and standard deviation for the South American samples (N=6) and North American samples (N=5). DMT concentrations ranged from 0.26 – 0.78 mg/ml, a 3 fold difference in concentration across all 11 samples. However, there were no significant differences between the mean DMT concentration between North (0.49 mg/ml + 0.18) and South (0.45 mg/ml + 0.71) American teas. Higher levels of 5-OH-DMTwere found in South American samples (p= 0.048) compared to North American samples (Table A.2). DMT concentration among ayahuasca teas made and used in healing ceremonies in South America or North America are remarkably similar in concentrations of the main psychoactive alkaloids present in ayahuasca tea (DMT, harmine, harmol and tetrahydroharmine).

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Concentrations of two of the three minor beta-carbolines (harmaline and harmalol) were statistically different. North American tea samples were higher in harmaline and harmalol. Table A.1. Concentration of Major Alkaloid Constituents in Ayahuasca Teas Collected in South and North America Sample #

Source Raw Plant Materials

Location of Ceremony

Ethno Tradition

DMT

Harmine mg/ml

Harmaline mg/ml

THH mg/ml

Harmol mg/ml

Harmalol mg/ml

THHO mg/ml

NMT mg/ml

5-OHDMT mg/ml

South America AY025

Brazil

Brazil

Barquinha Church

0.260

1.302

0.049

0.765

1.084

0.000

0.020

0.008

0.000

AY003A3

Peru

US Mainland

Shipibo

0.646

1.406

0.064

0.656

1.159

0.004

0.000

0.008

0.000

AY009A3

Peru

US Mainland

Shipibo

0.371

2.512

0.223

2.070

1.657

0.030

0.127

0.025

0.005

AY010A3

Peru

US Mainland

Shipibo

0.277

2.028

0.172

1.872

1.467

0.021

0.116

0.021

0.003

AY011A3

Peru

US Mainland

Shipibo

0.413

2.004

0.157

1.475

1.427

0.026

0.077

0.018

0.006

AY024

Peru

US Mainland

Shipibo

0.708

2.423

0.178

1.580

1.598

0.023

0.080

0.017

0.004

Mean

0.446

1.946

0.141

1.403

1.399

0.018

0.070

0.016

0.003

Std. Deviation

0.189

0.503

0.069

0.577

0.232

0.013

0.051

0.007

0.002

N=6

North America AY001B3

Hawaii

Hawaii

Non-denom

0.317

1.706

0.281

1.877

1.293

0.002

0.081

0.022

0.001

AY005B3 AY026

Hawaii Hawaii

Hawaii US Mainland

Non-denom Non-denom

0.784 0.393

2.343 1.114

0.315 0.215

1.898 1.441

1.717 0.980

0.000 0.000

0.036 0.045

0.023 0.016

0.000 0.000

AY027

Hawaii

US Mainland

Non-denom

0.524

2.063

0.348

2.137

1.612

0.002

0.052

0.027

0.000

AY020

Hawaii

Hawaii

Non-denom

0.425

3.916

0.228

2.116

2.743

0.007

0.097

0.031

0.000

Mean

0.489

2.229

0.277

1.894

1.669

0.002

0.062

0.024

0.000

Std. Deviation

0.181

1.049

0.057

0.280

0.666

0.003

0.026

0.005

0.001

N=5

A.3.2 DMT, harmine, THH and harmol concentration Figure A.1 shows the mean concentration of the four alkaloids found in highest concentration in ayahuasca teas made in South and North America: DMT, harmine, THH and harmol. None of the differences in concentration were statistically significant.

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4

South America North America

mg/ml + SD

3

2

1

0

DMT

Harmine

THH

Harmol

Figure A.1. Mean concentration in mg/ml of DMT, harmine, THH and harmol in ayahuasca teas made for ceremonial use in South American (n=6) compared to North American (n=5). There were no significant differences.

A.3.3 Beta-carboline alkaloid concentrations in ayahuasca teas The six beta-carbolines from the B. caapi vine were measured in the 11 ayahuasca tea samples and ranked by greatest to least concentration present in each tea sample. In South American teas harmine > harmol > THH > harmaline > THHO > harmalol. In North American teas harmine > THH > harmol > harmaline > THHO > harmalol. Harmine was found in highest concentration in all of the South American samples and three of the five North American samples. However, there were no statistical differences in mean harmine concentration between South (1.95 + 0.51) and North American (2.23+ 1.05) tea samples (Table A.2). Concentrations of the minor beta-carboline alkaloids present in ayahuasca (harmaline, harmalol and THHO) were found in lower concentrations compared to the major beta-carbolines 115

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(harmine, harmol and THH). There was more variability in the minor beta-carbolines among tea samples. For example, harmaline varied widely among all 11 samples, ranging from 0.049 – 0.348 (a 7 fold difference). Harmaline was found in higher concentrations in North American versus South American samples and this difference was statistically significant (p = 0.006). Higher levels of harmalol were found in South America than in North American samples (p=0.029). There were no significant differences in concentrations of the other beta-carbolines, harmine, harmaline, THH, Harmol, or THHO.

Table A.2. Comparison of active constituents of ayahuasca tea samples from South America versus North America using T tests.

DMT Harmine Harmaline THH Harmol Harmalol THHO NMT 5-OH DMT

South America 0.4459 1.9457 0.1406 1.4031 1.3988 0.0175 0.0699 0.0163 0.0028

North America 0.4888 2.2286 0.2773 1.8938 1.6691 0.0021 0.0623 0.0237 0.0003

t -.382 -.589 -3.551 -1.728 -.936 2.942 .301 -1.972 2.511

P 0.711 0.571 0.006 0.118 0.374 0.029 0.770 0.080 0.048

A.3.4 Bivariate and multivariate relationships among the 11 ayahuasca alkaloids Because the DMT present in ayahuasca tea derives only from the P. viridis plant admixture, we did not expect, nor detect, any significant correlations of DMT with the concentration of the beta-carbolines measured. Concentrations of 5-OH-DMT or Nmethyltryptamine (precursor of DMT) concentrations did not correlate with DMT (Table A.3).

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Table A.3. Correlations of DMT concentration with the other eight alkaloid constituents present in 11 ayahuasca tea samples Harmine Harmaline DMT

THH

Harmol Harmalol THHO

NMT

5-OH-DMT

r

.168

.228

-.018

.163

-.108

-.387

-.003

-.123

p

.621

.501

.958

.632

.753

.239

.993

.719

N

11

11

11

11

11

11

11

11

Table A.3 shows that there were no significant bivariate relationships between DMT and any of the other eight alkaloids present in ayahuasca, including 5-OH-DMT. This later result is surprising in that DMT and 5-OH-DMT are constituents in P. viridis and might be expected to co-vary. However, a regression where DMT is the dependent variable and the other eight alkaloid constituents as predictors turned out to be significant in that, other things being controlled, harmaline and harmalol had both a positive predictive relationship with DMT. Higher DMT concentrations were associated with higher harmaline and harmalol, which are two of the less significant beta carbolines present in ayahuasca tea. DMT levels did not correlate with concentrations of the major beta-carbolines, harmine, harmol or THH. There were also marginal significant negative relationships between NMT and DMT, and 5-OH-DMT with DMT (Table A.4).

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Table A.4. Regression coefficients where DMT is the constant dependent variable with each of the concentration of the other eight ayahuasca alkaloids Unstandardized Coefficients B (Constant)

Standardized Coefficients

Std. Error

Beta

.283

.199

Harmine

-.239

.386

Harmaline

8.656

t

P

1.422

.291

-1.034

-.619

.599

1.868

4.563

4.634

.044

-1.040

.422

-3.013

-2.468

.132

1.167

.745

3.117

1.567

.258

31.401

5.947

2.141

5.280

.034

4.165

3.036

.935

1.372

.304

NMT

-81.934

24.015

-3.257

-3.412

.076

5-OH-DMT

-94.689

26.626

-1.180

-3.556

.071

THH Harmol Harmalol THHO

To better understand the relationships among the nine active constituents of ayahuasca teas collected from 11 ceremonies, we performed a Principal Component Analysis ( PCA), which produced a map to determine how closely related were the samples and their relationship with the chemical components (Figure A.2).

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Figure 3.2. Ayahuasca Alkaloid Chemical Components by 11 tea samples 1.00

AY005B3 AY027

AY003A3

Alkaloid concentration

AY020

-1.50

AY026

0.50 DMT Harmaline

NMT -1.00

Harmol

THH

Harmine

AY025

AY001B3 0.00

-0.50

0.00

0.50

1.00

1.50

AY024 -0.50

AY010A3 AY011A3

THHO

AY009A3 -1.00 Harmalol

5-OH-DMT

-1.50 Geographic location

Figure A.2. Principal Components Analysis of 11 ayahuasca tea samples collected in either South or North America. Red represents the nine alkaloids. Green represents the North American samples and Yellow represents the South America samples. North American samples tend to fall on the upper left quadrant, the South American tend to fall at the upper right or lower middle of the map. Component 1 (geographical location of ceremony) and component 2 (psychoactive alkaloid concentrations) describe the 11 samples well. Geographical location (South versus North America) and psychoactive alkaloid concentrations, account for 76.2% of the variability.

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We compared results from our 11 samples to results published by Callaway et al (1996) [A.9] who measured DMT, harmine, harmaline and THH in an ayahuasca tea used in a União do Vegetal Church ceremony (UDV) in the Brazilian Amazon rain forest near Manaus [A.8]. The UDV ayahuasca tea contained DMT at 0.24 mg/ml, harmine at 1.7 mg/ml, harmaline at 0.2 mg/ml and THH at 1.07 mg/ml. The 11 samples measured in the current study that were collected from 2000-2011 had a higher average concentration of DMT (0.47 + 0.18) compared to the 1996 data, but had similar concentrations of the beta-carbolines harmine (2.1 + 0.77 mg/ml), harmaline (0.21 + 0.09 mg/ml) and tetrahydroharmine (1.63 + 0.51 mg/ml). A.4 Discussion Given the variation between South America and Hawaii in climate, seasonality of harvest and variations in ayahuasca tea formulas used cross-culturally, we expected to observe greater differences in concentration of the major known active constituents. Mean concentrations of DMT, the main psychoactive alkaloid present in ayahuasca tea, did not differ between South and North American tea samples. Concentrations of the major beta-carbolines (harmine, harmol and THH) also did not statistically differ between South and North American samples. Of the eight beta-carbolines measured in the 11 tea samples, only harmaline and harmalol concentrations significantly differed. Harmaline was nearly two-fold higher in North American tea samples suggesting that relatively more B. caapi vine is added to the decoctions made in North America compared to South America. However, this conclusion is complicated by our data showing that harmalol, a low concentration beta-carboline present in B. caapi, was found in much higher average concentration (nine-fold) in South American samples. Concentrations of 5-OH-DMT (with no known CNS effects) and harmalol were higher in South American samples.

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The difference between South and North American ayahuasca tea samples may be due to variations in season of harvest, strain and maturity of plant used, climate, tea recipe used, and freshness of decoction. North American samples were decocted within 24 hours of the time that the sample was frozen. We do not have information regarding the data of decoction of Brazilian or Peruvian samples. A.5 Conclusions DMT concentration among ayahuasca teas made and used in healing ceremonies in South America or North America are remarkably similar in concentrations of the main psychoactive alkaloids present in ayahuasca tea (DMT, harmine, harmol and tetrahydroharmine). Concentrations of two of the three minor beta-carbolines (harmaline and harmalol) were statistically different. North American tea samples were higher in harmaline and harmolol suggesting that the tea formula made and used in North America may include a higher ratio of B. caapi vine relative to P. viridis leaves or possibly a different strain of B. caapi. Seasonal, growth and or methodological practices may also account for some differences. A.6 Future Ayahuasca Characterization Publications We have further collaborated with Dr. Standish using the established ayahuasca method on two future ayahuasca characterization publications and two studies preparing ayahuasca for clinical trials. The data for these studies has already been collected and analyzed and the manuscripts are currently in the process of being written. The first examined anatomical distribution of DMT in P. viridis and beta-carbolines in B. caapi and was conducted by Reardon, McIlhenny, Barker, and Standish. We found no DMT present in B. caapi in any anatomical structure. Harmine concentrations were highest in B. caapi leaves, with lesser in the glands at the 121

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base of the leaves, still less in petiole, and the least amount was found in the woody vine. Interestingly the traditional brewing of ayahausca only included the vine of B. caapi and no parts of the leaf were included. Both the leaf and meristematic nodes in P. viridis contain DMT at similar levels. The second characterization study was a comparison of psychoactive alkaloids in ayahuasca tea formulations that varied in P. viridis and B. caapi weight ratios conducted by Standish, Reardon, McIlhenny, and Barker. We found that systematic increase of P. viridis and B. caapi weight ratios from 1:2 to 1:16 resulted in a fairly linear curve of ratio to concentration of harmine while DMT concentrations remained fairly constant. A.7 Future Publications to Prepare Ayahuasca for Clinical Trials The first study intended to prepare for potential ayahuasca clinical trials examined chemistry, manufacturing and controls and was conducted by Standish, Martzen, Reardon, McIlhenny, and Barker. We found that ayahuasca alkaloid concentration and therefore dose potency can be controlled by decocting the two plants separately then recombining and that ayahuasca extracts are stable when stored as there appears to be no loss of DMT or harmine after 12 months of refrigeration or freezing at - 80oC. It was further observed that ayahuasca can also be sterilized by lyophilization or autoclave without loss of psychoactive alkaloids and that the experimental ayahuasca extracts examined were free of heavy metals. The second clinical preparation was an ayahuasca dose consideration for planning a phase I dose escalation safety and tolerability study in healthy adults and was conducted by Standish, Reardon, Martzen, McIlhenny, and Barker. We asked the question: What Phase I doses should be used in the dose escalation? We found that an initial water volume of 12 liters may be boiled 122

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down to at least 833 ml to result in a potent ayahuasca extract that would be similar to what has been used as traditional medicine: 833 ml to provide 16 doses at 50 ml volume per dose. Dosing may start at DMT 0.025, 0.05, 0.10, 0.25. 0.50, and 1.0 mg/ml. Since harmine posed fewer safety concerns we will retain the same concentration of harmine at 1.5 mg/ml. Extracts may be made with B. caapi vine 3-5 cm in diameter cut fresh within one week and refrigerated from time of harvest to time of decoction. Extracts may be made with distal P. viridis leaves until we know DMT concentration in mature leaves. Extracts can be sterilized using 0.22 micron filtration and placed into 100 ml sterile brown bottles for freezing until administration when the extract will be removed from the freezer and rendered liquid again in a dedicated secure refrigerator. A.8 References [A.1] M. Yritia, J. Riba, J. Ortuno, A. Ramirez, A. Castillo, Y. Alfaro, R. De La Torre, M.J. Barbano. Determination of N,N-dimethyltryptamine and beta carboline alkaloids in human plasma following oral administration of Ayahuasca. Journal of Chromatography B 2002, 779, 271-281. [A.2] D.J. McKenna, G.H.N. Towers, F. Abbott. Monoamine oxidase inhibitors in South American hallucinogenic plants: tryptamine and beta-carboline constituents of ayahuasca. Journal of Ethnopharmacology 1984, 10, 195-223. [A.3] J.C. Callaway, D.J. McKenna, C.S. Grob, G.S. Brito, L.P. Raymon, R.E. Poland, E.N. Andrade, E.O. Andrade, D.C. Mash. Pharmacokinetics of Hoasca alkaloids in healthy humans. Journal of Ethnopharmacology 1999, 65, 243-256. [A.4] W.M. Abi-Saab, M. Bubser, R.H. Roth, A.Y. Deutch. 5-HT2 receptor regulation of extracellular GABA levels in the prefrontal cortex. Neuropsychopharmacology 1999, 20, 92-96. [A.5] J. Riba, A. Rodríguez-FornellS, G. Urbano, A. Morte, R. Antonijoan, M. Montero, J.C. Callaway, M.J. Barbanoj. Subjective effects and tolerability of the South American psychoactive beverage Ayahuasca in healthy volunteers. Psychopharmacology(Berlin) 2001, 154, 85-95. [A.6] M.M Airaksinen, I. Kari. Beta-carbolines, psychoactive compounds in the mammalian body. Part I: Occurrence, origin and metabolism. Med Biol 1981, 59, 21-34. [A.7] E.H. McIlhenny, K.E. Pipkin, L.J. Standish, H.A. Wechkin, R.J. Strassman, S.A. Barker. Direct analysis of psychoactive tryptamine and harmala alkaloids in the Amazonian 123

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botanical medicine ayahuasca by liquid chromatography-electrospray ionization-tandem mass spectrometry. Journal of Chromatography A 2009, 1216, 8960-8968. [A.8] E.H. McIlhenny, J. Riba, M.J. Barbanoj, R. Strassman, S.A. Barker. Methodology for determining major constituents of ayahuasca and their metabolites in blood. Biomed Chromatogr. 2012, 26, 301-313. [A.9] J.C. Callawa, L.P. Raymon, W.L. Hearn, D.J. McKenna, C.S. Grob, G.S. Brito, D.C. Mash. Quantitation of N,N-dimethyltryptamine and harmala alkaloids in human plasma after oral dosing with ayahuasca. Journal of Analytical Toxicology 1996, 20, 492-497.

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Appendix B. Metabolism and disposition of N,N-dimethyltryptamine and harmala alkaloids after oral administration of ayahuasca*

Jordi Riba1,2, Ethan H. McIlhenny3, Marta Valle2,4, José Carlos Bouso1,2, Steven A. Barker3 1

Human Experimental Neuropsychopharmacology. Institute for Biomedical Research IIB Sant Pau. Sant Antoni María Claret, 167. Barcelona 08025, Spain.

2

Centre d’Investigació de Medicaments, Servei de Farmacologia Clínica, Hospital de la Santa Creu i Sant Pau. Sant Antoni María Claret, 167. Barcelona 08025, Spain. Departament de Farmacologia, de Terapèutica i de Toxicologia, Universitat Autònoma de Barcelona. Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM.

3

Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803 USA.

4

Pharmacokinetic and Pharmacodynamic Modelling and Simulation. Institute for Biomedical Research IIB Sant Pau. Sant Antoni María Claret, 167. Barcelona 08025, Spain.

