Drug-Dietary Supplement Interactions What Have We Learned?

Transcription

1 Drug-Dietary Supplement Interactions What Have We Learned? John S. Markowitz, Pharm.D. Professor University of Florida 35 min

2 Overview The use of Dietary Supplements in the US What makes Botanical-Drug Interactions(BDIs) especially challenging to study? Basic categorization of drug interactions Conventional means and limitations of in vivo assessments The unique complexities of in vitro BDI assessment Conclusions

3 Drug Interactions with Dietary Supplements The actual prevalence of clinically meaningful botanical-drug interactions (BDIs) is unknown. What is known; ~ 50% of all adults in the US report having used at least one dietary supplement in the past month and use rates are even higher in in selected patient populations ~70% do not inform their health care provider of use and routinely combine supplements with conventional medications The estimated number of dietary supplement products on the market has increased from ~4000 in 1994 to >55,000 in Barnes PM and Bloom B (2008) Department of Health and Human Services. CDC, National Center for Health Statistics. Number 12: December 10, 2008.

4 The 20 top-selling Herbal Supplements in the US in 2014 HerbalGram. 2015:107;52-59

5 Medication Categories that Appear to be More Frequently Associated with BDIs Tsai H-H, et al. Int J Clin Pract 2012;66:

6 Individual Medications that Appear to be More Frequently Associated with BDIs

7 CHALLENGES of BDIs in General: Unlike conventional agents, dietary supplements/extracts are complex mixtures Phenols Volatile Oils Flavonoids Anthocyanins Tannins /proanthocyanidins Isoflavonoids others Glucosinilates Saponins Anthraquinones Polysaccharides Coumarins Macronutrients lipids, amino acids Vitamins, minerals Bitters BDI= Botanical Drug Interactions

8 Individual Botanicals are complex mixtures Goldenseal (Hydrastis canadensis) Hydrastine Berberine Canadine 3 -hydroxy-n,ndimethylcoclaurine 1-Demethyl-N,Ndimethyllincarpine 1,2-Dihydronorreticuline 4 -Demethoxyltembetarine Magnocurarine Magnoflorine N-methylcocluarine Canelilline Tembetarine Stepholidine Reticuline Hydrastinine Isohydrastidine Scoulerine Discretamine 20-Hydroxyecdysone 1-Hydroxyhydrastine Hydrastine methiodine Dehydrodiscretamine Canadinic acid 1-Hydroxyhydrastine Demethyleneberberine Tetrahydro-jatrorrhizine 13-hydroxyberberine Jatrorrhizine isomer Columbamine Hydrastidine Thalinfendin Tetrahydroberberastine Canadaline 13-Methylcanadine 13-Hydroxycanadine 5,6-Dehydroberberine 13-Methylberberine 13-Methoxyl 5,6- dehydroberberine 8-Oxotetrahydrothalinfendin 8-Oxotetrahydroberberine 8-Oxoberberine 8-Oxo 5,6-dehydroberberine

9 Proposed phase I and II metabolism of hydrastine in humans after administration of Goldenseal Extracts Gupta PK, et al. DMD 2015;43:

10 Botanical extracts are often sold as combinations; This further increases the number of potential perpetrators

11 Major Categories of Drug Interactions Pharmaceutic -e.g. physiochemical incompatibilities: rarely a clinical issue with botanical supplements Pharmacodynamic -PD interactions can be additive, antagonistic, synergistic by e.g. the anticoagulant action of warfarin is purportedly enhanced ginkgo biloba constituents Pharmacokinetic -Absorption, Distribution, Metabolism (e.g.cyp450), and Excretion (ADME) *PK interactions can be the most convincingly demonstrated by drug concentration measurements

12 Categorizing Drug-Drug Interaction Significance with Conventional Agents Major Clinical Significance- relatively well documented and potentially life threatening or harmful to patients (these are rare) Moderate Clinical Significance- more documentation desirable and potential harm to the patient is less Minor Clinical Significance- may occur but documentation is lacking, potential for harm is slight, the interaction is rare, or all of the aforementioned

