aTen!on, impulsivity, hyperac!vity, memory, and concentra!on of diagnosed pa!ents. Psychos!mulants, including methylphenidate and amphetamines are the.
Examining the Safety of Herbal Medicine Use and Herb-Drug Interac=ons in Pediatric APen=on-Deficit Hyperac=vity Disorder Mazhar, Hajra. (a,b)*, Guntaka, Sai P. (a), Galeffi, Raphael. (a), Liu, Rui. (a), Robaey, Philippe. (b), Harris, Cory. (a). (a) Department of Biology, Faculty of Science, University of O#awa, O#awa, Canada, (b) Children’s Hospital of Eastern Ontario Research Ins&tute, O#awa, Canada
INTRODUCTION
RESULTS AERs involving ADHD drugs
40 30
AERs involving other drugs
20
D
10 0
a. CES-Carboxylesterase , b. CYP-Cytochrome
Herbal Medicine
OBJECTIVES
METHODS
Incubation Time (mins)
20
60
15
Figure 1: Total adverse event reports and adverse event reports involving ADHD drugs for common herbal medicines used by pediatric ADHD pa&ents (age 0-18), as iden&fied through the FDAble database (h#p://www.fdable.com)5.
Figure 4 HPLC-DAD Chromatogram for rhodiola &ncture sample at 280nm. Panel A represents standard compound mixture and panel B represents rhodiola &ncture at 10mg/ml. 1=Salidroside, 2=Tyrosol, 3=Rosarin, 4=Rosavin, 5=Rosin.
Screening of herbal extracts in CES1, CYP2D6, and CYP3A4 assay A 100
Figure 5 HPLC-DAD Chromatogram for Linden &ncture sample at 350nm. Panel C represents standard compound mixture and panel D represents linden &ncture at 10mg/ml. 1=Ru&n, 2=Naringin, 3=Quercetrin, 4=Querce&n.
Dose response curves of select herbal medicines A 100
80 60 40 20 0
B
80 60 40 20 0
B
C
100 80 60 40 20 0
100
% Inhibi=on
Pediatric pa&ents with ADHD are commonly administered natural health products by parents, including herbal medicines, at &mes concurrently with prescrip&on drugs. A recent Canadian study reported that 48%-78% of pediatric psychiatry pa&ents had used complementary and alterna&ve medicine (CAM), and 46% had concurrently used CAM with prescrip&on medicine3. OPen, these CAM include herbal medicines which can contain raw or processed substances, that can elicit pharmacokine&c and pharmacodynamic herb-drug interac&ons. Concomitant use of prescrip&on drugs with natural health products can pose a risk for herbdrug interac&ons and adverse events4. Given their popularity and the poten&al risk, the safety of herbal medicines commonly used for ADHD must be examined with ADHD prescrip&on medica&ons. 1. To determine the prevalence of herb-drug interac&ons causing clinical adverse events in ADHD pa&ents 2. To determine poten&al risk of herb-drug interac&ons from concurrent use of herbal medicines and prescrip&on drugs in pediatric ADHD pa&ents using in-vitro methods. Searching for Clinical Evidence of Adverse Events We systema&cally searched the database (h#p://fdable.com) for adverse event reports (AERs) involving one or more of 22 herbals. We used the advanced search op&on to target each herbal by common and scien&fic name and recorded results for pa&ents aged 0-18 years. Extract Prepara=on for Enzyme Assays 30mL of the nineteen sample &nctures were put in the rotary evaporator to evaporate the solvent. Samples were freeze dried to remove water content and stored in -20oC freezer for further use. CYP2D6 & CYP3A4 Screening and IC50 Assay 10mg/ml of sample was resolubilized in methanol and further diluted with water to obtain a 1mg/ml working solu&on. In Corning Costar 96-well Micro&ter Plate, 10µL of each extract was added, giving a 50µg/mL in-well concentra&on. Respec&ve posi&ve control and substrate (table 2) were used with methanol as vehicle control with potassium phosphate buffer and NADPH. Plates were incubated at 37oC in Applied Biosensor Cytofluor 4000 plate reading system for different incuba&on &mes (table 2). Select samples were diluted with methanol further to obtain 6 concentra&on points and tested in the method above to obtain dose response curves. CES1 Screening Assay 10mg/ml of sample was resolubilized in 20% acetone and further diluted with water to obtain a 2mg/ml working solu&on. In Corning Costar 96-well Micro&ter Plate, 10µL of each extract was added, giving a 200µg/mL in-well concentra&on. Posi&ve control and substrate (table 2) were used with 20% acetone as vehicle control with 50mM Tris buffer. Table 2 Assay informa&on for CYP2D6 and 3A4, and CES1. CYP 3A4 CYP 2D6 CES1 + Control Ketoconazole Quinidine JZL184 Substrate DBF AAMC N-nitrophenyl acetate