________________________________________________________________________ *Reprinted with the permission of John Wiley and Sons and the Journal of Drug Testing and Analysis 125

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B.1 Introduction Ayahuasca is a psychotropic plant tea obtained from the stems of the jungle liana Banisteriopsis caapi and usually the leaves of Psychotria viridis or Diplopterys cabrerana [B.1, B.2]. The tea is used by many Amazonian peoples to attain a modified state of consciousness, which is a central element of rites of passage, religious ceremonies and shamanic medicine [B.2]. In recent years, the firmly established ancestral uses of ayahuasca have given way to new forms of consumption. Syncretic religious groups using ayahuasca as a sacrament have appeared and have expanded their activities to the urban areas of South America and also to Europe and North America. An increasing number of foreigners travel to the Amazon to participate in ayahuasca retreats and traditional healers travel to Europe to organize ayahuasca ceremonies. The growing attention ayahuasca is attracting worldwide has raised public health concerns [B.3]. The powerful psychotropic effects of ayahuasca arise from the pharmacological interaction between the β-carboline alkaloids present in B. caapi and the tryptamines found in P. viridis and D. cabrerana. On the one hand, the β-carbolines, mainly harmine, harmaline and tetrahydroharmine, are reversible inhibitors of the enzyme monoamine-oxidase A (MAO-A) [B.4, B.5]. On the other hand, P. viridis and D. cabrerana contain DMT [B.5], a potent psychedelic [B.6-B.8], which is a priori not active orally [B.7] due to extensive first pass metabolism by MAO-A. Both the β-carbolines, also known as harmala alkaloids, and the DMT present in the plants are extracted into the ayahuasca infusion and ingested by users [B.9]. The blockade of visceral MAO brought about by the β-carbolines is believed to render DMT orally active, allowing its access to systemic circulation and subsequently to the central nervous system [B.10]. There, DMT interacts with serotonergic 5-HT2A, 5-HT1A and 5-HT2C and other receptor sites [B.11-B.15] eliciting psychedelic effects in humans [B.16-B.17]. 127

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Figure B.1. Chemical structures of ayahuasca alkaloids and their metabolites. 128

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Early studies involving the administration of pure DMT to humans had already observed that it lacked psychoactive effects after oral administration [B.7]. Following parenteral DMT, Szára failed to find the unmetabolized drug in urine and identified indole-3-acetic acid (IAA), formed by oxidative deamination, as the drug's degradation product [B.6]. Kaplan and coworkers found that following an intramuscular injection, DMT disappeared from plasma very rapidly. They reported that less than 0.1% was recovered in 24 h urine but they did not attempt to identify the putative metabolites [B.18]. The role of MAO in the metabolic breakdown of DMT has been stressed in the literature based in the aforementioned presence of IAA in urine after DMT and in the efficacy of the harmala alkaloids and other MAO-inhibitors to render DMT psychoactive per os [B.19]. However, oxidative deamination by MAO may not be the sole metabolic pathway in humans. In vitro and animal studies have described N-oxidation, N-demethylation and cyclization as alternative metabolic routes, [B.20-B.22] as depicted in Figure B.2.

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Figure B.2. Metabolic pathways of N,N-dimethyltryptamine. MAO=monoamine-oxidase; ADH= aldehyde-dehydrogenase.

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To date no study has addressed the fate of DMT and the harmala alkaloids when administered in combination in ayahuasca. In a preliminary assessment conducted by our group in the course of analytical method validation, DMT-N-oxide (DMT-NO) and harmol and harmalol, the O-demethylation products of the harmine and harmaline, respectively, were detected in the urine and blood of three individuals after ayahuasca intake [B.23, B.24]. The present manuscript describes the assessment of the metabolism and urinary disposition of DMT and the harmala alkaloids in a group of healthy volunteers following ayahuasca administration. B.2 Materials and Methods

B.2.1 Volunteers Ten young healthy male volunteers were recruited. Participants were experienced psychedelic drug users. The most commonly used substances were psilocybin mushrooms and LSD, followed by ketamine, peyote and mescaline. None of the participants had used ayahuasca before. Volunteer mean age was 29.0 years (range 20-38); mean weight was 67.0 kg (range 6085); and mean height was 1.77 m (range 1.69-1.96). Volunteers underwent a structured psychiatric interview (DMS-IV) to exclude current or past history of Axis-I disorders and alcohol or other substance dependence. General good health was confirmed by medical history, laboratory tests and ECG. The study was conducted in accordance with the Declarations of Helsinki and Tokyo concerning experimentation on humans, and was approved by the hospital's ethics committee and the Spanish Ministry of Health. All volunteers gave their written informed consent to participate.

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B.2.2 Drugs Ayahuasca was administered orally as an encapsulated lyophilizate. The freeze-dried material was obtained from a Brazilian batch of ayahuasca and contained 8.33 mg DMT, 14.13 mg harmine, 0.96 mg harmaline and 11.36 mg tetrahydroharmine (THH) per gram. The lyophilizate was also tested for harmol and harmalol and was found to contain 0.30 mg/g harmol and 0.07 mg/g harmalol. Freeze-dried ayahuasca was administered in doses equivalent to 1.0 mg DMT/kg body weight. B.2.3 Study Design and Sample Collection Urine samples were obtained in the course of a clinical trial involving three experimental sessions. In a double-blind crossover balanced design, participants received in each experimental session one of the following treatments: a lactose placebo, 20 mg d-amphetamine, and 1.0 mg DMT/kg body weight ayahuasca. In addition to urine collection, the study involved the measurement of various pharmacodynamic parameters including subjective, neuroendocrine and immunomodulatory data. A detailed description of the methods used and the results concerning these variables have been published elsewhere [B.25]. In the present article we report only the data obtained from the analyses of urine samples collected following ayahuasca administration. The amounts of harmine, harmaline and tetrahydroharmine recovered in urine are reported together with the amounts of their potential O-demethylated metabolites, harmol, harmalol and tetrahydroharmol (7-hydroxy-tetrahydroharmine). Samples were also analyzed for DMT and its potential biotransformation products IAA, DMT-NO, N-methyltryptamine (NMT) and 2-methyltetrahydro-betacarboline

(2MTHBC).

Additionally,

samples

collected

after

placebo

administration were also quantified for IAA, which is known to be excreted under normal physiological conditions. In each experimental session, 24h urine was collected, subdivided into 132

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the following time intervals relative to ayahuasca (and placebo) administration: 0-4h, 4-8h, 8-16h and 16-24h. The collected urine volume at each time interval was noted, the pooled urine was well mixed and 50 ml aliquots were separated and stored at -80 ºC until analysis. Samples underwent a single freeze-thaw cycle prior to analysis. Samples were analyzed with and without enzyme hydrolysis. Enzyme hydrolysis was achieved using β-glucuronidase/sulfatase from limpets (Patella vulgata) Type L-II (Sigma-Aldrich, St. Louis, MO, USA) as described by McIlhenny and coworkers [B.23]. B.2.4 Analytical Method Urine sample analyses were conducted by the methods of McIlhenny and coworkers, which uses HPLC with electrospray ionization and tandem mass spectrometry [B.23]. Thus, 100 µl of well mixed urine were diluted to a volume of 1.0 ml (900 µl of LC mobile phase; 97:3 water with 0.1% formic acid:acetonitrile with 0.1% formic acid) and filtered [B.23]. A volume of 10 µl was injected for the analysis. The LC/MS/MS method had been validated for the determination of the following compounds: DMT, IAA, DMT-NO, NMT, 5-hydroxy-DMT, dimethylkynuramine, 2MTHBC, 5methoxy-DMT, harmine, harmaline, tetrahydroharmine, harmol, harmalol and tetrahydroharmol. Thus, analyses were conducted using an Agilent 1200 series LC system (Agilent Technologies, Palo Alto, CA, USA) equipped with an Agilent G1367A HiP ALS autosampler, an Agilent G1311A Quaternary micropump, and an Agilent G1332A degasser. An Agilent G131gA TCC column oven operating at 25o °C was interfaced to a TSQ Quantum Access 1.5 SP1 tandem MS (Thermo Fisher Scientific, Waltham, MA, USA) with electrospray ionization (ESI) operated in the positive ion mode.

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Chromatographic separation was achieved on a 1.8 m 4.6 x 50 mm (i.d.) Agilent ZORBAX Eclipse Plus C18 rapid resolution HT threaded column with an Alltech DirectConnect Column 2 m pre-filter (Deerfield, IL, USA) using gradient elution. The MS/MS analysis was performed using selected reaction monitoring (SRM) of the protonated molecular ions for the analytes. The spray voltage was 4000 V, sheath gas (nitrogen) pressure 50 psi, capillary temperature 310o °C, and collision pressure was 1.5 psi. of high purity argon. Generation of detection data and integration of chromatographic peaks were performed by Xcalibur 2.0.7 Thermo Fisher Scientific (Waltham, MA, USA) LCquan 2.5.6 QF 30115 software. Identification of the compounds was based on the presence of the molecular ion at the correct retention time, the presence of three transition ions and the correct ratio of these ions to one another (+/- 25% relative). The proven limit of quantitation (LOQ) was 5 ng/ml for all compounds. The limits of detection for the compounds examined were comparable to results previously attained, [B.23] ranging from 0.07 ng/ml for DMT-NO to 0.57 ng/ml for harmol. Tetrahydroharmol was observed to have a LOD of 0.17 ng/ml. B.2.5 Statistics Descriptive statistics (mean and standard deviation) were used to report the amounts of the different compounds measured. Percentage recoveries were calculated relative to the amount of parent compound administered. Differences in percentage recoveries between enzymaticallytreated and non-treated samples were analyzed using paired-samples t-tests. Pearson's correlation coefficient was used to explore potential linear relationships between measures. All comparisons were considered statistically significant for p values The residue was reconstituted in 25 lJL methanol for injection (2 IJL) into the instrument.

Results and Discussion Components of harmala alkaloids were identified by retention times and by co-injection of standards with selected experimental samples (50 ng/mL of added harmine or THH or 10 ng/mL harmaline) and further verified by their fluorescent characteristics using the two sets of established wavelengths. DMT was detected and quantitated in plasma samples by liquid-liquid extraction with DPH as an internal standard followed by GC-NPD. Spiked solvent samples were injected on a DB-1 column, and good chromatographic results for DMT were seen. However, when spiked control plasma samples were used, DMT coeluted with a large peak identified as caffeine by GC-MS (base peak at mtz 194, data not shown). Because caffeine would be a common contaminant in plasma samples, better separation of DMT from caffeine was achieved by using the more polar DB-17 column. The internal standard, DPH, which is also a tertiary amine, was chosen based on its retention time relative to DMT. Figure 2 illustrates two representative chromatograms from human plasma samples; Figure 2A is a chromatogram of a sample taken before administration of oral doses ofayahuasca, which shows a signal only for the internal

Journal of Analytical Toxicology, Vol. 20, October 1996

standard, and Figure 2B is a chromatogram of a sample taken 120 min after the consumption of ayahuasca (the ingested equivalent of 38.4 mg DMT),which demonstrates signals corresponding to plasma concentrations of 25.5 ng/mL DMT and 25 ng/mL DPH. DMT and the internal standard, DPH, had relative retention times of 5.3 and 5.7 min, respectively. Method validation Recovery. Harmala alkaloid recoveries were calculated from protein precipitation of 10 spiked plasma samples at 10 and 250 ng/mL for harmine and THH, respectively, and 1 and 15 ng/mL for harmaline. DMT recoverywas calculatedfrom the one-step alkaline liquid-liquid extraction of 10 spiked plasma samples at 10 and 20 ng/mL. The analyte recoverieswere quantitative at both concentrations for all alkaloids. For DMT,quantitative recoverieswere obtained provided the evaporation temperature was no higher than 40~ (data not shown). Sensitivity. The sensitivityof an analytical method is defined by its limit of detection (LOD) and its limit of quantitation (LOQ). The LODs for the harmala alkaloids in the present HPLC assay were as follows: harmine, 0.1 ng/mL; harmaline, 0.05 ng/mL; and THH, 0.1 ng/mL. For DMT, the LOD with GC-NPD was 0.5 ng/mL. These values were determined from averages of 10 samples and were statistically significant with greater than 95% probability. For quantitative method validation, the LOQwas considered to be the lowestconcentration of analyte in which the percent coefficient of variation (%CV) did not exceed20%. The LOQs for the harmala alkaloids in the present HPLC assay were as follows: harmine, 2 ng/mL; harOPH

A mV

s.~,7 rain

DPH

B DMT

mV

5

5.74

Figure 2. Representativechromatograms using gas chromatography with a nitrogen-phosphorus detector for analysis of N,N-dimethyltryptamine (DMT) from experimentalplasma samples:(A) an extractedplasmasample before administration of ayahuascawith 25 ng/mL diphenhydramine (DPH) as an internal standard and (B) a sample from the same individual 120 min afteringestion,again with 25 ng/mL DPH as an internal standard and a signal corresponding to 25.5 ng/mL DMT.

maline, 1.0 ng/mL; and THH, 1.9 ng/mL. The LOQ of DMT for the GC-NPD assay was 5 ng/mL. Precision. The intra-assay precision for the harmala alkaloids, determined by a comparison of the %CVsof 10 replicate calculated concentrations, and all standards were calculated from three separate experiments. For harmine, the values obtained by HPLC were 3.54 and 7.23% at 10 and 250 ng/mL, respectively; harmaline values were 13.34 and 2.97% at 1 and 25 ng/mL, respectively;and THH values were 5.68 and 13.22% at 10 and 250 ng/mL, respectively.For the analysis of DMT by GC-NPD, the values were 6.84 and 8.48% at 10 and 20 ng/mL, respectively.The HPLC interassay precision values for harmine were 10.57 and 5.63% at 10 and 250 ng/mL, respectively;harmaline values were 13.33 and 2.73% at I and 25 ng/mL; and THH values were 5.37 and 11.59% at 10 and 250 ng/mL, respectively. For the analysis of DMT, these values were determined to be 2.47 and 6.11% for 10 and 20 ng/mL, respectively. Accuracy. The accuracies of the assays were determined in parallel with the precision studies described. Ten replicate samples gave a mean of 10.8 • 0.3 ng/mL and 265.21 _+ 1.3 ng/mL at 10 and 250 ng/mL, respectively, for harmine; 1.1 + 0.2 ng/mL and 15.34 + 0.3 ng/mL at 1 and 15 ng/mL, respectively, for harmaline; 9.83 _+0.2 ng/mL and 273.93 + 3.5 ng/mL at 10 and 250 ng/mL, respectively,for THH, for the HPLC analyses, and 11.8 + 0.6 ng/mL and 22.2 +_0.4 ng/mL at 10 and 20 ng/mL, respectively,for DMT.The variation was less than 20%.

tinearity The linearity was determined for the three harmala alkaloids by diluting 100-mg/mLsolutions of each alkaloid in the mobile phase and preparing spiked plasma samples from these solutions to concentrations of 10, 25, 50, 125, and 250 ng/mL for harmine and THH and 1, 2.5, 5, 10, and 25 ng/mL for harmaline. Each standard was taken through the preparation procedure and assayed by injecting 50 IJL. Initial calculations gave recoveries just over 100% when spiked plasma samples were compared with pure alkaloids diluted in mobile phase, perhaps because of an overestimation based on a slight loss in precipitated protein from the plasma samples. For DMT analysis by GC-NPD, the concentration-response relationship was defined by a linear regression of peak area versus concentration of DMT. All standard curves were prepared by spiking known amounts of DMT in blank plasma samples and coextracting them with the experimental samples. For assay method validation, the linearity was measured initially up to 1000 ng/mL. Standard curves were generated with the following concentrations: 5, 10, 50, 100, 500, and 1000 ng/mL; the internal standard, DPH, had a concentration of 250 ng/mL. The standard curves were found to be linear (coefficient of determination [r2], 0.996) across this concentration range. However,the plasma samples taken for the study had DMT concentrations that ranged between 10 and 25 ng/mL. Based on this initial assessment, the working range was chosen to be 5-50 ng/mL, and the internal standard was at 25 ng/mL. Least-squares regression analysis gave an r 2value of 0.994. The linear relationship between concentration and response was reproducible across all experimental runs. 495

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Two detectors in tandem

Although the Perkin-Elmer LC 240 fluorescence detector can be programmed to change wavelengths during chromatographic analysis, it can only monitor one set of wavelengths (emission or excitation) at any given time. Using two detectors allowed for the simultaneous quantitation of harmine with either THH (emission/excitation wavelength, 232/351 nm) or harmaline (340/495 nm). Only harmaline was quantitated at emission/excitation wavelengths of 340/495 nm, and harmine was found to be approximately 5 times more sensitive at emission/excitation wavelengths of 232/351 nm. Figure 3 shows typical chromatograms of an actual sample (Figure 3A and 3C) and standards (Figure 3B and 3D) observed at emission/excitation wavelengths of 232/351 nm (Figure 3A and 3B) and 340/495 nm (Figure 3C and 3D). DMTwas found to be a major component of this beverage. In the HPLC analysis, DMT could be resolved as a single peak, although it was not strongly fluorescent at emission/excitation wavelengths of 232/351 nm (LOD, 20 ng/mL) and was essentially undetected at emission/excitation wavelengths of 340/495 nm. Like harmaline, the DMT concentration was relatively low in the ayahuasca tea. Moreover, the retention time of DMT (6.2 rain) did not coincide with any of the signals for the harmala alkaloids under these conditions. Although DMT was not detected in any of the plasma samples, it was quantitated

from the single batch of tea that was used in this study and verified by GC-NPD (data not shown).