13 General Scheme of Drug Metabolism

14 Estimated contribution of phase I and phase II enzymes to drug metabolism

15 What about Drug/Physiological Transporters? Amino acid transporters, LAT SLC7A5 LAT1 (BBB, Placenta), L-DOPA SLC7A8 LAT2 (many tissues), basic and neutral amino acids Bile acid transporter SLC10A1 NTCP (Liver), bile acids SLC10A2 ASBT (ileum), bile acids Peptide transporters, PEP SLC15A1/2 PEPT1/2 (gut/kidney) oligopeptides, ß-lactams Monocarboxylate transporters, MCT SLC16A1 MCT1( gut, BBB etc.) SLC16A7 lactate, benzoate Organic anion transporting polypeptides, OATP SLCO2A1 PGT (lung et al., PG) SLCO1A2 OATP-A, OATP (Brain, anions) SLCO1B1 OATP-C, LST1 (Liver specific) SLCO1B3 OATP-8 (liver specific) SLCO2B1 OATP-B (liver, gut etc.) SLCO3A1 OATP-D (many tissues) SLCO4A1 OATP-E (many tissues) SLCO1C1 OATP-F SLCO4C1 OATP-R (Kidney) digoxin, Organic ion transporters, OCT, OCTN, OAT SLC22A1 OCT1 (liver, cations, TEA, MPP + ) SLC22A2 OCT2 (kidney, TEA, dopamine) SLC22A3 OCT3 (placenta, brain) SLC22A4 OCTN1 (kidney, blood cell, cations) SLC22A5 OCTN2 (kidney etc., carnitine) SLC22A6 OAT1 (kidney, PAH) SLC22A7 OAT2 (liver, PAH, MTX, camp) SLC22A8 OAT3 (kidney, PCG, cimetidine) SLC22A OAT4 (placenta, PAH, ochratoxin A) SLC22A OAT5 (liver) SLC22A12 URAT1 (kidney, uric acid) Nucleoside transporter, CNT, ENT SLC28A1,2 CNT1,2 (many tissues), nucleoside concentrative transporters (active) SLC29A1,2 ENT1,2 (many tissues, nucleoside) equilibrium transporters (facilitative) ABC/ATP-dependent transporters ABCA1 Cholesterol ABCB1 P-glycoprotein (many tissues) ABCC1 MRP1 (many tissues, anionic?) ABCC2 MRP2 (liver, gut etc., anions) ABCC3 MRP3 (liver, gut etc., anions) ABCC4 MRP4 (lung etc. antiviral drugs, anions) ABCG2 BCRP(placenta, liver etc. anions)

16 Relative Abundance of CYP450s in Humans Zanger & Schwab. Pharmacol Ther 2013

17 Mechanisms underlying metabolic interactions between botanical constituents and medications Brantley SJ, et al. DMD 2014;42:301-17

18 Choosing Bioassays to Study BDIs in vivo In the living body, referring to tests conducted in living animals-normal subject studies are ideal. * Animal models, particularly rodents, though relatively inexpensive, have a number of significant translational limitations in the study of DDIs in vitro In an artificial environment- i.e. test tube or culture media ex vivo Usually refers to conducting experiments or tests on a tissue(s) taken from a living organism

19 in vivo Probe Drug or Cocktail Methodology Combinations of probe drugs metabolized by specific known pathways are administered simultaneously to healthy volunteers both before/after exposure to an agent of interest * Standard PK parameters (e.g. Cmax, AUC) for the probe drug and/or metabolite(s) are then calculated pre- and post exposure * Significance of the results are based upon the magnitude of the effect, and a substrates potential toxicity at higher Cp (if inhibition) or consequences of therapeutic failure at lower Cp (if induction) Examples of Probe Drugs Used: CYP450 Assessed: dextromethorphan, debrisoquine : CYP2D6 midazolam, dapsone: CYP3A4 caffeine: CYP1A2 mephenytoin, omeprazole: CYP2C19 tolbutamide, diclofenac: CYP2C9 chloroxazone: CYP2E1

20 Typical Cocktail Methodology: CYP3A4 and CYP2D6 Assessing BDIs in Healthy Volunteers Baseline CYP450 assessments followed by a minimum 14 day botanical exposure and then re-assessment is a typical study paradigm Research volunteer has consented to photography

21 Alprazolam Concentration (ng/ml) Alprazolam Pharmacokinetics: St. John s wort 100 Alprazolam Pharmacokinetics Baseline After SJW C max (ng/ml) T max (hours) SJW Baseline AUC (ng hour ml -1 ) ß 1/2 (hour) The AUC and T 1/2 were significantly different at p< The C max and T max were not significantly different after SJW treatment Time (hours) After SJW treatment only 7 subjects had measurable levels of ALPZ at 36 hours, and no SJW treated subject had measurable levels of ALPZ at 48 hours. Markowitz et al, JAMA 2003;290:1500.

