Drug-Dietary Supplement Interactions

What Have We Learned?

John S. Markowitz, Pharm.D. 35 min

Professor

University of Florida

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

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 2012.

Barnes PM and Bloom B (2008) Department of Health and Human Services.

CDC, National Center for Health Statistics. Number 12: December 10, 2008.

The 20 top-selling Herbal Supplements in the US in 2014

HerbalGram. 2015:107;52-59

Medication Categories that Appear to be More

Frequently Associated with BDIs

Tsai H-H, et al. Int J Clin Pract 2012;66:1056–1078

Individual Medications that Appear to be

More Frequently Associated with BDIs

CHALLENGES of BDIs in General:

Unlike conventional agents, dietary

supplements/extracts are complex mixtures

Phenols

Volatile Oils

Flavonoids

Anthocyanins

Tannins

/proanthocyanidins

Isoflavonoids

others

Glucosinilates

Bitters

Saponins

Anthraquinones

Polysaccharides

Coumarins

Macronutrients

lipids, amino acids

Vitamins, minerals

BDI= Botanical Drug Interactions

Individual Botanicals are complex mixtures Goldenseal (Hydrastis canadensis)

Hydrastine

Berberine

Canadine

3′-hydroxy-N,N-

dimethylcoclaurine

1-Demethyl-N,N-

dimethyllincarpine

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

Proposed phase I and II metabolism of hydrastine in

humans after administration of Goldenseal Extracts

Gupta PK, et al. DMD 2015;43:534-552

↑

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

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

e.g. the anticoagulant action of warfarin is purportedly enhanced by 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

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

General Scheme of Drug Metabolism

Estimated contribution of phase I and

phase II enzymes to drug metabolism

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)

Zanger & Schwab. Pharmacol Ther 2013

Relative Abundance of CYP450s in Humans

Mechanisms underlying metabolic interactions

between botanical constituents and medications

Brantley SJ, et al. DMD 2014;42:301-17

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

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

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

Alprazolam Pharmacokinetics: St. John’s wort

The AUC and T1/2 were significantly different at p<0.001. The Cmax and Tmax were not significantly different after SJW treatment.

Alprazolam

PharmacokineticsBaseline After SJW

Cmax (ng/ml) 34 + 9 31 + 7

Tmax (hours) 1.1 + 0.5 1.3 + 0.6

AUC

(ng • hour • ml

-1)

522 + 103 254 + 67

ß1/2 (hour) 12.4 + 3.9 6.0 + 2.4

1

10

100

0 12 24 36 48 60

Time (hours)

Alp

razola

m C

oncentr

ation (

ng/m

l)

Baseline

SJW

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.

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, 14-28 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

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

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, cDNA-expressed CYP enzymes, and specific probe substrates are valuable tools in the assessment of a drug's potential to inhibit different CYP enzymes in vitro.

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, non-

hepatic 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

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

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., 2004.

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

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

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).

↑ ↑

Analysis of Milk Thistle Capsule (Legalon®)

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

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)

J Chromatogr. B 902 (2012) 1– 9

NOTE: In vivo, the Cmax values of free

(unconjugated) silybin A were ~ 2-3-fold

higher than those observed for silybin B, as

well as the much greater AUC0–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; 41:743–751.

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

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.

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.

R21 AT002817-01: “Pharmacokinetics and Drug Interactions with Milk Thistle” NIH National Center for Complementary and Alternative Medicine (NCCAM), Markowitz JS (PI).

Wild Flowers Worth Knowing by Neltje Blanchan (1934)

EXTRA SLIDES

An exploratory ex vivo approach

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.

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.

Comparison of in vitro and potential value of

ex vivo studies

in vitro Studies with Botanical Supplements

Advantages Limitations

Easy/fast to perform Unknown absorption or bioavailability

Controlled environment Single constituent used

Relatively inexpensive Product-to-product variability=reproducibility problems

Ethical considerations Metabolites poorly characterized=role in DDI

*Results not always confirmed by in vivo studies

ex vivo (plasma) vs in vitro (in buffer) Advantages Limitations

Plasma: all constituents and metabolites in the circulation Time consuming

Clinically relevant concentrations Somewhat more expensive

Endogenous plasma compounds

Involves human subjects although fewer, with a single study phase (no pre- and post exposure),

and much less intense blood sampling

Accounts for protein binding Far less drug analysis

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;41:1679-85

Selected Drug Transporters implicated in DDIs of

Interest to the US FDA

FDA Draft Guidance 2012

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

stereoselective metabolism