Application of the analytical methods The analytical procedures were applied to a pharmacokinetic study of DMT and harmala alkaloid concentrations in plasma from 15 healthy male volunteers after they ingested 2 mL/kg body weight from the same batch ofayahuasca. The ayahuascaused in this study was analyzed for concentrations of harmine (1.70 mg/mL), harmaline (0.2 mg/mL), THH (1.07 mg/mL), and DMT (0.24 mg/mL) using the methods described here. Thus, for a 59-kg individual, the average oral doses of ayahuasca alkaloids ingested were as follows: harmine, 204.0 mg; harmaline, 24.0 rag; THH, 128.4 mg; and DMT,28.8 mg. These concentrations in the tea resulted in the followingpeak plasma concentrations (Cma~)in a representative individual volunteer: harmine, 92.3 ng/mL; harmaline, less than 1.0 ng/mL; THH, 82.2 ng/mL; and DMT, 12.4 ng/mL (Figure 4). The range and average values for the 15 subjects are shown in Table I. Although harmaline was detected in some volunteers, values for Cmaxwere only reliably determined in six of the 15 volunteers. This finding most likely reflects the low levels of harmaline contained in the tea (0.2 mg/mL) and, perhaps in part, to individual differences in absorption and metabolism. In previous reports (6,14), peak plasma levels of DMT after intravenous administration occurred after 2 rain, then rapidly dropped to gr baseline levels by 30 rain. Peak plasma DMT levels were reported to correspond with peak psychoactivity after intravenous ad~ ministration (15). In the present study, we observed a similar range for DMT levels in plasma after oral ingestion of the tea. AlA though the psychoactivity was reportedly much less intense, the psychoactive effects lasted for a longer duration with oral doses than with intravenous administrations. These preliminary observations suggest that the time course for the appearance of the subjective effects most closely follows the DMT pharmacokinetic profile shown in Figure 4. Unlike the reported "rush" associated with the rapid rise in plasma DMT concentrations after intravenous doses, the oral doses produced mild responses that were typically associated with the known spectrum of psychedelic effects demonstrated with intravenous administrations of DMT (6,15). Harmala alkaloids are wellknown inhibitors of MAO-A enzymes, for which DMT is a substrate (10). The more extended profiles seen for DMT levels in the 3 _ plasma after ingestion of ayahuasca are best explained by the actions of harmala alFigure 3. Chromatograms of harmala alkaloids detected at (A and B) an excitation wavelength of 232 nm and an emission wavelength of 351 nm and (C and D) an excitation wavelength of 340 nm and kaloids, which inhibit the metabolism of an emission wavelength of 495 nm, showing (A and C) an actual sample taken 120 rain after the inDMT by MAO-A. gestion of ayahuascaand (B and D) a sample of spiked plasma.

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Conclusion Evaluation of the effects of ayahuascain human volunteers required the development of analytical methods to quantitate DMT and harmala alkaloids in plasma samples. The analytical methods described here provide accurate and reliable quantitative assays of DMT and harmala alkaloids in human plasma samples and may be applicable to other matrices, including whole blood, urine, and tissue homogenates. Hallucinogens produce a unique syndrome of psychological effects in humans and cause alterations in perceptual and cognitive processes that make it difficultto maintain a clear sensorium (6). The application of the analytical procedures for measurement of DMT,hatmaline, and THH will contribute to the understanding of the subjective dose effects and the pharmacodynamic and pharmacokinetic profiles ofayahuascaalkaloids in human subjects. Because of the rising popularity of ayahuasca and other plant inebriant beverages and the current widespread use of Prozac and other SSRIsin North America and Europe, there is the possibility of inadvertent coadministration of MAOIswith an SSRI; therefore, there is a concern about potentially serious toxicities associated with the emergence of a serotonin syndrome. This potentially lethal condition is especially insidious 10o 0

DIKr

-~-

THH

~9 8 0 r 0

60

8 -~ 40 M

,~ 20 o

! 1O0

200 Time

300

400

500

(mln)

Figure 4. Representativepharmacokinetic profiles of plasma alkaloids over time from the sameindividual. Thisvolunteer(59 kg) ingested120 mE of ayahuasca,whic[~ correspondsto a total consumption of 204.0 mg harmine, 24.0 mg harmaline, 128.4 mg tetrahydroharmine (THH), and 28.8 mg N,N-dimethyltryptamine (DMT). Table I. Range and Average (Plus or Minus Standard Error) Values for Peak Concentrations of DMT and Harmala Alkaloids in 15 Men after Oral Doses of

Ayahuasca

DMT* (ng/mt) Range

THH t (ng/mt)

11.5-25.5 49.2-134.5

Average 15.8+_.I.1

90.8_+5.9

* D M T = N,N-Dimethyltryptamine. * THH = Tetrahydroharmine.

Harmaline (ng/mL)

Harmine (ng/mL)

0.020

H

H 3C

N-methyltyramine

12.43 (± 2.7, 0.98)

6.61 (± 10.6, 0.91)

0.532

H 3C

H 3C

N,N’-dimethyltyramine

38.27 (± 10.3, 0.98)

64.29 (± 43.0, 0.94)

1.680

N-ethyltyramine

1.62 (± 0.5 0.97)

R2

Tyramine

H

Me

A

O OM e

N

>30,000

H

R1 N

I 125

O

N/A

N/A

I 125

N

B

O

N3

N3

O

[125I]-IAF

[125I]-IACoc Tryptamine (µM) -

P

N-Me Tryptamine (µM)

Tryptamine (µM)

DMT (µM)

10 50 100 10 50 100 10 50 100

Percent intensity sigma-1 (%)

-

-

140 120 100 80 60 40 20 0

H

100 80 60 40 20 0

100

0

111 74

81 121 136 72 124 39 0.02 100

Percent intensity sigma-1 (%) 100 0

N-Me Tryptamine (µM)

P 10 50 100 10

DMT (µM)

50 100 10

66

70

50 100

13 100 81 65

69

69 57

31

42 53 22

72 55 40 58 29

18

kDa

kDa 26

26

18

18

Percent intensity sigma-2 (%) 100 0

0

21

20 Fig. 2. Tryptamine, N-methyltryptamine, and DMT inhibition of photolabeling. Rat liver membranes 40 (100 mg per lane) were suspended in the presence or absence of the protecting drugs. Samples were 60 photolyzed with (A) 1 nM carrier-free [125I]-IACoc or (B) 1 nM carrier-free [125I]IAF. Ten micromolar (+)80 100 pentazocine (P) protected sigma-1 receptor photolabeling, whereas 10 mM haloperidol (H) protected both sigma-1 and sigma-2 receptors. Percent band intensities are shown as compared to controls performed in the absence of protecting ligand (−).

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936

WT Veh WT DMT KO Veh KO DMT

Distance traveled (cm)

A 1750 1500

*** ***

1250 1000 750 500 250 0 0

10

20

30

40

50

60

70

80

90

Time (min)

B Distance traveled (cm)

Fig. 4. DMT-induced hypermobility abrogated in the sigma-1 KO mouse. (A) Distances traveled by WT and KO mice were measured in an open-field assay in 5-min increments. Pargyline was injected 2 hours before DMT or vehicle (Veh) ip injection. Bars represent mean T SEM (n = 8 to 14 mice). WT mice showed a significant (***P < 0.0001) increase in mobility in response to DMT as compared to KO mice. (B) Total distance traveled over 30 min after DMT, vehicle (Veh), or methamphetamine (Meth, n = 6 mice) injection in WT and KO mice. (C) Methamphetamine serves as a positive control for hypermobility in KO mice.

DMT or Veh

26000

***

18000 10000 8000 6000 4000 2000 0

C Distance traveled (cm)

and N-methyltryptamine protected minimally against sigma-1 receptor [125I]-IACoc photolabeling, even at these high concentrations (Fig. 2A). Similarly, [125I]IAF photolabeling of the sigma-1 [Kd = 194 nM (18)] receptor showed that DMT was the most potent protector. Ten micromolar DMT provided 31% protection, whereas 50 and 100 mM DMT provided 43 and 69% protection, respectively (Fig. 2B). With the exception of N-methyltryptamine, protection of [125I]IAF sigma-2 [Kd = 2780 nM (18)] receptor photolabeling paralleled the sigma-2 binding data. Tryptamine afforded the greatest protection of sigma-2 receptor photolabeling, with values of 47, 78, and 79% for 10, 50, and 100 mM, respectively (Fig. 2B). An important biological activity of sigma receptor activation is the inhibition of ion channels, which operates through protein-protein interactions without mediation by G proteins and protein kinases (20–22). In addition to modulating various types of voltage-activated K+ channels (21, 23, 24), the sigma-1 receptor associates with the Kv1.4 K+ channel in posterior pituitary nerve terminals, as well as in Xenopus oocytes (22). Sigma receptor ligands also modulate N-, L-, P/Q-, and R-type Ca2+ channels in rat sympathetic and parasympathetic neurons (25). Sigma receptor ligands modulate cardiac voltage-gated Na+ channels (hNav1.5) in human embryonic kidney 293 (HEK293) cells, COS-7 cells, and neonatal mouse cardiac myocytes (26). To evaluate the capacity of DMT to induce physiological responses by binding to sigma receptors, we examined the action of DMTon voltageactivated Na+ current. Patch-clamp recordings from HEK293 cells stably expressing the human cardiac Na+ channel hNav1.5 revealed voltage-activated Na+ currents (INa) in response to voltage steps from –80 to –10 mV (Fig. 3B). Application of 100 mM DMT inhibited INa by 62 T 3% (n = 3 HEK293 cells), which reversed upon DMT removal. With hNav1.5 transiently transfected into COS-7 cells,

6000

WT Meth

5000

KO Meth

4000 3000 2000 1000 0 0 10 20 30 40 50 60 70 80 90 100110120130

Time (min)

Meth

100 mM DMT inhibited INa by only 22 T 4% (n = 3 COS-7 cells), but photolabeling has shown that these cells have much lower concentrations of endogenous sigma-1 receptors compared to HEK293 cells (fig. S1 and Fig. 3B). The difference between DMT inhibition of INa in HEK293 and COS-7

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cells (Fig. 3B, P < 0.03) thus demonstrates the dependence of INa inhibition on sigma-1 receptors. Experiments in cardiac myocytes demonstrated the same DMT action in a native preparation (Fig. 3C) and enabled further demonstration of sigma-1 receptor dependence by using a sigma-1 receptor

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Percent Inhibition

Percent Inhibition

* Fig. 3. Sodium chanA B COS-7 HEK293 70 nel inhibition by DMT. [125I]-IAF 60 (A) In the presence or WT Sigma-1 KO absence of 10 mM hal50 operidol, wild type (WT) 40 + kDa 10 µM Haloperidol + or sigma-1 receptor knock30 out (KO) mouse liver ho20 66 mogenates (200 mg/lane) 10 were photolabeled with 0 45 125 1 nM [ I]IAF. (B) ExamHEK293 COS-7 ples of INa evoked by C 31 * 35 Sigma-1 KO WT steps from −80 to −10 mV Sigma-1 26 kDa 30 in HEK293 or COS-7 cells 21.5 25 expressing hNav1.5 chan- Sigma-2 18 kDa 20 nel in the absence (con14.4 15 trol, black), presence (DMT, 10 red), and after wash out 5 (recovery, blue) of 100 mM DMT. Average inhibition by DMT was determined by measuring peak INa. Bars rep0 WT KO resent mean T SEM (n = 3 cells). INa inhibition in HEK293 cells differed significantly from that in COS-7 cells (*P < 0.03). (C) Examples of INa evoked as described in (B) in neonatal cardiac myocytes from WT and KO mice in the absence (control, black), presence (DMT, red), and after wash out (recovery, blue) of 100 mM DMT. Current inhibition in WT was significantly different from that in KO (*P < 0.002, n = 7 neonatal cardiac myocytes).

REPORTS relevant, because sigma-1 receptors, which are observed in the endoplasmic reticulum, associate with plasma membrane Kv 1.4 channels (22) and may serve as a molecular chaperone for ion channels. Furthermore, the behavioral effect of DMT may be due to activation or inhibition of sigma-1 receptor chaperone activity instead of, or in addition to, DMT/sigma-1 receptor modulation of ion channels. These studies thus suggest that this natural hallucinogen could exert its action by binding to sigma-1 receptors, which are abundant in the brain (1, 27). This discovery may also extend to N,Ndimethylated neurotransmitters such as the psychoactive serotonin derivative N,N-dimethylserotonin (bufotenine), which has been found at elevated concentrations in the urine of schizophrenic patients (10). The finding that DMT and sigma-1 receptors act as a ligand-receptor pair provides a long-awaited connection that will enable researchers to elucidate the biological functions of both of these molecules. References and Notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

T. Hayashi, T. P. Su, CNS Drugs 18, 269 (2004). P. Bouchard et al., Eur. J. Neurosci. 7, 1952 (1995). T. P. Su, A. D. Weissman, S. Y. Yeh, Life Sci. 38, 2199 (1986). T. P. Su, E. D. London, J. H. Jaffe, Science 240, 219 (1988). R. A. Wilke et al., J. Physiol. 517, 391 (1999). R. A. Glennon et al., J. Med. Chem. 37, 1214 (1994). F. F. Moebius, R. J. Reiter, M. Hanner, H. Glossmann, Br. J. Pharmacol. 121, 1 (1997). S. A. Barker, J. A. Monti, S. T. Christian, Int. Rev. Neurobiol. 22, 83 (1981). F. Franzen, H. Gross, Nature 206, 1052 (1965). M. S. Jacob, D. E. Presti, Med. Hypotheses 64, 930 (2005). J. Axelrod, Science 134, 343 (1961). J. M. Saavedra, J. Axelrod, Science 175, 1365 (1972). J. M. Beaton, P. E. Morris, Mech. Ageing Dev. 25, 343 (1984). S. A. Burchett, T. P. Hicks, Prog. Neurobiol. 79, 223 (2006).

15. B. Borowsky et al., Proc. Natl. Acad. Sci. U.S.A. 98, 8966 (2001). 16. L. Lindemann et al., Genomics 85, 372 (2005). 17. J. R. Kahoun, A. E. Ruoho, Proc. Natl. Acad. Sci. U.S.A. 89, 1393 (1992). 18. A. Pal et al., Mol. Pharmacol. 72, 921 (2007). 19. Y. Chen, A. R. Hajipour, M. K. Sievert, M. Arbabian, A. E. Ruoho, Biochemistry 46, 3532 (2007). 20. P. J. Lupardus et al., J. Physiol. 526, 527 (2000). 21. H. Zhang, J. Cuevas, J. Pharmacol. Exp. Ther. 313, 1387 (2005). 22. E. Aydar, C. P. Palmer, V. A. Klyachko, M. B. Jackson, Neuron 34, 399 (2002). 23. R. A. Wilke et al., J. Biol. Chem. 274, 18387 (1999). 24. C. Kennedy, G. Henderson, Neuroscience 35, 725 (1990). 25. H. Zhang, J. Cuevas, J. Neurophysiol. 87, 2867 (2002). 26. M. A. Johannessen, A. Ramos-Serrano, S. Ramachandran, A. E. Ruoho, M. B. Jackson, “Sigma receptor modulation of voltage-dependent sodium channels,” Program No. 466.22, Annual Neuroscience Meeting, San Diego, CA, 5 November 2007. 27. F. Langa et al., Eur. J. Neurosci. 18, 2188 (2003). 28. P. Jenner, C. D. Marsden, C. M. Thanki, Br. J. Pharmacol. 69, 69 (1980). 29. R. R. Matsumoto, B. Pouw, Eur. J. Pharmacol. 401, 155 (2000). 30. T. Hayashi, T. P. Su, Cell 131, 596 (2007). 31. We thank the Corinna Burger laboratory for use of their mouse behavior equipment, and A. Paul and T. Mavlyutov for providing [125I]IAF and [125I]-IACoc, respectively. Supported by the Molecular and Cellular Pharmacology (MCP) Graduate Program training grant from NIH T32 GM08688 and by the NIH Ruth L. Kirschstein National Research Service Award (NRSA) (F31 DA022932) from the National Institute on Drug Abuse (to D.F.). This work was funded by NIH grants R01 MH065503 (to A.E.R.) and NS30016 (to M.B.J.).

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knockout mouse (27). [125I]IAF photolabeling of liver homogenates from wild-type (WT) and sigma-1 receptor knockout (KO) mice indeed showed the absence of sigma-1 receptor (26 kD) in the KO samples (Fig. 3A). In WT neonatal cardiac myocytes, 100 mM DMT reversibly inhibited INa by 29 T 3% (n = 7 WT myocytes), whereas INa was reduced by only 7 T 2% (n = 7 KO myocytes) in KO myocytes (Fig. 3C, P < 0.002). Both DMT and sigma receptor ligands influence animal behavior. DMT injection induces hypermobility in rodents concurrently treated with the monoamine oxidase inhibitor pargyline (28), and this action is not antagonized by blockers of dopamine or serotonin receptors, but is potently inhibited by haloperidol (28). Although haloperidol is thought to act in part through the dopamine D2 receptor system, it is also a potent sigma-1 receptor agonist [sigma-1 inhibition constant (Ki) = 3 nM (29); sigma-2 Ki = 54 nM (29)] when inhibiting voltage-gated ion channels (5, 25). Haloperidol reduces brain concentrations of DMT (8) and DMT inhibits haloperidol binding in brain tissue more robustly than the dopamine agonist apomorphine (8). On the basis of these findings, which were discovered before sigma receptor identification, DMT has been hypothesized to act through an unknown “hallucinogen” receptor (8). We confirmed results (28) that intraperitoneal (ip) administration of DMT (2 mg per kilogram of body weight) 2 hours after pargyline (75 mg/kg, ip) injection induced hypermobility in WT mice (7025 T 524.1 cm, n = 12 WT mice) in an open-field assay. Identical drug treatments in sigma-1 receptor KO mice had no hypermobility action (2328 T 322.9 cm, n = 12 KO mice, P < 0.0001; Fig. 4, A and B). This result is particularly important to our understanding of sigma-1 receptor biological function because the KO mice are viable and fertile (27). The sigma-1 receptor dependence of DMT-induced hypermobility parallels that induced by the sigma-1 receptor ligand (+)-SKF10047 in WT but not in KO mice (27). As a positive control, methamphetamine, which is thought to act through catecholaminergic systems, induced hypermobility in both WT and KO mice (3 mg/kg, ip, n = 6 mice; Fig. 4, B and C) with a reduced onset rate compared with that seen for DMT (Fig. 4, A and C). This indicates that behavioral actions of DMT depend on the sigma-1 receptor, which may provide an alternative research area for psychiatric disorders that have not been linked to dopamine or N-methyl-Daspartate systems. The binding, biochemical, physiological, and behavioral studies reported here all support the hypothesis that DMT acts as a ligand for the sigma-1 receptor. On the basis of our binding results and the sigma-1 receptor pharmacophore, endogenous trace amines and their N-methyl and N,N-dimethyl derivatives are likely to serve as endogenous sigma receptor regulators. Moreover, DMT, the only known mammalian N,N-dimethylated trace amine, can activate the sigma-1 receptor to modulate Na+ channels. The recent discovery that the sigma-1 receptor functions as a molecular chaperone (30) may be

Supporting Online Material www.sciencemag.org/cgi/content/full/323/5916/934/DC1 Materials and Methods Fig. S1 and scheme S2 References 18 September 2008; accepted 10 December 2008 10.1126/science.1166127