22 Limitations of in vivo (i.e. human) studies Very expensive to conduct Protracted time-line for completion e.g. 8hr research unit study day x 2, day exposure periods between study visits, etc Inter-individual variability in PK Requisite analytical capability (e.g.lc-ms/ms) Problems with generalizability of results Most studies do not include measures of systemic botanical exposure Potential risk to Human Subjects

23 in vitro Tools to Predict Metabolic Clearance * Liver microsomes high throughput and most commonly employed mostly oxidative (e.g. CYP 450) * S9 fraction Supernatant fraction obtained from an organ (usually liver) homogenate by centrifuging at 9000 g x 20 min in a suitable medium; this fraction contains cytosol and microsomes high throughput Phase I & Phase II metabolism * Hepatocytes low throughput cell membrane/transporters intracellular concentration Phase I & Phase II metabolism/induction

24 in vitro Screening for BDIs * Generally Accepted Advantages High through-put, and may be carried out in most labs Non-invasive, Specific mechanism(s) evaluated in controlled system Potential to identify perpetrating components In principle, can forecast the magnitude of an intx Information from enzyme inhibition studies is extremely valuable as it can allow extrapolation of the data to other compounds and of DDIs in organs other than liver. The availability of human liver tissue, cdnaexpressed CYP enzymes, and specific probe substrates are valuable tools in the assessment of a drug's potential to inhibit different CYP enzymes in vitro.

25 in vitro Screening for BDIs * Potential Limitations In the evaluation of the potency of DDIs, estimation of the inhibitor concentrations at the target site is essential, but extremely difficult since its direct measurement is almost always impossible. Does not account for the contribution of first-pass effects, hepatic blood flow, protein binding, nonhepatic elimination Unique to Botanical Assessments; Some constituents found within plant extracts may not be absorbed or attain meaningful concentrations Metabolites of botanical extracts are poorly characterized for most extracts and could potentially contribute to the net inhibitory or inductive effects

26 in vitro Screening for BDIs Potential Limitations Unique to Botanical Assessments Cont. The commercial availability of many phytochemical is limited which precludes their initial screening using in vitro systems. (this is improving!) There is a known large product to product variability and known difficulties in characterization and standardization of products with complex phytochemical profiles. This may lead to difficulties in reproducibility of experiments Confounding physiochemical issues solubility, stability, purity, solvent effects, buffer effects? differential contribution of stereoisomers is rarely considered

27 PROBLEM: In vitro screening studies suggesting BDI are often not confirmed in clinical confirmatory studies: e.g. Milk Thistle (Silybum marianum) In vitro: Concentration-dependent inhibition of CYP2D6, CYP2E1, and CYP3A4 by silymarin and silybin and mechanism-based inactivation of CYP3A4 and CYP2C9 by silybins and silymarin extracts have also been reported Beckmann-Knopp et al., 2000; Venkataramanan et al., 2000; Zuber et al., 2002; and Sridar et al., In vivo: Formal pharmacokinetic studies in humans have failed to confirm in vitro predictions of metabolic inhibition Piscitelli et al., 2002; DiCenzo et al., 2003; Gurley et al., 2004, 2006, 2008; Mills et al., 2005; van Erp et al., 2005; Fuhr et al., 2007; Rao et al., 2007; Deng et al., 2008; and Kawaguchi-Suzuki et al., 2014

28 Milk Thistle (Silybum marianum) Extract content vs in vivo disposition

29 Milk Thistle (Silybum marianum) Extract content vs in vivo disposition NOTE: Among all evaluated P450 isoenzymes, CYP2C9 appears to be the isoform most sensitive to inhibition by flavonolignans. In a human liver microsome incubation study, silybin B was determined to be the most potent flavonolignan for the inhibition of CYP2C9 with an IC50 value of 8.2 mm, followed by silybin A (Brantley et al., 2010).