50
% Inhibi=on
Clinical Adverse event reports from FDAble Number of Adverse Event Reports
A#en&on Deficit Hyperac&vity Disorder (ADHD) affects the a#en&on, impulsivity, hyperac&vity, memory, and concentra&on of diagnosed pa&ents. Psychos&mulants, including methylphenidate and amphetamines are the first-line of pharmacotherapy prescribed. In some cases, non-s&mulants including atomoxe&ne and guanfacine are also prescribed2. ADHD medica&on have varying pathways of metabolism (Table 1). Table 1 Metabolism informa&on of commonly prescribed ADHD drugs. Drug Metabolizing Enzyme Major Metabolite Methylphenidate CES1a Ritalnic Acid Amphetamine CYP2D6b 4-Hydroxyamphetamine Atomoxetine CYP2D6b 4-Hydroxyatomoxetine b Guanfacine CYP3A4 3-Hydroxyguanfacine
C
0.5
1
1.5
2
2.5
0
0.5
1
-0.3
0.2
1.5
2
2.5
D
80 60 40 20 0 0
E
C 100
0.5
1
1.5
2
100
2.5
-0.8
0.7
1.2
1.7
F
80
80 60 40 20 0
60 40 20 0 0
0.5
1
1.5
2 0.4
0.8
1.2
1.6
2
Log of concentra=on (ug/ml) Figure 6 Dose response rela&onships of (A) Rhodiola: IC50 = 1.21 µg/mL and (B) Ginkgo biloba: IC50 = 23.51 µg/mL with CES1. C) Rhodiola: IC50 = 12.22 µg/mL and D) St. John’s Wort: IC50 = 7.09 µg/mL with CYP3A4. E) Goldenseal: IC50 = 12.22 µg/mL and F) Rosemary: IC50 = 17.43 µg/mL with CYP2D6. At each concentra&on, n= 3.
Herbal Medicine Figure 2 Mean % inhibi&on by herbal medicines used by ADHD pa&ents of A) CES1 at 200µg/mL, B) CYP3A4 at 50xµg/mL, C) CYP2D6 at 50µg/mL. n=3 (+/- SEM).
DISCUSSION & CONCLUSIONS
Phytochemistry of herbal extracts
EA
EP
• Some herbs used by ADHD pa&ents show high inhibi&on poten&al for ADHD drug metabolizing enzymes • Rosemary, ginkgo biloba, ginger, rhodiola, goldenseal show high inhibi&on and are good candidates for future studies in the field • Potent herbal: rhodiola-CES1, St. John’s Wort-CYP3A4, goldenseal-CYP2D6 • Linden needs more research as it showed high inhibi&on and is scarcely studied • Healthcare prac&&oners should ini&ate conversa&on about use of CAM with pa&ents to discuss poten&al benefits and adverse effects • Pharmacology of ADHD drugs needs more research. Specifically, CES1 is rarely studied in combina&on with natural health products
ACKNOWLEDGEMENTS
EC
I would like to thank: • Dr. Philippe Robaey • Dr. Cory Harris • Dr. Brian Foster
• Dr. John Arnason • Rui Liu • St. Francis Herbal Farm
• University of O#awa • NSERC • CHEO
REFERENCES 1. 2.
Figure 3: HPLC chromatograms of EA: Echinacea angus,folia, EP: Echinacea purpurea, EC: Combina&on &ncture of EA and EP at 260nm and 330nm.
3. 4. 5.
Biederman J. A#en&on-deficit/hyperac&vity disorder: a selec&ve overview. Biol Psychiatry. 2005 Jun 1;57(11):1215-20. Wagner DJ, Vallerand IA, McLennan JD. Treatment receipt and outcomes from a clinic employing the a#en&on-deficit/hyperac&vity disorder treatment guideline of the children's medica&on algorithm project. J Child Adolesc Psychopharmacol. 2014 Nov;24(9):472-80. Galicia-Connolly E et al. CAM use in pediatric neurology: an explora&on of concurrent use with conven&onal medicine. PLoS One. 2014;9(4):e94078. World Health Organiza&on (WHO), Na&onal Policy on Tradi&onal Medicine and Regula&on of Herbal Medicines., in Report of a World Health Organiza&on Global Survey. 2005, WHO: Geneva, Switzerland. Mazhar H, Harkin EF, Foster BC, Harris CS. Complementary and Alterna&ve Medicine use in Pediatric A#en&on-Deficit Hyperac&vity Disorder (ADHD): Reviewing the Safety and Efficacy of Herbal Medicines. Curr Dev Disod Rep. 2016; 3(1):15-24.