When Your Gain Is My Pain and Your Pain Is My Gain: Neural Correlates of Envy and Schadenfreude Hidehiko Takahashi,1,2,3* Motoichiro Kato,4 Masato Matsuura,2 Dean Mobbs,5 Tetsuya Suhara,1 Yoshiro Okubo6 We often evaluate the self and others from social comparisons. We feel envy when the target person has superior and self-relevant characteristics. Schadenfreude occurs when envied persons fall from grace. To elucidate the neurocognitive mechanisms of envy and schadenfreude, we conducted two functional magnetic resonance imaging studies. In study one, the participants read information concerning target persons characterized by levels of possession and self-relevance of comparison domains. When the target person’s possession was superior and self-relevant, stronger envy and stronger anterior cingulate cortex (ACC) activation were induced. In study two, stronger schadenfreude and stronger striatum activation were induced when misfortunes happened to envied persons. ACC activation in study one predicted ventral striatum activation in study two. Our findings document mechanisms of painful emotion, envy, and a rewarding reaction, schadenfreude. nvy is one of the seven biblical sins, the Shakespearian “green-eyed monster,” and what Bertrand Russell (1) called an unfortunate facet of human nature. It is an irrational, unpleasant feeling and a “painful emotion” (2)

E

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characterized by feelings of inferiority and resentment produced by an awareness of another’s superior quality, achievement, or possessions (3). Understanding envy is important because of its broad implications, ranging from individual mat-

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Drug and Alcohol Dependence 111 (2010) 30–37

Contents lists available at ScienceDirect

Drug and Alcohol Dependence journal homepage: www.elsevier.com/locate/drugalcdep

Dimethyltryptamine (DMT): Subjective effects and patterns of use among Australian recreational users Vince Cakic a,∗ , Jacob Potkonyak b , Alex Marshall a a b

School of Psychology, University of Sydney, Sydney, NSW 2006, Australia School of Psychology, Macquarie University, Sydney, NSW 2109, Australia

a r t i c l e

i n f o

Article history: Received 16 February 2010 Received in revised form 7 March 2010 Accepted 9 March 2010 Available online 31 May 2010 Keywords: Dimethyltryptamine DMT Ayahuasca Hallucinogens Psychedelics

a b s t r a c t Dimethyltryptamine (DMT) is an endogenous hallucinogen with traditional use as a sacrament in the orally active preparation of ayahuasca. Although the religious use of ayahuasca has been examined extensively, very little is known about the recreational use of DMT. In this study, Australian participants (n = 121) reporting at least one lifetime use of DMT completed an online questionnaire recording patterns of use, subjective effects and attitudes towards their DMT use. Smoking DMT was by far the most common route of administration (98.3%) with a comparatively smaller proportion reporting use of ayahuasca (30.6%). The reasons for first trying DMT were out of a general interest in hallucinogenic drugs (46.6%) or curiosity about DMT’s effects (41.7%), while almost one-third (31.1%) cited possible psychotherapeutic benefits of the drug. An increase in psychospiritual insight was the most commonly reported positive effect of both smoked DMT (75.5%) and ayahuasca (46.7%), a finding that is consistent with other studies examining the ritualised use of ayahuasca in a religious context. Although previous studies of DMT use have examined ayahuasca use exclusively, the present study demonstrates the ubiquity of smoking as the most prevalent route of administration among recreational DMT users. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Dimethyltryptamine (DMT) is a naturally occurring hallucinogen similar in structure to both serotonin and psilocybin. Like other hallucinogens, DMT is a serotonin 5-HT2A receptor agonist (Smith et al., 1998), in addition to being a ligand of both trace amine-associated receptor 1 (TAAR1; Bunzow et al., 2001) and the sigma-1 (␴-1) receptor (Fontanilla et al., 2009). It is found ubiquitously across the plant and animal kingdoms (McKenna, 2004) and, although it is a relatively obscure drug, its presence in the popular media (Otis, 2009; De Conceicao, 2009; Grigoriadis, 2006; Cox, 2009) suggests that interest or use in Western countries is increasing. Szára (1956) was the first to report its hallucinogenic effects, however, its consumption in the hallucinogenic plant beverage ayahuasca dates to pre-Colombian times and continues to the present day (Pomilio et al., 1999; Grob et al., 1996). Ayahuasca, which translates to “the vine of the souls” by the Amazon-dwelling Quechua people among whom it is traditionally used, is consumed as a sacrament throughout the Amazon Basin by indigenous populations (Dobkin de Rios, 1972; McKenna, 2006). Although it is an umbrella term used to describe any orally active DMT preparation, ayahuasca is usually prepared by infus-

∗ Corresponding author. E-mail address: [email protected] (V. Cakic). 0376-8716/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.drugalcdep.2010.03.015

ing the DMT-bearing leaves of Psychotria viridis with the stems of the Banisteriopsis caapi vine (McKenna, 2004). The latter contains ␤-carboline alkaloids, chiefly harmine, tetrahydroharmine, and harmaline. Ordinarily, DMT is rapidly metabolised by gut and liver monoamine oxidase (MAO), making it orally inactive. However, the ␤-carbolines display MAO inhibition and their co-administration enables DMT to reach the CNS intact. The hallucinogenic effects of ayahuasca commence within an hour of its consumption and last approximately 4 h. In contrast to oral ingestion, the effects of smoked DMT commence almost immediately, peak within several minutes and typically resolve within 30 min (Turner, 1994; Strassman, 2001). Smokeable freebase DMT may be obtained from a variety of flora through a simple extraction process, the instructions for which are readily available on the Internet (Halpern and Pope, 2001). Owing to the brevity and intensity of its effects, smoked DMT has been facetiously coined the “businessman’s lunch trip” (Turner, 1994) and this contrasts the duration of LSD, whose effects last 8–12 h (Rothlin, 1957). In the Amazon Basin, DMT is legal and its use, in the form of ayahuasca, is a key component of the religious practices of several syncretic churches including the União do Vegetal (UDV) and Santo Daime (MacRae, 1998). In the Western world, however, DMT remains a controlled substance, although recent rulings by the United States Supreme Court now protect the religious use of ayahuasca in the United States under the Religious Freedom

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Restoration Act (1993; Bullis, 2008). To date, several studies have examined the religious use and effects of ayahuasca among adolescent (Da Silveira et al., 2005; Doering-Silveira et al., 2005a,b), as well as first-time (Trichter et al., 2009) and long-term (Grob et al., 1996; Halpern et al., 2008) ayahuasca users. These studies have generally found ayahuasca to be psychologically beneficial, or at worst, lacking in deleterious effects when consumed in an appropriate religious context. Users generally report that ayahuasca facilitates an experience characterised by complex and semantically rich visual hallucinations of personal or spiritual significance (Shanon, 2002) and this is consistent with findings that the chemically related hallucinogen psilocybin may evoke religious experiences in hallucinogen-naive subjects (Griffiths et al., 2006, 2008). In recent years there appears to have been growing interest in hallucinogens such as DMT in countries such as Australia and the United States (Otis, 2009; De Conceicao, 2009; Grigoriadis, 2006; Cox, 2009). This may be due to the emergence of the outdoor rave or ‘doof’ music subculture (Luckman, 2003; Tramacchi, 2000) and the proliferation of neo-shamanic practises in the West (Tupper, 2008, 2009). There is an apparent increase in Internet resources surrounding the use of hallucinogens (Halpern and Pope, 2001; Boyer et al., 2005) as well as the expansion of online “head shops” legally trading in the sale of psychoactive plant material, including ayahuasca ingredients (Dalgarno, 2008). Interest may exist in particular for DMT given its endogenous presence in humans (Barker et al., 1981) and its unique and bizarre phenomenology (Shanon, 2002; Strassman, 2001). Despite the aforementioned studies of ayahuasca, recreational1 use of DMT in Western countries remains largely unexamined. Previous research has investigated the recreational use of the shortacting hallucinogen Salvia divinorum (Gonzalez et al., 2006; Lange et al., 2008), yet a similar study of DMT has not previously been undertaken. In his ethnographic study of Australian DMT users, Tramacchi (2006) examined the phenomenological aspects of the DMT experience, however, little is known regarding the patterns of DMT use among recreational users. Furthermore, data regarding the use of smokeable DMT remain scant. The present study seeks to address the paucity of such data by examining Australian recreational DMT users. The use of DMT within Australia may be of particular interest given the presence of DMT in several native species of Acacia (Fitzgerald and Sioumis, 1965; Rouvelli and Vaughan, 1967) as well as the veritable existence of an electronic ‘bush doof’ music subculture with which hallucinogen use is commonly associated (Tramacchi, 2000; Luckman, 2003). In the absence of existing literature regarding recreational DMT use, this study is exploratory in nature and seeks to identify the demographics and general pattern of use, as well as subjective effects and attitudes towards DMT in a sample of recreational users of the drug. 2. Method 2.1. Sample Individuals who were Australian residents aged 18 years and over and who had used DMT at least once in their lifetime were recruited for the study. Data were collected between July and August 2009, with participants recruited via ‘snowballing’ (Biernacki and Waldorf, 1981) involving individuals known to the researchers, and an advertisement in an Australian hallucinogen-related internet newsletter that contained a link to an online questionnaire. Respondents were assured of their anonymity and the study conformed to National Health and Medical Research Council ethics guidelines for human research.

1 Given its traditional use as a sacrament it is likely that DMT use deemed to be ‘recreational’ involves the use of this drug for religious purposes. Although it is acknowledged as a misnomer, in the present study the term ‘recreational’ will imply the illicit use of DMT outside of a recognised religious institution such as Sainto Daime.

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2.2. Questionnaire The self-administered online questionnaire included both closed and openended items and took approximately 25 min to complete. The questionnaire comprised four main sections: (1) demographic information: including age, gender, postcode, religious affiliation, education and occupation; (2) history of other drug use: including previous drugs consumed, and drugs consumed in the previous 12 months, 6 months, and 30 days; (3) history of DMT use: including age of initiation, number of occasions used, routes of administration, sources for obtaining DMT, and locations consumed; (4) patterns of use and attitudes about DMT: including reasons for first consuming DMT, positive and negative aspects of both smoked DMT and ayahuasca, patterns of use of each route of administration, and perceived risks and harm reduction regarding DMT use. Items regarding DMT use were distinguished via the two main routes of administration, smoked and ayahuasca. Respondents were asked to complete sections relevant to their reported route of administration; those who had consumed DMT via both routes were required to complete both sections. Additionally, cannabis dependence was measured using the Severity of Dependence Scale (SDS) adapted for cannabis use in the previous 30 days. The SDS is a 5-item questionnaire with reliability in identifying cannabis dependence (Hildes et al., 2007; Martin et al., 2005). Although a cut-off SDS score of ≥3 is considered optimal (Swift et al., 1998), the present study used a cut-off of ≥4 in order to increase measure specificity. 2.3. Data analyses The majority of the analyses were descriptive in nature. Percentages were reported for categorical variables. For continuous normally distributed variables, mean, standard deviation and range were reported and t-tests were employed with a 0.05 level of significance. For skewed continuous variables, median and range were reported and the Mann–Whitney U-test was used. Qualitative responses for openended questions were coded according to themes and percentages reported. Unless otherwise noted, the response rate of each item was n = 121.

3. Results 3.1. Demographic characteristics A total of 121 lifetime DMT users were recruited, the majority of whom were male (86%). Respondents ranged in age from 18 to 68 years, with a median age of 28 years. The majority of respondents were Australian-born (86.8%) and one reported being of indigenous Australian descent. Almost three-quarters (73.6%) reported having no religious affiliation, and only 4.1% belonged to a mainstream monotheistic religion such as Christianity. The remainder cited pantheistic religions (e.g. shamanism; 11.6%), esoteric or non-specific beliefs (e.g. “non-religious, intuitively spiritual”; 8.3%), or Eastern mysticism (e.g. Buddhism; 2.5%). Mean of total years of formal education was 15.9 (S.D. 2.9; range 10–24), with 60.3% of the sample having completed some university or higher. Onethird (35.5%) reported full-time employment, with another third reporting part-time or casual employment (34.8%), 14.8% identifying themselves as students and 10.7% as unemployed. Another 4.1% of the sample listed home duties as their primary occupation. 3.2. Overall pattern of drug use Lifetime history of drug use was extensive among the sample (Table 1). A total of 92 discrete drugs had been used by the respondents. The mean number of drugs ever tried was 17.3 (maximum allowed 35, range 4–34.). Although the use of other hallucinogens was typical, drug use extended to a disparate array of drug classes including stimulants, opiates, depressants and anaesthetics. Use of various mildly psychoactive plants such as damiana (Turnera diffusa) was also reported but excluded from this analysis. All but two respondents (98.3%) reported lifetime use of alcohol and cannabis, and 89.3% and 73.6% had consumed these drugs in the previous 30 days, respectively. Almost one-third of the sample (31.4%) had consumed five or more standard drinks of alcohol in a single sitting on five or more occasions in the past 30 days. Using a cut-off score of ≥4 in the SDS for cannabis use in the previous 30 days, 26.4% of the sample appeared to be cannabis dependent.

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Table 1 Patterns of drug use of DMT users in the study (n = 121). Drug class

Ever used (%)

Used last 6 months (%)

Used last 30 days (%)

Days used last 30 days (median)a

DMT Cannabis Alcohol LSD Tobacco MDMA Psilocybin Meth/amphetamine Nitrous oxide Cocaine Salvia divinorum Mescaline Amyl nitrite Benzodiazepinesb Ketamine Other opiatesb 2C-B MDA Peganum harmala Banisteriopsis caapi Other stimulantsb DXM Heroin GHB

100.0 98.3 98.3 96.7 95.9 94.2 91.7 83.5 79.3 72.7 67.8 64.5 60.3 58.7 56.2 50.4 44.6 43.0 42.1 38.8 38.8 29.8 26.4 24.8

68.6 82.6 91.7 63.6 74.4 55.4 50.4 26.4 33.9 24.8 14.9 21.5 8.3 18.2 14.0 12.4 17.4 2.5 18.2 23.1 8.3 3.3 5.8 1.7

34.7 73.6 89.3 30.6 66.9 31.4 29.8 14.0 14.9 12.4 6.6 8.3 5.0 9.9 5.0 8.3 4.1 1.7 10.7 4.1 3.3 1.7 1.7 0.8

2 15 8 1 29 1 1.5 3 1 1 1 1 1.5 2.5 1 2.5 1 2 1 1 2.5 1.5 11.5 1

a b

Among those who had ever used the drug. Involving non-medical use.

Including alcohol and tobacco, the median number of discrete drugs that had been consumed by the sample in the previous 30 days was 6 (range 0–16). Hallucinogens most widely consumed included LSD (96.7%), psilocybin mushrooms (91.7%), S. divinorum (67.8%), mescaline (64.5%) and ketamine (56.2%). Several plant-based drugs associated with DMT use were also frequently reported by the sample; the MAO inhibitors Peganum harmala (Syrian rue; 42.1%) and B. caapi (38.8%), as well as the compounds 5-methoxy-dimethyltryptamine (5-MeO-DMT) and bufotenin, which are present in Anadenanthera peregrina snuffs (11.6%; Ott, 1996). Respondents reported use of ‘party drugs’ such as MDMA (94.2%), meth/amphetamine (83.5%), cocaine (72.7%), amyl nitrite (60.3%) and GHB or related compounds (24.8%). Over one-quarter reported previous use of heroin (26.4%), while past non-medical use of opiates (50.4%), benzodiazepines (58.7%) and stimulants such as methylphenidate (38.8%) was also commonplace. Several plant-based psychoactives such as kava (Piper methysticum; 9.1%), Amanita muscaria (6.6%), ergine containing species such as Ipomoea violacea and Argyreia nervosa (6.6%), opium (6.6%), and anticholinergic deleriants such as Datura spp. and Brugmansia spp. (6.6%) were also reported. Interestingly, over 25 synthetic tryptamine and phenethylamine ‘research chemicals’ (Shulgin and Shulgin, 1991, 1997) were also widely reported. The most commonly used compounds of this class included 2C-B (44.6%), 2C-I (13.2%), 2C-E (9.1%) and 2C-T-7 (7.4%). More than a quarter (28.1%) reported having injected drugs intravenously for non-medical purposes on at least one occasion, and 7.4% had previously injected drugs with a needle that had been used by another person. Refer to Table 1 for frequency of drug use in the previous 6 months and 30 days, in addition to median number of days used in the past 30 days. 3.3. DMT use The median age of initiation of DMT use was 24 years (range 15–65 years). The median total number of occasions respondents had used DMT was 10 times (range 1–400 times). The most common route of administering DMT was smoking (98.3%), fol-

lowed by oral administration in the form of ayahuasca (30.6%). Those who reported having previously consumed ayahuasca took DMT on significantly more occasions than those who did not (median = 30 versus 6, U = 732, p < 0.001). Less common routes of administration included insufflation (5%) and injection (2.5%). Eleven participants (9.1%) had consumed DMT on only one occasion; 34.7% had used it on 20 or more occasions. Approximately two-thirds (68.6%) of respondents had used DMT in the past 6 months, while 86.7% had used it in the past 12 months. Median duration of DMT use was 2 years (range 6 months to 31 years). The most frequently cited sources for first having heard about DMT was primarily through friends (47.9%), the Internet (24.8%) and print media (22.3%). The median number of friends that the respondent had stated had also taken DMT was 15 (range 1–500). There was a strong positive correlation between the number of friends that had taken DMT with the respondent’s total occasions used (r = 0.67, p < 0.001). The median number of people that the respondents had given DMT to was 3 (range 0–200). 3.4. Obtaining DMT The reported usual sources for obtaining or purchasing DMT included friends (60.3%) and acquaintances (9.1%), friends that were dealers (14.9%), known dealers (5.8%) and rarely, dealers that were strangers (2.5%). Moreover, approximately one-quarter (26.4%) reported that they extract DMT themselves from plant material that they find, grow or purchase. Obtaining or purchasing the necessary plant material was rated as very easy (26.4%), somewhat easy (30.6%), somewhat difficult (20.7%), very difficult (9.1%), and unsure (13.2%). Almost half of respondents (48.8%) reported not knowing the street price of 1 g of ready-toconsume DMT. Of those that did, the median quoted price was $150/g (range $35–1000/g). The ease with which DMT could be obtained was rated as very difficult (19.8%), somewhat difficult (36.4%), somewhat easy (26.4%), very easy (11.6%), and unsure (5.8%).