30 Analysis of Milk Thistle Capsule (Legalon )

31 Silymarin Constituents in Human Plasma 1.5hrs post dose (two 175 mg Legalon capsules)

32 LC-MS/MS Analysis of Silymarin Constituents in vitro analysis of Legalon capsule Plasma concentrations 1.5 h after a single dose (two 175 mg capsules) NOTE: In vivo, the C max values of free (unconjugated) silybin A were ~ 2-3-fold higher than those observed for silybin B, as well as the much greater AUC 0 α and lower CL/F values for silybin A. This finding is consistent with the known stereoselective (i.e. favored) glucuronidation of silybin B* *Jancová et al. Evidence for differences in regioselective and stereoselective glucuronidation of silybin diastereomers from milk thistle (Silybum marianum) by human UDP glucuronosyltransferases. Xenobiotica 2011; 41: J Chromatogr. B 902 (2012) 1 9

33 There are limitations with in vitro testing for BDIs whether testing single or multi-constituent extracts in vitro studies assessing single phytoconstituents extracts do not accurately reflect in vivo exposures does not account for metabolites or co-administered phytoconstituents when an extract is taken in vitro studies assessing whole phytoconstituent extracts will not accurately reflect in vivo exposures Assumes all constituents in a given extract are bioavailable and what would be presented to the liver

34 Overall Summary and Conclusions A number of limitations should be recognized in the use of in vitro methodologies to assess of BDIs In spite of these limitations, in vitro methods remain the most powerful and cost-effective tool for initial screening procedures as well as in other applied experiments. Semi-quantitative predictions of drug interactions many unknown factors human ADME properties in vivo Models provide numbers that must be placed in context with multiple factors: therapeutic area, therapeutic index, route of administration The relative contribution of stereoisomers should not be ignored in in vitro studies or in bioanalysis.

35 Acknowledgments Hao-Jie Zhu, Ph.D. Xinwen Wang, M.S. William J. Gurley, Ph.D. Brian Brinda, Pharm.D. David I. Appel, MD Juliana Munoz, Pharm.D. Wild Flowers Worth Knowing by Neltje Blanchan (1934) R21 AT : Pharmacokinetics and Drug Interactions with Milk Thistle NIH National Center for Complementary and Alternative Medicine (NCCAM), Markowitz JS (PI).

36 EXTRA SLIDES

37 An exploratory ex vivo approach

38 An exploratory ex vivo approach This study aimed to develop a novel ex vivo approach differing from conventional in vitro methods in that rather than botanical extracts or individual constituents being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects previously studied was utilized to assess BDIs METHODS The clinical samples utilized were sourced from banked residual blood samples (stored -70 C) from completed normal volunteer PK studies of milk thistle (MT) extracts and goldenseal (GS).Silybin A, silybin B and hydrastine and berberine were selected to represent the principal components of MT and GS, respectively. Pooled HLMs were pre-incubated with an NADPH generating system in the presence and absence of various concentrations of the phytoconstituents in phosphate buffer at 37 C for 10 min. The reactions for the inhibitory effect assessment of MT and GS were initiated by adding the probe substrates of CYP2C9 (tolbutamide )and CYP3A4 (midazolam) For the ex vivo study, plasma samples containing principal phytochemical constituents and their metabolites from 5 healthy volunteers who had participated in PK studies of characterized MT and GS supplements.

39 ex vivo: Results/Discussion Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybin A and B and hydrastine and berberine on CYP2C9 and CYP3A4/5, respectively, with results which more closely mirrored those generated in clinical study. Data from conventional buffer-based in vitro studies were actually less predictive than the ex vivo assessments. Thus, this novel ex vivo approach may be a promising approach predicting clinically relevant BDIs than conventional in vitro methods.

40 Comparison of in vitro and potential value of ex vivo studies in vitro Studies with Botanical Supplements Advantages Easy/fast to perform Controlled environment Relatively inexpensive Ethical considerations Limitations Unknown absorption or bioavailability Single constituent used Product-to-product variability=reproducibility problems Metabolites poorly characterized=role in DDI *Results not always confirmed by in vivo studies ex vivo (plasma) vs in vitro (in buffer) Advantages Plasma: all constituents and metabolites in the circulation Time consuming Limitations Clinically relevant concentrations Somewhat more expensive Involves human subjects although fewer, with a single study phase (no pre- and post exposure), Endogenous plasma compounds and much less intense blood sampling Accounts for protein binding Far less drug analysis

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42 Mean free plasma concentrations of silybin A (A), silybin B (B), isosilybin A (C), isosilybin B (D) after single oral doses of one (175 mg), two (350 mg), and three (525 mg) milk thistle extract (Legalon ) capsules in volunteers (n=13) Drug Metab Dispos 2013;41:

43 Selected Drug Transporters implicated in DDIs of Interest to the US FDA FDA Draft Guidance 2012

44 Non-Agreement of in vitro predictions vs in vivo study Consideration to bioavailability and the potential influence of stereoselective metabolism