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3.5. Reasons for trying

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Participants were asked why they initially tried DMT, the reasons for which were subsequently coded according to themes (n = 103). The most commonly cited reasons were out of a general interest in hallucinogenic or plant-based drugs (46.6%) and curiosity about DMT’s effects (41.7%). Interestingly, almost one-third (31.1%) stated that they tried DMT for its potential psychotherapeutic benefits, such as personal insight or self exploration. Of a similar theme was the potential spiritual benefits of DMT (29.1%) such as “finding God” or “finding spiritual meaning”. Being given DMT or having it recommended by others was also mentioned (18.4%) as a reason for trying the drug. Only three (2.9%) respondents stated that they first consumed DMT for fun.

tions produced under its influence (21.4%) were also mentioned as positive effects. When asked what the negative aspects of smoked DMT were almost half (49.6%) reported difficulties with the act of smoking the drug. This included the bad taste, coughing and possible respiratory damage from smoking. Other negative aspects included psychological or spiritual conflicts (23.5%). Descriptions included an overwhelming change in psychological or spiritual outlook or worldview that may be difficult to integrate once the effects of DMT had worn off. Approximately 1 in 10 (10.9%) also noted an experience under the influence of DMT characterised by marked anxiety or stress. Interestingly, the brief duration of smoked DMT was also cited as a negative aspect of the experience (10.9%), as was its illegality (5.9%).

3.6. Location of usage

3.8. Ayahuasca

Respondent’s usual places of DMT use were: their own home (81.8%), outdoors (55.4%), a friend’s home (52.1%), doofs (24.8%), private parties (16.5%), public places (12.4%) and the home of an acquaintance (5%). Users generally took DMT in a small group of up to four friends (76.9%), alone (52.1%), with their partner (26.4%), in a large group of five or more friends (9.1%) or in ayahuasca ceremonies (5%). Those who reported commonly taking DMT alone used DMT on significantly more occasions than those who did not (median = 20 versus 5, U = 971, p < 0.001).

Thirty-seven (30.6%) respondents reported having consumed ayahuasca on at least one occasion. Interestingly, only two ayahuasca users reported not having smoked DMT. Thus, although many DMT smokers had not consumed ayahuasca, the vast majority (94.6%) of ayahuasca users had also smoked DMT. Analysis indicated that ayahuasca users had smoked DMT on more occasions than those who reported only smoking DMT (median 20 versus 7, U = 851.5, p < 0.001). The median number of occasions ayahuasca had been consumed was four times (range 1–180). As previously noted, an MAO inhibitor must be consumed for DMT to be orally active (McKenna, 2004). The most commonly reported MAO inhibitors were B. caapi (73%) and P. harmala (54.1%). In contrast to smoking DMT, users reported not usually combining ayahuasca with any other drugs (73%). This was generally independent of whether or not the same individual reported smoking DMT with other drugs. In the 10 users who commonly used other drugs with ayahuasca, the most frequently used other drugs were cannabis (7 users) and psilocybin (3). As with smoked DMT, respondents were asked to cite the positive (n = 30) and negative (n = 25) aspects of ayahuasca (Table 2). Almost half (46.7%) felt that the ayahuasca experience offered them personal insight. One-third also stated that the experience was more psychologically cleansing or cathartic in comparison to smoked DMT. The longer duration of ayahuasca relative to smoked DMT (30%) and a somewhat smoother onset of action and more gentle experience (30%) were also noted. The unusual psychic phenomena characteristic of ayahuasca such as apparent near-death or out-of-body experiences (Shanon, 2002) was also mentioned as a positive aspect of the preparation (20%). Ayahuasca’s putrid taste and the associated nausea characteristic of the experience were by far the most commonly reported negative aspect (44%). One-fifth (20%) also reported ayahuasca’s

3.7. Smoked DMT All but two of the 121 respondents (98.3%) reported having previously smoked DMT on at least one occasion. The median number of occasions users had smoked DMT was 8 (range 1–400). By far the most common methods for smoking were through a waterpipe or ‘bong’ (55.5%), glass pipe (54.6%), or in a cigarette or joint (29.4%). Other methods included non-glass pipes (10.1%) and vaporisers (5%). Approximately two-thirds (68.1%) of DMT smokers reported commonly smoking DMT with other drugs. In those who did, cannabis was the most commonly combined drug (53.1%), followed by LSD (28.4%), alcohol (27.2%), psilocybin (19.8%) and MDMA (8.6%). Moreover, 42% of DMT smokers reported concomitant use of an MAO inhibitor such as B. caapi or P. harmala. Respondents were asked to nominate up to five positive (n = 98) and five negative aspects (n = 103) of smoked DMT and responses were coded according to themes (Table 2). Three-quarters (75.5%) of the sample reported a positive aspect of smoked DMT to be a personally meaningful or insightful experience, and over half (54.1%) cited spiritual experiences. Feelings of euphoria (29.6%), the brief duration of smoked DMT (27.6%) and the intense visual hallucina-

Table 2 Five most frequently mentioned positive (n = 98) and negative (n = 103) aspects of smoked DMT, and positive (n = 30) and negative (n = 25) aspects of ayahuasca. Positive aspects

Percentage (%)

Negative aspects

Percentage (%)

Smoked DMT Meaningful or insightful Spiritual experiences Euphoria Short duration Visual hallucinations

75.5 54.1 29.6 27.6 21.4

Bad taste or coughing Psychospiritual distress Anxiety or stress during Short duration Legal status

49.6 23.5 10.9 10.9 5.9

Ayahuasca Meaningful or insightful Cleansing or cathartic Long duration Smooth, gentle experience Unusual psychic phenomena

46.7 33.3 30.0 30.0 20.0

Nausea, taste, or purge Long duration Psychospiritual distress Intensity Poorly prepared brews

44.0 20.0 16.0 12.0 12.0

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long duration. It was commonly reported that unlike smoked DMT, the longer duration of ayahuasca meant that the trip could not end shortly thereafter should a negative experience occur. As with smoked DMT, psychospiritual conflict was also mentioned (16%) as a negative aspect of ayahuasca. Ayahuasca’s intensity (12%) and poorly prepared brews (12%) were also noted as negative aspects. 3.9. Perceived dangers Participants rated on a 5-point scale how safe they thought it was to use DMT for them personally. The majority of the sample generally considered their use to be safe, with most deeming it to be either very safe (54.5%), quite safe (38%) or moderately safe (6.6%). No participant reported having sought medical attention as a consequence of their DMT use, and only one stated having previously driven whilst under the influence of ayahuasca (2.7% of ayahuasca users). Respondents were asked to state what they felt were the three main risks about DMT use in general (n = 111). The most frequently reported risk for DMT was having a bad trip (50.5%), or a frightening experience while under the influence of DMT. Commonly cited was the potential for psychospiritual problems arising as a consequence of DMT use (39%). Responses generally reflected the belief that DMT could produce an overwhelming change in spiritual or psychological outlook or worldview that may be difficult to integrate once the effects of the drug had worn off. Over a quarter (26.3%) also stated aversive physiological reactions from consuming DMT. In the case of smoked DMT, respiratory irritation from inhaling smoke was the primary complaint while nausea was the main physical concern for ayahuasca use. Other dangers included falls or burns arising as a consequence of not remaining seated under its influence (22.1%) as well as the disrespectful or irreligious use of DMT (17.9%). When asked how the risks associated with DMT could be reduced (n = 103), approximately half (50.5%) mentioned the need for greater education regarding safe use. The presence of another individual was deemed important (34.7%) as well as the need to consume DMT with an appropriate mindset (28.4%) and in a suitable context or environment (28.4%). The legalisation of DMT (32.6%) and limiting one’s use of the drug (29.5%) were also mentioned. Ensuring purity of the DMT consumed (9.5%) was also noted as an important factor in harm minimisation. 4. Discussion This study provides the first comprehensive investigation of recreational DMT use by examining 121 Australian DMT users. Sample demographics indicate that DMT users are generally employed or undertaking higher education, and are less likely to be subject to the social and economic marginalisation characteristic of other drug using populations (Room, 2005). The main routes of administration for recreational use were smoking DMT and ayahuasca, with smoking being the most common method. Given that almost all ayahuasca users had also smoked DMT, but not all DMT smokers had used ayahuasca, the data suggest that the typical pattern of use commences with smoked DMT. Thereafter, some but not all users will progress to ayahuasca use, but clearly this requires further investigation. 4.1. Pattern of use 4.1.1. Initiation and use. Consumption of DMT appears to be mediated by peers. Friends were the primary source in regard to first hearing about DMT and were a common source for providing the drug. This suggests that a friend will initiate an individual to DMT, who will subsequently introduce it to others. The high correlation

between the number of friends who had taken DMT and the respondent’s own times used further supports this. The influence of peers in drug initiation and use is well characterised (Doherty et al., 2000; Andrews et al., 2002; Kandel et al., 1978) and this appears to be the case with DMT. An interest in hallucinogens and curiosity about the effects of DMT seemed to be the overarching reasons for initiation of use. Although it was not examined in the present study, it would be of benefit to determine the drugs used preceding initiation of DMT use. Of particular relevance was the fact that many sought to consume DMT for psychospiritual benefits. This is consistent with the ritualised use of ayahuasca by members of the UDV and Sainto Daime (McKenna, 2004; Shanon, 2002). In our unpublished qualitative data, the belief in the psychotherapeutic or spiritual benefits of the drug appears to be a recurrent factor influencing continued use of DMT. 4.1.2. Smoked DMT. Smoking was the most common route of administration and had been employed by almost all respondents. Smoked DMT is frequently combined with cannabis or other hallucinogens. Anecdotally, DMT users sprinkle DMT over cannabis which is subsequently smoked through a bong (Turner, 1994). Of note is the concomitant use of plant-based MAO inhibitors such as B. caapi, which would presumably prolong the duration of smoked DMT by inhibiting its metabolism (McKenna, 2004). The co-administration of these plants may suggest a purposeful attempt to extend the duration of the experience. However, the motivations underpinning concomitant use of MAO inhibitors requires further investigation before any stringent conclusions can be drawn. 4.1.3. Ayahuasca. Ayahuasca was less commonly used, having been consumed by 30.6% of the sample. As previously noted, DMT’s short duration when smoked was cited as a negative aspect of the drug, and it is possible that smokers who subsequently progress to ayahuasca use do so from a desire for a prolonged drug effect. It is also of note that ayahuasca users had smoked DMT on more occasions than those who had not. This may indicate that in a subset of DMT users, individuals who develop a predilection for the drug may then progress to ayahuasca use. This, however, remains entirely speculative. Aside from the necessary use of MAO inhibitors to make DMT orally active, users of ayahuasca generally do not mix it with other drugs. This is an important departure from smoked DMT. The use of MAO inhibitors in conjunction with other drugs interacting with monoamine neurotransmitters such as antidepressants may result in potentially fatal serotonin syndrome (Callaway and Grob, 1998). Owing to the intensity of the ayahuasca experience (Shanon, 2002), the absence of concomitant drug use among those consuming the preparation may reflect an aversion towards intensifying what may already be an overwhelming experience, or perhaps an unwillingness to risk dangerous drug interactions associated with MAO inhibitor use. Investigating users’ awareness of such risks may warrant attention in future studies, as it is arguably the most pressing health-related risk associated with ayahuasca use. 4.1.4. Positive and negative aspects. Regardless of the route of administration, respondents cited an increase in personal or spiritual insight as a positive aspect of their DMT use. This is consistent with reports of ayahuasca users in formal religious groups such as the UDV and Sainto Daime (Shanon, 2002; Grob et al., 1996). Naturally occurring hallucinogens such as ayahuasca have a long history of religious use, and the recent rise in neo-shamanism reflects a resurgence of Western interest in these compounds (Tupper, 2008, 2009). For instance, psilocybin has been found to produce religious experience in healthy volunteers (Griffiths et al., 2006).

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Of further interest is the antipodal attitude towards drug duration. For both DMT smokers and ayahuasca users, the short and the long duration of each route respectively was perceived as both a good and bad aspect of the drug depending on the respondent. For example, while some were amenable to the short duration of smoked DMT, others considered this to be a shortcoming. Similar considerations can be made of ayahuasca. The degree to which duration of effects influences patterns of DMT use requires further investigation, although ayahuasca users appeared to use this preparation less frequently, possibly due to the nausea also associated with such use (Shanon, 2002). The most commonly cited negative aspect of both routes of administration was unique to the route of administration. Smoked DMT causes coughing and is oftentimes described as having a harsh, plastic-like taste (Turner, 1994), while ayahuasca commonly produces nausea and emesis after administration (Shanon, 2002). Among traditional users of ayahuasca, emesis or ‘la purga’ is said to be both cathartic and restorative. It remains entirely possible that this unpleasant experience of nausea may be a contributing factor to the ‘cleansing’ aspects reported regarding ayahuasca usage in this sample. Psychospiritual conflict is a commonly cited negative aspect of the DMT experience, regardless of the method of administration. This is discussed in greater detail below. 4.1.5. Perceived dangers and risks. The majority of DMT users recruited for this study believed their use of DMT to be safe. Although previous studies examining supervised religious use of ayahuasca found associated harms to be negligible (e.g. Halpern et al., 2008), a level of concern may be directed at the finding that approximately half of the sample commonly consume DMT alone. This is despite general acknowledgement that consuming DMT in the company of others would minimise the risks of an adverse drug effect. Moreover, the safety profile of illicit DMT remains unclear. While pure DMT and ayahuasca are well-tolerated (Strassman, 2001; Gable, 2007) the majority of DMT available in Australia appears to be extracted from plant matter by individuals typically lacking any formal qualification in organic chemistry (Tramacchi, 2006). Aside from DMT, the presence of other psychoactive tryptamines such as 5-MeO-DMT in the relevant plant matter (Ott, 1996) suggests that it is unlikely that DMT users consume the pure compound, but rather a mixture of DMT and related analogues (Tramacchi, 2006). This raises potentially serious questions as to the safety and purity of illicit DMT, a point widely acknowledged by the present sample. Other, more obvious physiological harms such as respiratory damage from smoking are acknowledged by users, although the extent to which such risks influence patterns of DMT use remains unclear. In addition to physical harm, the present sample perceived psychological trauma as a risk of DMT use. Hallucinogens in general have been reported as potential catalysts of rapid and profound changes in psychological or spiritual outlook (Grof, 2008; Hofmann, 2005), and although such changes may be beneficial for some users, in others they may produce considerable distress. When used in an unsupportive environment the likelihood of negative psychological impacts from many hallucinogens increases, an observation readily noted in the present sample. The perceived risks from DMT are predominantly psychological in nature, from the frightening experience of acute intoxication – a ‘bad trip’ – to psychospiritual disturbances that may accompany and outlast the primary drug effects. Although we did not explicitly examine the frequency with which this occurred in the sample, this is an important question for future research. The need to remain informed about the risks inherent in DMT use was the most frequently cited harm minimisation strategy. Common responses about harm minimisation also centred around

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using DMT in an appropriate “set and setting” – the physical and social environment in which a drug is consumed as well as the subject’s emotional state, intentions, expectancies and beliefs concerning the use of a psychoactive substance (Faillace and Szára, 1968). The practise of this would also minimise the possibility of falls or burns that may occur due to DMT intoxication. The addition of a ‘sitter’, or another individual who may render assistance if required, was also stated as a factor in using the drug safely. 4.2. Pattern of other drug use In this sample DMT is part of an extensive pattern of drug use involving a diverse class of drugs. In addition to substances such as alcohol, cannabis, psychostimulants, depressants and narcotics, the use of a wide array of otherwise obscure drugs was reported. These drugs included plant-based psychoactives as well as novel tryptamine and phenethylamine-based hallucinogens described by Shulgin and Shulgin (1991, 1997). Although multiple drug use is commonplace among drug users (Wilkinson et al., 1987), the degree to which this was observed in the present sample is exceptional. For example, in his study of 135 Scottish MDMA users, Forsyth (1996) found that 51 discrete drugs had been used by respondents; in the present study, nearly double that number (92) was reported. Moreover, while Forsyth found the mean number of discrete drugs used to be 10.7, in this sample this was 17.3. Although there appears a need to raise concern within our sample, this level of concern is perhaps best directed at the history of drug use, rather than with DMT use per se. For example, the use of cannabis seems ubiquitous, and approximately a quarter of the sample may be cannabis dependent. In addition, over onequarter of the sample reported lifetime use of intravenous drugs. Thus, although hallucinogens appeared to be favoured by the sample, to describe the sample primarily as DMT or hallucinogen users would be inaccurate. Future studies would benefit from determining the degree to which users favour DMT relative to other drugs they consume. 5. Conclusions This sample of recreational DMT users was recruited primarily by snowball sampling. Snowballing has the advantage of using social networks to gain access to ‘hidden’ populations such as illicit drug users (Thompson and Collins, 2002; Biernacki and Waldorf, 1981). This is particularly advantageous given that DMT remains an infrequently used drug in most Western countries including Australia, and it is doubtful that such a large sample could be obtained without doing so. However, the very benefits of snowball sampling may lead to potential problems in sample biasing. For instance, the majority of respondents in the sample were male and it remains unclear whether the data are a true representation of Australian DMT users in general, or a bias in the sample. It is also necessary to note that aside from containing DMT, plant extractions of DMT are likely to comprise other psychoactive tryptamines such as 5-MeO-DMT and bufotenin (Ott, 1996). Thus, to consider this to be a study of DMT use exclusively is akin to presuming that ecstasy use constitutes the use of unadulterated MDMA. In the absence of forensic data of seized DMT, the exact purity and chemical constituents of material purported to be DMT remains unknown. Despite evidence that the use of DMT in Australia and other Western countries is gaining in popularity (e.g. Grigoriadis, 2006; Cox, 2009; Greenhouse, 2006), it is prudent to note that DMT presently remains an uncommon drug. There are several reasons for this. First, as is generally the case with other hallucinogens, DMT is neither physically nor psychologically addictive (Callaway et al.,

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1999; Strassman, 2001). Moreover, many are drawn to DMT out of curiosity, yet the very effects to which users are attracted are also likely to moderate its frequency of use. Its intense and unpredictable effects, and individual susceptibility to having a frightening experience or bad trip (Strassman, 2001) are likely to dissuade many recreational users from consuming it on more than one occasion. Although largely exploratory, this study offers a framework within which to approach future studies of recreational DMT use. First, it demonstrates that smoking is the most common route of administration for recreational users, whereas studies previous to this have predominantly investigated ayahuasca use (e.g. Grob et al., 1996; Shanon, 2002; Trichter et al., 2009). Although recruiting DMT users through formal religions such as the UDV and Sainto Daime offers a readily accessible sample of DMT users, future studies may benefit from examining DMT use beyond these institutions. Even beyond these churches, ‘recreational’ DMT use appears to reflect an earnest attempt at gaining psychospiritual insight through means of altered states of consciousness. Future studies should seek to examine the psychological characteristics of recreational DMT users, their attitudes and motivations, and actual benefits and harms, if any, associated with DMT use. Role of funding source Nothing declared.

Contributors V.C. and J.P. developed the questionnaire and recruited participants. A.M. conducted data analysis and literature review. V.C. wrote the first draft of the manuscript, and J.P. and A.M. subsequently edited and finalised drafts. All authors contributed to and have approved the final manuscript.

Conflict of interest No conflict declared.

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Published in final edited form as: Sci Signal. ; 2(61): pe12. doi:10.1126/scisignal.261pe12.

When the Endogenous Hallucinogenic Trace Amine N,NDimethyltryptamine Meets the Sigma-1 Receptor Tsung-Ping Su1,*, Teruo Hayashi1, and D. Bruce Vaupel2 1Cellular Pathobiology Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, 333 Cassell Drive, Baltimore, MD 21224, USA 2Neuroimaging

Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, 333 Cassell Drive, Baltimore, MD 21224, USA

Abstract NIH-PA Author Manuscript

N,N-dimethyltryptamine (DMT) is a hallucinogen found endogenously in human brain that is commonly recognized to target the 5-hydroxytryptamine 2A receptor or the trace amine– associated receptor to exert its psychedelic effect. DMT has been recently shown to bind sigma-1 receptors, which are ligand-regulated molecular chaperones whose function includes inhibiting various voltage-sensitive ion channels. Thus, it is possible that the psychedelic action of DMT might be mediated in part through sigma-1 receptors. Here, we present a hypothetical signaling scheme that might be triggered by the binding of DMT to sigma-1 receptors.

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Some amino acid metabolites are biogenic amines that, unlike the major neurotransmitter amines, such as dopamine, nore-pinephrine, and 5-hydroxytryptamine (5-HT), are typically present at low concentrations and accumulate in high amounts only if the amine-digestive enzyme monoamine oxidase is inhibited. These trace amines (TAs) include βphenylethylamine, tyramine, octopamine, synephrine, and tryptamine, as well as some of their metabolites or derivatives. TAs are purported to be involved in several human diseases (1). Here, we focus on findings related to N,N-dimethyltryptamine (DMT), a tryptamine metabolite with psychedelic effects. DMT is the main ingredient in the hallucinogenic beverage called “ayahuasca,” which has been brewed (by boiling the bark of Banisteriopsis caapi together with the leaves of Psychotria viridis) and used by indigenous people around the South American Amazon basin (2, 3). Using purified DMT, Szara and colleagues first reported the psychoactive effect of the compound in humans (4, 5). Saavedra and Axelrod then demonstrated the formation of DMT in rat and human brain (6), leading both groups to propose that DMT was an endogenous hallucinogen (4–6). Several studies have since confirmed the psychedelic properties of DMT in humans (7–14). DMT is generally believed to exert its psychedelic effects through the 5-HT receptor, specifically the 5-HT2A subtype, which was identified by using the semisynthetic hallucinogen lysergic acid diethylamide (LSD) (15). However, certain behaviors seen in rats treated with DMT (0.5 to 35 mg/kg administered intraperitoneally), such as jerking, retropulsion, and tremor, do not involve the 5-HT system or other monoaminergic systems (16). Micro-molar concentrations of DMT enhances phosphatidylinositol production in a

Copyright 2008 by the American Association for the Advancement of Science; all rights reserved. * Corresponding author. Cellular Pathobiology Section, IRP, NIDA, NIH Suite 3304, 333 Cassell Drive, Baltimore, MD 21224, USA. Telephone, 443-740-2804; fax, 443-740-2142; [email protected]

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manner that is not blocked by the 5-HT2A receptor antagonist ketanserin (17), which suggests that part of the action of DMT is not mediated through 5-HT receptors. With the discovery of the G protein–coupled TA-associated receptors (TAARs), which activate adenylyl cyclase and cause cyclic adenosine monophosphate (cAMP) accumulation (18, 19), it was speculated that TAARs mediated part of the pharmacological or psychedelic effect of trace amines, including DMT, as well as LSD. (19). Although DMT at 1 μM is as potent in eliciting cAMP accumulation as the prototypic trace amine tryptamine or LSD (19), it is unclear whether TAARs mediate the psychedelic effect of trace amines, including DMT, because TAAR antagonists have not been tested in humans in this regard. Furthermore, gene association studies attempting to link TAARs and psychiatric disturbances have generated conflicting results as to whether TAARs are involved in schizophrenic symptomatologies, including hallucination. TAAR1 knockout mice display a deficit in “prepulse inhibition” (PPI), or the ability to suppress the magnitude of startle induced by an incoming acoustic signal that had been previously experienced (20). They are therefore a relevant animal model for schizophrenia because the PPI is typically impaired in schizophrenic patients (20). In addition, a genetic study has demonstrated associations between polymorphisms in the TAAR4 subtype with susceptibility to schizophrenia (21); however, conflicting reports later emerged that demonstrated a lack of association between the TAAR4 or TAAR6 gene and schizophrenia (22, 23). Thus, it remains to be fully established whether TAARs mediate the psychotomimetic action of DMT.

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A report now demonstrates that DMT targets a receptor called the sigma-1 receptor (Sig-1R) (24). DMT binds to the Sig-1R with a moderate affinity at about 14 μM (24). Although this affinity is not impressive when compared to other Sig-1R ligands, such as (+)pentazocine (which has an affinity in nanomolar range), high concentrations of DMT (100 μM, about 7 times as high as its affinity for Sig-1R) could nonetheless inhibit voltage-gated sodium channels (24), a hallmark action of Sig-1R ligands and Sig-1Rs (25). Sig-1R knockout mice, which reacted normally to the locomotor stimulating effect of methamphetamine, did not become hyper-active in response to DMT (24), a phenomenon also observed with the prototypic Sig-1R agonist N-allylnormetazocine, an opiate analog better known as SKF-10047 (26). Furthermore, the locomotor-stimulating action of DMT resembles that of SKF-10047 (24, 26). These results definitively link the action of DMT to the Sig-1R.

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The Sig-1R was originally thought to be the opiate receptor subtype that mediated the psychotomimetic or drug-induced psychotic-like effect of SKF-10047 in animals (27). However, the same laboratory later found that the psychotomimetic effect of SKF-10047 was not reversed by naloxone, a universal antagonist for all opiate receptor subtypes (28). Thus, the Sig-1R was recognized to be a nonopiate receptor (29–31) that might mediate the psychotomimetic effect not only of SKF-10047 but also of the dissociative anesthetic phencyclidine (PCP) (28, 32). However, PCP is thought to induce its mind-altering effect through the N-methyl-D-aspartate (NMDA) receptor, and systematic behavioral studies are needed to differentiate between the SKF-10047– and PCP-induced effects mediated by the Sig-1R versus the NMDA receptor. In addition to their postulated psychotomimetic action, Sig-1Rs have been implicated in diseases such as addiction, depression, amnesia, pain, stroke, and cancer (33). Sig-1Rs localize at the interface between the endoplasmic reticulum (ER) and mitochondrion, which is known as the mitochondria-associated ER membrane (MAM). Sig-1R agonists at affinity concentrations (i.e., close to their Ki values) cause Sig-1Rs to disassociate from another ER chaperone, binding immunoglobulin protein (BiP), allowing them to act as molecular chaperones to inositol 1,4,5-trisphosphate (IP3) receptors. By stabilizing IP3 receptors, Sig-1Rs at the MAM enhance Ca2+ signaling from the ER into mitochondria (34, 35), thereby activating the tricarboxylic acid (TCA) cycle and increasing

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the production of adenosine triphosphate (ATP) (35) (Fig. 1). Although Sig-1Rs reside primarily at the ER, they can translocate from the MAM to the plasma membrane (also termed the plasmalemma) or the subplasma membrane area when stimulated by higher concentrations (e.g., at approximately 10-fold Ki) of Sig-1R ligands or when Sig-1Rs are overexpressed in cells (36–38) (Fig. 1). This may explain why higher concentrations of Sig-1R ligands result in the inhibition of various ion channels at the plasma membrane and, in particular, why the channel-inhibiting concentration of DMT is almost 10 times as high as its affinity concentration (24). By triggering the translocation of Sig-1Rs from the MAM to the plasma membrane or subplasma membrane, high concentrations of Sig-1R ligands may allow Sig-1Rs to directly interact with and inhibit channel proteins (24, 38). High concentrations of Sig-1R ligands tonically inhibit the small conductance K+ (SK) channel, which in turn leads to the potentiation of NMDA receptors (39). The NaV1.5 channel (24, 25), the KV1.4 channel (38), the voltage-gated N-, L-, and P/Q-type Ca2+ channels (40), the acid-sensing ion channel (41), and the volume-regulated Cl− channel (42) are also inhibited by high concentrations of Sig-1R ligands.

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So, do sigma-1 receptors mediate the psychedelic effect of DMT? First, we need to specify that Sig-1Rs have not been firmly established as being involved in causing psychotomimesis. Secondly, moderate concentrations of selective Sig-1R ligands, including (+)pentazocine and PRE-084, are not reported to cause psychotomimetic-like effects in animals (43). However, the possibility that Sig-1Rs are involved in psychotomimesis cannot be totally excluded at present. We therefore speculate that Sig-1Rs may partially mediate the psychotomimetic effects of DMT, such as visual hallucinations in humans (7–14). PCP and SKF-10047 cause animals to behave as if they are hallucinating (they move their heads and eyes as if they are tracking objects in the air) (28). Could the psychotomimetic effect caused by PCP and SKF-10047 in animals (28) be explained by PCP or SKF-10047 blocking NMDA receptors and not by their binding to Sig-1Rs? It might not be, because it might be difficult to distinguish the psychedelic effect mediated by the NMDA receptor blockade from that mediated by Sig-1Rs in animal studies. A clearer differentiation of the effects mediated by the two different receptors might come only from human studies. Results from previously mentioned clinical studies, although not designed to answer this question, might provide some interesting clues.

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DMT, which we know now is also a Sig-1R ligand, has been used as a 5-HT2A agonist by Gouzoulis-Mayfrank et al. to compare the psychedelic effect of DMT with that of ketamine, which is also an NMDA receptor blocker like PCP (11–14). DMT effects relate more to the paranoid-type psychoses with particular positive formal thought disorders—including loosening of associations, derailment, and distractibility—than to the neurocognitive impairment seen with ketamine (11, 14). Thus, DMT effects in humans might be mediated through Sig-1Rs or 5-HT2A receptors and not through blockade of NMDA receptors. In this regard, it would be interesting to examine whether Sig-1R antagonists block the psychedelic effect of DMT in humans. Based on the current understanding of the cell biological actions of Sig-1Rs (34, 35, 38), we propose a hypothetical scheme for the molecular mechanism by which DMT signals through sigma-1 receptors (Fig. 1). Like other Sig-1R agonists (34), DMT at affinity concentrations (14 μM) (24) might cause the dissociation of Sig-1Rs from the Sig-1R-BiP complex (34, 35) (Fig. 1A), and at higher concentrations (100 μM) (24) might cause Sig-1Rs to translocate from the MAM to the plasma membrane (36, 37) (Fig. 1B). By doing so, DMT might first unleash the chaperone activity of the free form of Sig-1Rs at the MAM (34) and then cause the receptors to translocate (36, 37) to the plasma membrane to inhibit voltage-gated ion channels (24, 38–42). We do not know at present whether the chaperone activity of Sig-1Rs contributes to ion channel inhibition or whether Sig-1Rs associate with ion channels at the

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subplasma ER membrane or at the plasma membrane. Nor do we know whether the chaperone-unleashing action seen at affinity concentrations of DMT or the ion channel– inhibiting action caused by high concentration of DMT relate to the psychedelic effect induced by DMT. More studies are needed to provide answers to these questions. Almost 30 years after the initial description of a psychotomimesis-related sigma receptor (27–32), investigators have identified DMT as an endogenous hallucinogen that targets a new site of action (24). The characterization of Sig-1Rs as ligand-regulated chaperone receptors (34) and the discovery of the endogenous hallucinogen DMT as a Sig-1R ligand (24) represent potential breakthroughs in drug abuse research. Yet many questions remain, the most important being: What is the physiological importance or relevance of the DMT signaling through Sig-1Rs? Furthermore, Sig-1Rs are present not only in the central nervous system, but also in peripheral organs such as the liver, heart, lung, adrenal gland, spleen, and pancreas (34). For example, the enzyme that synthesizes DMT from its precursor tryptamine (44) and Sig-1Rs (34) are particularly abundant in lung tissue. Thus, it will be important to delineate the roles of Sig-1Rs and their associated ligands, including DMT, within the context of the physiology or pathophysiology of human diseases related to those organs. It is hoped that future research will increase our understanding of these roles.

Acknowledgments NIH-PA Author Manuscript

This work was supported by the Intramural Research Program of the National Institute on Drug Abuse, NIH, Department of Health and Human Services of the United States.

References and Notes

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11. Gouzoulis-Mayfrank E, Heekeren K, Neukirch A, Stoll M, Stock C, Obradovic M, Kovar KA. Psychological effects of (S)-ketamine and N,N-dimethyltryptamine (DMT): A double blind, crossover study in healthy volunteers. Pharmacopsychiatry. 2005; 38:301–311. [PubMed: 16342002] 12. Heekeren K, Neukirch A, Daumann J, Stoll M, Obradovic M, Kovar KA, Geyer MA, GouzoulisMayfrank E. Prepulse inhibition of the startle reflex and its attentional modulation in the human Sketamine and N,N-dimethyltryptamine (DMT) models of psychosis. J Psychopharmacol. 2007; 21:312–320. [PubMed: 17591658] 13. Heekeren K, Daumann J, Neukirch A, Stock C, Kawohl W, Norra C, Waberski TD, GouzoulisMayfrank E. Mismatch negativity generation in the human 5HT2A agonist and NMDA antagonist model of psychosis. Psychopharmacology (Berlin). 2008; 199:77–88. [PubMed: 18488201] 14. Daumann J, Heekeren K, Neukirch A, Thiel CM, Moller-Hartmann W, Gouzoulis-Mayfrank E. Pharmacological modulation of the neural basis underlying inhibition of return (IOR) in the human 5HT2A agonist and NMDA antagonist model of psychosis. Psychopharmacology (Berlin). 2008; 200:573–583. [PubMed: 18649072] 15. Bennett JP Jr, Snyder SH. Serotonin and lysergic acid diethylamide binding in rat brain membranes: Relationship to post-synaptic serotonin receptors. Mol Pharmacol. 1976; 12:373–389. [PubMed: 6896] 16. Jenner P, Marsden CD, Thanki CM. Behavioural changes induced by N,N-dimethyltryptamine in rodents. Br J Pharmacol. 1978; 63:380P. 17. Deliganis AV, Pierce PA, Peroutka SJ. Differential interactions of dimethyltryptamine (DMT) with 5-HT1A and 5-HT2 receptors. Biochem Pharmacol. 1991; 41:1739–1744. [PubMed: 1828347] 18. Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, Durkin MM, Lakhlani PP, Bonini JA, Pathirana S, Boyle TA, Pu X, Kouranova E, Lichtblau H, Ochoa FY, Branchek TA, Gerald C. Trace amines: Identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci USA. 2001; 98:8966–8971. [PubMed: 11459929] 19. Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI, Darland T, Suchland KL, Pasumamula S, Kennedy JL, Olson SB, Magenis RE, Amara SG, Grandy DK. Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the cate-cholamine neurotransmitters are agonists of a rat trace amine receptor. Mol Pharmacol. 2001; 60:1181–1188. [PubMed: 11723224] 20. Wolinsky TD, Swanson CJ, Smith KE, Zhong H, Borowsky B, Seeman P, Branchek T, Gerald CP. The trace amine 1 receptor knockout mice: An animal model with relevance to schizophrenia. Genes Brain Behav. 2007; 6:628–639. [PubMed: 17212650] 21. Duan J, Martinez M, Sanders AR, Hou C, Saitou N, Kitano T, Mowry BJ, Crowe RR, Silverman JM, Levinson DF, Gejman PV. Polymorphisms in the trace amine receptor 4 (TAAR4) gene on chromosome 6q23.2 are associated with susceptibility to schizophrenia. Am J Hum Genet. 2004; 75:624–638. [PubMed: 15329799] 22. Amann D, Avidan N, Kanyas K, Kohn Y, Hamdan A, Ben-Asher E, Macciardi F, Beckmann JS, Lancet D, Lerer B. The trace amine receptor 4 gene is not associated with schizophrenia in a sample linked to chromosome 6q23. Mol Psychiatry. 2006; 11:119–121. [PubMed: 16189505] 23. Vladimirov VI, Maher BS, Wormley B, O’Neill FA, Walsh D, Kendler KS, Riley BP. The trace amine associated receptor (TAAR6) gene is not associated with schizophrenia in the Irish casecontrol study of schizophrenia (ICCSS) sample. Schizophr Res. 2009; 107:249–254. [PubMed: 18973992] 24. Fontanilla D, Johannessen M, Hajipour AR, Cozzi NV, Jackson MB, Ruoho AE. The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator. Science. 2009; 323:934–937. [PubMed: 19213917] 25. Cheng ZX, Lan DM, Wu PY, Zhu YH, Dong Y, Ma L, Zheng P. Neurosteroid dehydroepiandrosterone sulfate inhibits persistent sodium current in rat medial prefrontal cortex via activation of sigma-1 receptors. Exp Neurol. 2008; 210:128–136. [PubMed: 18035354] 26. Langa F, Condony X, Tovar V, Lavado A, Gimenez E, Cozar P, Cantero M, Dordal A, Hernandez E, Perez R, Monroy X, Zamanillo D, Guitart X, Montoliu L. Generation and phenotypic analysis of sigma receptor type 1 (sigma 1) knockout mice. Eur J Neurosci. 2003; 18:2188–2196. [PubMed: 14622179]

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Fig. 1.

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Hypothetical scheme illustrating the signaling of N,N-dimethyltryptamine through sigma-1 receptors. (A) Sigma-1 receptors (Sig-1Rs) at the mitochondrion-associated endoplasmic reticulum (ER) membrane (MAM) function as ligand-activated molecular chaperones, particularly when ligands are present at concentrations close to their affinities (34). Sig-1R ligands, including DMT, at concentrations close to their Ki values, cause the dissociation of Sig-1Rs from another ER chaperone, binding immunoglobulin protein (BiP) (34), allowing Sig-1Rs to chaperone inositol 1,4,5-trisphosphate receptors (IP3Rs) at the MAM (34). This enhances Ca2+ signaling from the ER into mitochondria (34, 35), activates the tricarboxylic acid (TCA) cycle, and increases adenosine triphosphate (ATP) production (35). (B) Higher concentrations of DMT cause the translocation of Sig-1Rs from the MAM to the plasma membrane, leading to the inhibition of ion channels. Thus, Sig-1R ligands might shift the site of action of Sig-1R chaperones from the center of the cell to its periphery. In the present scheme, Sig-1Rs and related molecules or organelles are illustrated in the postsynaptic region for the sake of simplicity, although they may also be present presynaptically or in glia.

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Journal Code D T A

Article ID Dispatch: 30.01.12 4 2 2 No. of Pages: 19

CE: ME:

Drug Testing and Analysis

Review Received: 7 December 2011

Revised: 3 January 2012

Accepted: 3 January 2012

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/dta.422

A critical review of reports of endogenous psychedelic N, N-dimethyltryptamines in humans: 1955–2010 Steven A. Barker,a* Ethan H. McIlhennya and Rick Strassmanb Three indole alkaloids that possess differing degrees of psychotropic/psychedelic activity have been reported as endogenous substances in humans; N,N-dimethyltryptamine (DMT), 5-hydroxy-DMT (bufotenine, HDMT), and 5-methoxy-DMT (MDMT). We have undertaken a critical review of 69 published studies reporting the detection or detection and quantitation of these compounds in human body fluids. In reviewing this literature, we address the methods applied and the criteria used in the determination of the presence of DMT, MDMT, and HDMT. The review provides a historical perspective of the research conducted from 1955 to 2010, summarizing the findings for the individual compounds in blood, urine, and/or cerebrospinal fluid. A critique of the data is offered that addresses the strengths and weaknesses of the methods and approaches to date. The review also discusses the shortcomings of the existing data in light of more recent findings and how these may be overcome. Suggestions for the future directions of endogenous psychedelics research are offered. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: dimethyltryptamine; psychedelic; endogenous

Introduction Three indole alkaloids that possess differing degrees of psychotropic/ psychedelic activity have been reported as endogenous substances in humans. These compounds, all metabolites of tryptophan, are N,N-dimethyltryptamine (DMT, 1, Figure 1), 5-hydroxy-DMT (bufotenine, HDMT, 2), and 5-methoxy-DMT (MDMT, 3). Their presence has been reported in human cerebrospinal fluid (CSF), urine, and/or blood utilizing either paper and/or thin layer chromatography (TLC), direct ultraviolet (UV) or fluorescence (Fl) measurements, gas chromatography (GC) using various sensors (nitrogen-phosphorous detector (NPD); electron capture detector (ECD); mass spectrometry detector (MSD)), high-performance liquid chromatography (HPLC) using UV and/or Fl detection, HPLCradioimmunoassay, HPLC-electrochemical detection, and liquid chromatography-tandem mass spectrometry (LC-MS/ MS) (Tables 1–3, references[1–69]). Indeed, the review of the 55-year history of the development of methodology for the analysis of these compounds shows how closely it has paralleled the evolution of analytical technology itself, with each researcher seeking more specific and sensitive techniques. A renewed interest in these compounds as naturally occurring substances in humans has occurred, in part, due to DMT’s recent characterization as an endogenous substrate for the ubiquitous sigma 1 receptor[70] and for its possible action at trace amine receptors.[71] In both cases, the roles of DMT and the receptors themselves in regulating some aspect(s) of human physiology are poorly understood. Given their known psychedelic effects, there remains an interest in their possible role in naturally occurring altered states of consciousness, such as psychosis, dreams, creativity and imagination, religious phenomena, and even near-death

Drug Test. Analysis (2012)

experiences.[72] Although the vast majority of research into the presence of these compounds sought their role in mental illness, no definitive conclusions yet exist. A determination of the role of these compounds in humans awaits further research, much of which awaits the development of adequate analytical methodology. Interest in DMT has also increased because of the burgeoning use and popularity of the religious sacrament ayahuasca which contains DMT and several harmala alkaloids, which serve to make DMT orally active. Ayahuasca tourism in South America and the establishment of syncretic churches using ayahuasca as a sacrament[73,74] have stimulated research into the mechanisms of its effects and its possible use as a therapeutic.[75] The resumption of human research characterizing DMT’s psychopharmacology[76–84] and the ongoing use of pure DMT for therapeutic and recreational purposes have also focused interest on this and related psychedelics. The dimethylated-tryptamines (DMTs) increasing visibility within medical, non-medical, religious and/or recreational contexts[75] reinforce the importance of determining their endogenous role. This review addresses several fundamental issues regarding these three endogenous psychedelics. For example, are DMT,

* Correspondence to: Steven A. Barker, Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70806, USA. E-mail: [email protected] a School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA b School of Medicine, University of New Mexico, Albuquerque, and Cottonwood Research Foundation, Taos, New Mexico, USA

Copyright © 2012 John Wiley & Sons, Ltd.

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S. A. Barker, E. H. McIlhenny and R. Strassman

Figure 1. Structures of the compounds discussed.

HDMT, and/or MDMT truly present in humans?[85] Early criticisms of reports of endogenous psychedelics were directed at the fact that rather non-specific chemical tests were being applied, double-blind analyses were not always being performed, and dietary or medication sources were not always adequately ruled out as responsible for the identifications.[2,12] Further, it was claimed that possible artifacts produced from the extraction solvents and conditions of analysis may have led to misidentification of the DMTs in some early studies[20] and, more recently, that the use of halogenated solvents in the analysis may have affected their detection.[86] Biological factors that may have affected the detectabilty of these compounds in the periphery were also acknowledged, which included their rapid metabolism.[87,88] Finally, there have been concerns that the studies searching for their presence and an association with specific clinical disorders have failed to understand and fully characterize their metabolism or monitor their metabolites.[88–91] To address these issues, we have undertaken a critical review of 69 published studies reporting the detection or detection and quantitation of these compounds in human body fluids. In reviewing this literature, we address the methods applied and the criteria used in the determination of the presence of DMT, MDMT, and HDMT. We begin with the original report of the presence of bufotenin (HDMT) in human urine in 1955 using paper chromatography[1] and end with the most recent report concerning the presence of bufotenin (HDMT) in human urine using LC-MS/MS.[69] We will be addressing the following questions: How valid were early studies regarding the presence and/or quantities of these compounds in human cerebrospinal fluid (CSF), blood and/or urine? Were the analytical methodologies and the identification criteria adequate? Are they truly there? When present, are they of dietary origin? When and where in the human body are they produced? Can we influence their detection in biological samples by pharmacologically inhibiting their metabolism by monoamine oxidase (MAO)? How does turnover rate and metabolism of these substances influence their detectabilty? Have the precursors and/or metabolites of these compounds been adequately monitored? Is

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monitoring these compounds in biological samples such as CSF, blood and/or urine the best, or even most practical way to determine their role? What will such data tell us about the function of these compounds? Where does the research on endogenous psychedelics go from here?

Historical perspective The search for endogenous psychedelics soon followed the discovery of the psychedelic effects of mescaline and lysergic acid diethylamide (LSD) in humans. Observations of these effects gave rise to hypotheses that they were related to the symptomology observed in a heterogeneous group of mental disorders, especially psychoses – either mania or schizophrenia.[92] It was proposed that schizophrenics may biochemically produce similar compounds as ‘schizotoxins’.[93] A search for mescaline-like compounds proved unrewarding,[94] but in studies examining urine samples for serotoninlike compounds, researchers reported in 1955[1] and 1956,[2] the presence of 5-hydroxy-N,N-DMT (HDMT, bufotenin) in humans. Subsequently, Axelrod[95] reported the presence of an enzyme capable of N-methylating indole-ethylamines and producing DMTs. Following these reports, attention began to focus in earnest on the possible endogenous formation of the indole-ethylamine psychedelics. During the next 50 years, many studies reported finding DMT, HDMT, and/or MDMT in human CSF, urine, and/or blood. Most of these studies sought differences in levels between controls and psychiatric, especially psychotic, patients. Some studies claimed higher concentrations and significant differences in levels between the groups; some reported not finding the compounds at all in either patients or controls. It is of interest to note that in its original conception, the schizotoxin hypothesis proposed that the formation of an endogenous psychedelic schizotoxin would be an aberration of metabolism and that ‘normals’ would not form such compounds.[92] However, numerous studies subsequently reported finding one or more of these compounds in controls

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Bumpus and Page Rodnight[2] Fischer et al.[3] Fischer et al.[4] Feldstein et al.[5] Perry et al.[6]

Brune et al.[7] Perry[8] Sprince et al.[9] Perry and Schroeder[10] Franzen and Gross[11] Siegel[12] Nishimura and Gjessing[13] Takesada et al.[14] Runge et al.[15] Perry et al.[16] Heller[17] Fischer and Spatz[18] Kakimoto et al.[19] Tanimukai[20] Tanimukai et al.[21] Tanimukai et al.[22] Acebal and Spatz[23] Faurbye and Pind[24] Sireix and Marini[25] Spatz et al.[26] Fischer and Spatz[27] Saavedra and Udabe[28] Tanimukai et al.[29] Heller et al.[30] Narsimhachari et al.[31] Narasimhachari et al.[32] Fischer et al.[33] Himwich et al.[34] Narasimhachari et al.[35] Walker et al.[36]

1963 1963 1963 1963 1965 1965 1965 1965 1966 1966 1966 1967 1967 1967 1967 1967 1967 1968 1969 1969 1970 1970 1970 1970 1971 1971 1971 1972 1972 1973

[1]

Author

1955 1956 1961 1961 1961 1962

Year

Drug Test. Analysis (2012) HDMT; DMT HDMT; DMT DMT, HDMT HDMT DMT, HDMT HDMT HDMT HDMT HDMT DMT, HDMT HDMT HDMT DMT, HDMT HNMT, HDMT, NMT, DMT, MDMT HDMT HDMT HDMT HDMT HDMT HDMT HDMT HDMT HNMT, HDMT, DMT, MDMT DMT, MDMT, HDMT DMT, MDMT, HDMT NMT, DMT, MDMT HDMT, glucuronide HDMT, DMT, MDMT HDMT, DMT, MDMT DMT

HNMT, HDMT HNMT, HDMT HDMT HDMT HDMT HDMT, conjugate

Compounds Analyzed 24-hour urine 10 ml portions, HCl; urease 24-hour urine; 75–120 ml extracted 1 L of urine 1 L of urine 8 hour urines; IV/oral 14 C serotonin (130 mg) 24-36 hour urine; ext vol 500 mg creatinine; w/wo hydrolysis 24 hour urine 24 or 48 hour urine; ext vol 500 mg creatinine 24 hour urine 24-36 hour urine; ext vol 250–350 mg creatinine blood and urine (24 hour) fresh urine, 100 ml fresh urine vol 500–1,000 mg creatinine 24 hour urine 1 L of urine 48 hour urine 1 L of urine 100 ml fresh urine 24 hour urine; vol 600 mg creatinine analyzed 24 hour urine; 1/4th used in assay 24 hour urine;1/3 rd used in assay 24 hour urine; 1/4th used in assay 100 ml urine 24 hour urine, hydrolyzed at pH1.6 100 ml fresh urine 50 ml fresh urine; 100 ml fresh urine 50 ml fresh urine; acid hydrolysis 50 ml fresh urine; acid hydrolysis 24 hour urine; 1/4th used in assay fasting blood, oxalate tube; acid hydrolyzed 24 hour urine; 75% used in assay fasting blood, oxalate tube 50 ml morning urine; w/wo glucuronidase 24 hour urine 24 hour urine plasma; DMT stable for 60 days at 6 degrees C

Collection

pH 10, ethyl ether ext, evap, acetone Amberlite CG-120, CG-50; ethanol-acetone ppt pH 10, ethyl ether-butanone ext, evap, acetone Amberlite CG-120, CG-50; ethanol-acetone ppt Extensive multi-step extraction, ppt and clean-up pH 10, ethyl ether ext, evap, acetone Dowex 50, Amberlite CG 50, Ext, Dowex 50 column, alumina column pH 8–9, butanol ext, acetone ppt, acetone Dowex 50 W, Amberlite CG-50; HCl hydrolysis NaHCO3 sat., butanol, evap, acetone NaCO3, ether ext, evap, acetone Ext, Dowex 50 column, alumina column Dowex 50 W X2; w/wo HCl hydrolysis cation exchange resin; w/wo HCL hydrolysis Dowex 50 W X2; HCl hydrolysis NaCO3, ether ext, evap, acetone column chromatography, sublimation, paper/TLC NaCO3, ether ext, evap, acetone pH 10 NaOH, ethyl acetate; diazo-reagent or TLC pH 10 NaOH, ethyl acetate; diazo-reagent and TLC pH 10 NaOH, ethyl acetate; diazo-reagent and TLC Dowex 50 W X2; HCl hydrolysis Dowex 50; HCl ext and ethyl acetate at pH 10.2 Dowex 50; HCl ext and ethyl acetate at pH 10.2 Dowex 50; HCl ext and ethyl acetate at pH 10.2 Liquid-Liquid ext; w/wo glucuronidase treatment Dowex 50; HCl ext and ethyl acetate at pH 10.2 Franzen and Gross; HCl ext ethyl acetate at pH 10.2 HCL ext acid pH with CHCl3, pH 9, ext CHCl3, evap

Evap, Acetone, evap, MeOH, evap, AlO3 column Zeo-Karb 226 resin, EtOH/acetone ppt, evap NaHCO3 sat., butanol, evap, acetone NaOH pH 9, butanol, evap, acetone not described Amberlite CG-120, CG-50; ethanol-acetone ppt

Extraction Method

Table 1. Review of 69 studies regarding endogenous psychedelics showing the year, reference, compounds analyzed, type of sample and method of extraction. Acronyms and abbreviations; IV, intravenous; HNMT, 5-hydroxy-N-methyltryptamine; ext, extraction; vol, volume; w/wo, with or without; evap, evaporate; ppt, precipitate; sat., saturated; TLC, thin-layer chromatography; cent, centrifuge; TFAA, trifluoro-acetic anhydride; SPE, solid-phase extraction; LC, liquid chromatography.

Reports of endogenous psychedelic N, N-dimethyltryptamines in humans

Copyright © 2012 John Wiley & Sons, Ltd.

Drug Testing and Analysis

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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

4

wileyonlinelibrary.com/journal/dta

Copyright © 2012 John Wiley & Sons, Ltd. DMT, MDMT DMT DMT, NMT

DMT

DMT, HDMT DMT, MDMT DMT

Riceberg and Van Vunakis[52]

Corbett et al.[53] Walker et al.[54] Murray et al.[55]

Checkley et al.[56]

Raisanen and Karkkainen[57] Smythies et al.[58] Checkley et al.[59]

1978

1978 1979 1979

1979

1979 1979 1980

DMT, MDMT HDMT HDMT HDMT

DMT, HDMT, MDMT

Oon and Rodnight[51]

1977

Uebelhack et al.[60] Sitaram et al.[61] Raisanen et al.[62] Karkkainen et al.[63]

DMT, NMT

Wyatt et al.[37] Narasimhachari and Himwich[38] Lipinski et al.[39] Bidder et al.[40] Narasimhachari et al.[41] Carpenter et al.[42] Christian et al.[43] Narasimhachari and Himwich[44] Angrist et al.[45] Rodnight et al.[46] Murray and Oon[47] Huszka et al.[48] Cottrell et al.[49] Oon et al.[50]

1973 1973 1974 1974 1974 1975 1975 1975 1976 1976 1976 1976 1977 1977

1983 1983 1984 1988

DMT DMT, HDMT DMT DMT HDMT, DMT, MDMT DMT, HDMT DMT, MDMT DMT, HDMT DMT DMT DMT HDMT, DMT, MDMT HDMT DMT, NMT

Author

Year

Compounds Analyzed

Cerebrospinal fluid 12 hr specimens (8 pm-8 am); 200 ml assayed not stated morning urine samples

150 ml morning urine samples Cerebrospinal fluid Serial 24 hour urine; longitudinal study

24 hour urine; 50% used in assay

Cerebrospinal fluid 10 ml whole blood; arterial and venous 24-hour urine; 50% used in assay

24 hour urine; 300 ml used in assay 50 ml whole blood; plasma

plasma 24-hour urine plasma separated by centrifugation Heparinised plasma or whole blood; 24 hr urine 24 hour urine 24 hour urine, 90% used in assay Cerebrospinal fluid 24 hour urine, 80% used in assay non-fasting blood; heparin; 10 ml assayed 24-hour urine 24-hour urine 24 hour urine; 1/3 rd used in assay 24 hour urine 24-hour urine; 50% used; DMT, NMT stable 90 days at 15 C 24-hour urine; 50% used in assay

Collection

HCL ext acid pH with CHCl3, pH 9, ext CHCl3, evap Dowex 50; HCl ext and ethyl acetate at pH 10.2 HCL ext acid pH with CHCl3, pH 9, ext CHCl3, evap HCL ext acid pH with CHCl3, pH 9, ext CHCl3, evap Dowex 50; HCl ext and ethyl acetate at pH 10.2 Dowex 50; HCl ext and ethyl acetate at pH 10.2 Deproteinization, liquid-liquid ext, CH2Cl2 Dowex 50; HCl ext and ethyl acetate at pH 10.2 HCL ext acid pH with CHCl3, pH 9, ext CHCl3, evap Dowex 50; HCl ext and ethyl acetate at pH 10.2 Dowex 50; HCl ext and ethyl acetate at pH 10.2 Dowex 50 W X2; HCl hydrolysis HCL ext acid pH with CHCl3, pH 11, ext CHCl3, evap 50% concentrated and extracted with toluene purified by TLC, derivatized with TFAA 50% concentrated and extracted with toluene purified by TLC, derivatized with TFAA Urine (pH 10.5) ext with CHCl3 Whole blood lysed, protein ppt. with HClO4 extracted twice with chloroform Deproteinization, Liquid-Liquid ext, CH2Cl2 HCL ext acid pH with CHCl3, pH 9, ext CHCl3, evap acidified with HCl 50% concentrated and extracted with toluene purified by TLC, derivatized with TFAA acidified with HCl 50% concentrated and extracted with toluene purified by TLC, derivatized with TFAA pH 11, XAD resin, ethyl acetate elution, evap, TLC Deproteinization, liquid-liquid ext, CH2Cl2 acidified with HCl 50% concentrated and extracted with toluene purified by TLC, derivatized with TFAA Deproteinization, liquid-liquid ext, CH2Cl2 ion pair ext CHCl3, LC-silica column purification pH11, XAD resin, ethyl acetate elution, evap, TLC pH11, XAD resin, ethyl acetate elution, evap, TLC

Extraction Method

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Table 1. (Continued)

Drug Testing and Analysis S. A. Barker, E. H. McIlhenny and R. Strassman

Drug Test. Analysis (2012)

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

Drug Test. Analysis (2012) HDMT HDMT, HNMT DMT, MDMT, HDMT, NMT DMT, HDMT HDMT

Karkkainen et al.[65] Takeda et al.[66] Forsstrom et al.[67] Karkkainen et al.[68]

Emanuele et al.[69]

1995 1995 2001 2005

2010

individual urine samples; w /wo nialamide treatment morning urine samples; 50–100 ml morning urine samples morning and afternoon urines; 5 ml assayed urine (5 ml), plasma or serum (1 ml), stool; tissues (0.5-1.5 g) random urine samples

Collection

pH11, XAD resin, ethyl acetate elution, evap, TLC centrifugation, direct injection of 80 ml of urine urine centrifuged and ext on Oasis SPE cartridge urine cent and ext on Oasis HLB cartridge; Prep LC for blood urine cent and ext on Oasis HLB cartridge

pH11, XAD resin, ethyl acetate elution, evap, TLC

Extraction Method

Copyright © 2012 John Wiley & Sons, Ltd. paper chromatography, color reaction

HDMT

1965

paper chromatography (1 system), color reaction, bioassay paper chromatography (3 systems), color reaction, bioassay paper chromatography (1 system) paper chromatography (1 system) paper chromatography and auto-radiographs paper chromatography (2-D), color reaction paper chromatography (2-D), color reaction 2-D paper chromatography, color reaction 2-D paper chromatography, color reaction paper chromatography (3 systems) Fluorescence TLC (1 system), color reaction TLC (2-D), color reaction

HNMT, HDMT HNMT, HDMT HDMT HDMT HDMT HDMT, conjugate HDMT; DMT HDMT; DMT DMT, HDMT HDMT DMT, HDMT HDMT HDMT

Bumpus and Page[1] Rodnight[2] Fischer et al.[3] Fischer et al.[4] Feldstein et al.[5] Perry et al.[6] Brune et al.[7] Perry[8] Sprince et al.[9] Perry and Schroeder[10] Franzen and Gross[11] Siegel[12] Nishimura and Gjessing[13] Takesada et al.[14]

1955 1956 1961 1961 1961 1962 1963 1963 1963 1963 1965 1965 1965

Detection Methods

Compounds Analyzed

Author

Year

Rf and color (1 system) Rf and color (3 systems) Rf and color (1 system) Rf and color (1 system) Rf and color (1 system), radioactive spot Rf and color (2-D) Rf and color (2-D) Rf and color (2-D) Rf and color (2-D) Rf and color (3 systems) Fluoresence reading Rf and color (1 system) Rf and color (2-D) Rf and color

20 mg/24 hour

Confirmation Criteria

ND >5 mg/ 24 hour for HDMT ND ND ND ND 20 ng/ml ND ND ND 2 ng/ml 0.1 mg/100 ml ND

Limit of Detection

Table 2. Review of 69 studies regarding endogenous psychedelics showing the year, reference, compounds analyzed, detection methods, limits of detection (LOD) and confirmation criteria. Acronyms and abbreviations; HNMT, 5-hydroxy-N-methyltryptamine; TLC, thin-layer chromatography; 2-D, two dimensional; GC-FID, gas chromatography-flame ionization detector; derive, derivative; HFBI, heptafluoro-butyryl-imidazole; IS, internal standard; HPLC, high performance liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; ND, not determined; RT, retention time; UV, ultraviolet; TI, total ion; m/z, mass-to-charge ratio; CI, chemical ionization; IA, immunoassay; MRM, multiple reaction monitoring.

HDMT

Karkkainen and Raisanen[64]

Compounds Analyzed

1992

Year

Author

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Table 1. (Continued)

Reports of endogenous psychedelic N, N-dimethyltryptamines in humans

Drug Testing and Analysis

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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

6

wileyonlinelibrary.com/journal/dta TLC DACA and OPT spray on cellulose and silica; GC/MS, 58 m/z only TLC DACA and OPT spray on cellulose and silica; GC/MS, 58 m/z only GC-ECD; packed column TLC DACA and OPT spray on cellulose and silica; GC/MS, 58 m/z only

HDMT, DMT, DMT DMT DMT DMT, HDMT

DMT DMT

DMT, MDMT DMT, HDMT

Narasimhachari et al.[35] Walker et al.[36] Wyatt et al.[37] Narasimhachari and Himwich[38]

Lipinski et al.[39] Bidder et al.[40]

Narasimhachari et al.[41] HDMT, DMT, MDMT

DMT, HDMT

Heller et al.[30] Narsimhachari et al.[31] Narasimhachari et al.[32] Fischer et al.[33] Himwich et al.[34]

Carpenter et al.[42]

Christian et al.[43] Narasimhachari and Himwich[44]

1970 1971 1971 1971 1972

1972 1973 1973 1973

1974 1974

Copyright © 2012 John Wiley & Sons, Ltd.

1974

1975

1975 1975

GC-MS; 2 ft. SE-30 glass capillary column, DMT-d2 IS, TMS deriv GC-MS; 2 ft. SE-30 glass capillary column, DMT-d2 IS, TMS deriv

GC-FID, TLC, and Spectrofluorometry TLC and GC-FID, verified with spectrofluorometer TLC and GC-FID, verified with spectrofluorometer UV; paper chromatography, color reaction TLC (3 systems), color reaction; verified with spectrofluorometer paper and TLC (2-D); color reaction; GC-FID GC-MS; 2 ft. SE-30 glass capillary column, DMT-d2 IS, TMS deriv GC-MS; 2 ft. SE-30 glass capillary column, DMT-d2 IS, TMS deriv TLC DACA and OPT spray on cellulose and silica; GC/MS, 58 m/z only

paper chromatography, TLC (2-D), color reaction; GC-FID paper and TLC (2-D); color reaction; GC-FID of HDMT paper chromatography (2-D), color reaction paper chromatography and TLC, color reaction UV; paper chromatography, color reaction UV of diazo-deriv; paper chromatography, color reaction UV; TLC, color reaction UV; TLC, color reaction paper and TLC (2-D); color reaction; GC-FID of HDMT

Tanimukai et al.[21] Tanimukai et al.[22] Acebal and Spatz[23] Faurbye and Pind[24] Sireix and Marini[25] Spatz et al.[26] Fischer and Spatz[27] Saavedra and Udabe[28] Tanimukai et al.[29]

paper chromatography, color reaction paper chromatography (2-D), color reaction paper chromatography (2-D), color reaction paper chromatography (2-D), color reaction paper chromatography (3 systems), color reaction paper and TLC (2-D); color reaction; GC-FID of HDMT

1967 1967 1967 1968 1969 1969 1970 1970 1970

HDMT DMT, HDMT HDMT HDMT DMT, HDMT HNMT, HDMT, NMT, DMT, MDMT HDMT HDMT HDMT HDMT HDMT HDMT HDMT HDMT HNMT, HDMT, DMT, MDMT DMT, MDMT, HDMT DMT, MDMT, HDMT NMT, DMT, MDMT HDMT, glucuronide HDMT, DMT, MDMT

Detection Methods

Runge et al.[15] Perry et al.[16] Heller[17] Fischer and Spatz[18] Kakimoto et al.[19] Tanimukai[20]

Author

1966 1966 1966 1967 1967 1967

Year

Compounds Analyzed

DMT 10 pg/ml; MDMT 5 pg/ml 5 ng/ml HDMT; 1 ng/ml DMT

5 ng/ml HDMT; 1 ng/ml DMT

RT Rf and color (2-D); GC-RT; GC/MS 58 mz

TI spectrum match with DMT, HDMT Rf and color (2-D); GC-RT; GC/MS 58 mz

Rf and color (2-D); GC-RT; GC/MS 58 mz

TI spectrum match with DMT standard GC-RT, two ions and ratio GC-RT, two ions and ratio

Rf and color (2-D); GC-RT GC-RT, two ions and ratio GC-RT, two ions and ratio Rf and color (2-D); GC-RT; GC/MS 58 mz

0.05 mg/24 hour 0.5 ng/ml; m/z 202/204, 260/262 0.5 - 1.8 ng/ml; m/z 202/204, 260/262 5 ng/ml HDMT; 1 ng/ml DMT

0.5 ng/ml blood 0.05-2 ng/ml; urine 0.070.2 ng/ml 5 ng/ml HDMT; 1 ng/ml DMT

GC-RT and TLC or spectrofluorometer TLC and GC-FID, spectrofluorometer TLC and/or GC-FID, spectrofluorometer UV; Rf and color Rf, color and fluoresence

Rf and color (2-D); GC-RT Rf and color (2-D paper, TLC); GC-RT Rf and color (2-D) Rf and color (paper and 2-D TLC) Rf and color (2-D) UV; Rf and color UV; Rf and color UV; Rf and color Rf and color (2-D paper, TLC); GC-RT

>0.1 mg/24 hour ND ND >0.7 mg/24 hour ND ND ND ND ND 2 ng/ml 5 mg/ml per 24hour for DMT 2 ng/ml ND ND

Rf and color (2-D) Rf and color (2-D) Rf and color (2-D) Rf and color (2-D) Rf and color (3 systems) Rf and color (2-D paper, TLC)

Confirmation Criteria

ND 2 mg/24 hr for DMT and HDMT ND ND 10 mg/24 hour 5 ng/ml HDMT; 1 ng/ml others

Limit of Detection

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Table 2. (Continued)

Drug Testing and Analysis S. A. Barker, E. H. McIlhenny and R. Strassman

Drug Test. Analysis (2012)

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

Drug Test. Analysis (2012)

Copyright © 2012 John Wiley & Sons, Ltd. GC/MS selected ion monitoring; d4-DMT, d4-MDMT IS GC with nitrogen-sensitive detector GC-FID HPLC/fluoresence spectrum TMS derivatives; GC/MS, multiple ion detection TMS derivatives; GC/MS, multiple ion detection TMS derivatives; GC/MS, multiple ion detection TMS derivatives; GC/MS, multiple ion detection

DMT, NMT

DMT, HDMT, MDMT

DMT, MDMT DMT

DMT, NMT

DMT DMT, HDMT

DMT, MDMT DMT DMT, MDMT HDMT HDMT

HDMT

HDMT

HDMT

HDMT, HNMT DMT, MDMT, HDMT, NMT

DMT, HDMT HDMT

Oon and Rodnight[51]

Riceberg and Van Vunakis[52]

Corbett et al.[53] Walker et al.[54]

Murray et al.[55]

Checkley et al.[56] Raisanen and Karkkainen[57] Smythies et al.[58] Checkley et al.[59] Uebelhack et al.[60] Sitaram et al.[61] Raisanen et al.[62]

Karkkainen et al.[63]

Karkkainen and Raisanen[64] Karkkainen et al.[65]

Takeda et al.[66] Forsstrom et al.[67]

Karkkainen et al.[68] Emanuele et al.[69]

1977

1978

1978 1979

1979

1979 1979

1988

1992

1995 2001

2005 2010

1995

1979 1980 1983 1983 1984

GC-ECD; HFBI derivative GC/MS, Selective Ion Monitoring capillary column gas chromatography GC-NPD,TLC on cellulose; GC/MS 2 patients and pooled (10) extract GC with nitrogen-sensitive detector TMS derivatives; GC/MS, multiple ion detection

HDMT, DMT, MDMT HDMT DMT, NMT

Huszka et al.[48] Cottrell et al.[49] Oon et al.[50]

1976 1977 1977

HPLC/ESI-MS/MS HPLC/ESI-MS/MS

3-D-HPLC-electrochemical detection HPLC/ESI-MS-MS

Radioimmunoassay and HPLC (RIA-HPLC)

GC/NPD;GC/MS

DMT

Murray and Oon[47]

1976

GC-MS; 2 ft. SE-30 glass capillary column, DMT-d2 IS, TMS deriv GC-FID,TLC on cellulose; GC/MS 2 patients and pooled (10) extract GC-FID,TLC on cellulose; GC/MS 2 patients and pooled (10) extract TLC and GC-FID, verified with spectrofluorometer HFBI derivatives, GC-ECD GC/NPD;GC/MS

DMT DMT

Detection Methods

Angrist et al.[45] Rodnight et al.[46]

Author

1976 1976

Year

Compounds Analyzed

RT; MS of selected samples GC/MS RT, m/z 58 only

15fmol/ml DMT DMT 10 pg/ml; MDMT 5 pg/ml 10 pg/ml whole blood

RT and electrochemical response RT, Pseudo molecular ion, MRM

RT, Pseudo molecular ion, MRM RT, Pseudo molecular ion, MRM

0.1 ng/ml MDMT; 0.05 ng/ml NMT 0.3 ng/ml HDMT; 0.2 ng/ml DMT ND

RT, molecular ions or fragments

RT, molecular ions or fragments

RT, molecular ions or fragments

RT, ion fragments, ratios RT RT RT and fluoresence spectrum RT, molecular ions or fragments

RT RT, molecular ions or fragments

20 ng/24hour DMT; 50 ng/24 hour NMT 0.5 mg/ml per 24hour 0.1-0.15 ng/ml DMT; 0.25-0.3 ng/ml HDMT 70 pg/ml DMT, MDMT 0.5 mg/ml per 24hour ND >0.01 ng/ml per 12 hr 0.1-0.15 ng/ml DMT; 0.25-0.3 ng/ml HDMT 0.1-0.15 ng/ml DMT; 0.25-0.3 ng/ml HDMT 0.1-0.15 ng/ml DMT; 0.25-0.3 ng/ml HDMT 0.1-0.15 ng/ml DMT; 0.25-0.3 ng/ml HDMT 50 pg/ml 0.35 ng/ml HDMT; 0.1 ng/ml DMT

RT; MS of selected samples

RT; CI MS confirmation HPLC RT and IA response

TLC and GC-FID, spectrofluorometer RT RT; CI MS confirmation

Rf and color; GC-RT; GC-MS

RT, two ions and ratio Rf and color; GC-RT; matching TI MS

Confirmation Criteria

4 ng/ml 1 mg/24 hour

ND

Concentration

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Table 3. (Continued)

Reports of endogenous psychedelic N, N-dimethyltryptamines in humans

Drug Testing and Analysis

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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

10

wileyonlinelibrary.com/journal/dta

HDMT, DMT, MDMT HDMT

DMT, NMT

DMT, NMT

Huszka et al.[48]

Cottrell et al.[49]

Oon et al.[50]

Oon and Rodnight[51]

Riceberg and Van Vunakis[52]

1976 1976 1976

1976

1977

Copyright © 2012 John Wiley & Sons, Ltd.

1977

1977

1978

DMT, HDMT, MDMT

6 controls

69 patients, 24 normal

19 normal

20 psychiatric patients; 2 controls

7 schizophrenics, special diet; MAOI phenelzine

4/4 DMT, 2/4 MDMT, 4/4 HDMT, whole blood

3/4 DMT, 1/4 MDMT, 3/4HDMT, plasma

No diurnal variation, no dietary source 69/69 DMT; 17/24 DMT

15/20 HDMT; 0/2 HDMT; no DMT or MDMT detected 19/19 DMT; 19/19 NMT

No HDMT, DMT, MDMT detected

13/23 DMT; 7/17 DMT 37/122 DMT; 1/20 DMT 23/54 DMT; 1/14 DMT

23 psychiatric patients, 17 controls 122 psyciatric patients; 20 controls 54 psychiatric patients, 14 controls; 1 patient strict diet, 2 patients on neomycin

Christian et al.[43] Narasimhachari and Himwich[44] Angrist et al.[45] Rodnight et al.[46] Murray and Oon[47]

1975 1975

DMT DMT DMT

Carpenter et al.[42]

1975

positive for DMT, MDMT 24/47 HDMT, 10/47 DMT; 14/46 HDMT

Narasimhachari et al.[41]

1974

2/11 acute schizo DMT 2/38 blood DMT; 1/44 urine psychotic patients 6/6 HDMT;3/6 DMT; 0/6 MDMT

1 control cerebrospinal fluid 47 infantile autism, 46 controls

Lipinski et al.[39] Bidder et al.[40]

1974 1974

3/6 DMT, 6/6 HDMT

DMT, MDMT DMT, HDMT

HDMT, DMT, MDMT DMT, HDMT

Narasimhachari and Himwich[38]

1973

6 controls neg for all; 5/6 autistics positive for HDMT 4/6 schizo DMT, HDMT; 7/7 controls negative; 6/45 DMT 1/11 DMT; 1/29 DMT

Positive/Negative

4/26 DMT, 6/26 5-HDMT; 4/10 DMT, 8/10 HDMT

DMT DMT

Walker et al.[36] Wyatt et al.[37]

1973 1973

6 chronic schizophrenics, 7 controls; special diets, restricted meds 45 controls 11 controls, 29 psychiatric patients; no meds for 30 days 6 chronic schizophrenics

6 autistics, 6 controls; special diets

Subjects

7 control 6 chronic schizo, 11 acute schizo, 11 hepatic coma 34 with acute psychotic illness, 3 with non psychotic illness, 1 control 6 chronic schizophrenics highly restricted diet, no drug administration 4 weeks 26 acute schizophrenics; 10 controls; no meds for 3 weeks

DMT, HDMT

Narasimhachari et al.[35]

1972

HDMT, DMT, MDMT HDMT, DMT, MDMT DMT DMT

Himwich et al.[34]

Author

1972

Year

Compounds Analyzed 78 ng/ml

DMT range 0.1-4.5 mg/ 24 hr; DMT range 0.1-0.5 mg/ 24 hr HDMT 0.25-0.38pmol/ml, MDMT 0.09pmol/ml, DMT 0.77-3.69pmol/ml HDMT 0.11-2.64pmol/ml, MDMT 0.7-2.89pmol/ml, DMT 0.27-14pmol/ml

DMT range 20–2500 ng/24 hour; NMT range 121–3000 ng/24 hour

1-120 nmol HDMT/24 hour

0.05-0.79 ng/ml; 0.06-0.22 ng/ml >500 ng/24 hour DMT > 500 ng/24 hour, Mean range 226–1,717 ng/ 24 hour; control 228 ng/ 24 hour NA

HDMT mean 1.67 mg/24 hr schizo, 1.73 mg/24 hr controls; DMT not quantitated ND ND

(1) 6, (1) 1.8 (1) 2.5 ng/ml, (1) 4.6 ng/ml; 0.76 ng/ml 1-3 mg/24 hour; 500 ng/24 hr DMT mean 96 ng/g creatinine; HDMT mean 950 ng/g creatinine DMT range from