THERAPEUTIC PRODUCTS PROGRAMME

GUIDANCE DOCUMENT

Drug-Drug Interactions:
Studies In Vitro and In Vivo

September 21, 2000

 FOREWORD

Guidance documents are intended to provide assistance to industry and health care professionals
on how to comply with the Therapeutic Products Programme policies and governing statutes and
regulations. They also serve to provide review and compliance guidance to Therapeutic Products
Programme staff, thereby ensuring that the Programme’s mandate is implemented in a fair, consistent, and
effective manner.

Guidance documents are administrative instruments not having force of law and, as such, allow for
flexibility in approach. Alternative approaches to the principles and practices described in this document
may be acceptable provided that they are supported by adequate scientific justification. Alternative
approaches should be discussed in advance with the Programme to avoid the possible finding that
applicable statutory or regulatory requirements have not been met.

As a corollary to the above, it is equally important to note that the Programme reserves the right
to request information or material or define conditions not specifically described in this guidance, in order
to allow the Programme to adequately assess the safety, efficacy, or quality of a therapeutic product. The
Therapeutic Products Programme is committed to ensuring that such requests are justifiable and that
decisions are clearly documented.

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 TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Basic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Drug Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

STUDY OF METABOLIC DRUG INTERACTIONS IN VITRO . . . . . . . . . . . . . . . . . . . . . . . . . . 4

In Vitro Studies of Drug Metabolism and Enzyme Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . 4
Subcellular Fractions of Human Liver Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Whole Cell Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Heterologous Expressed and Purified Human Drug-Metabolizing Enzymes . . . . . . . . 6
Pharmacological Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Immunochemical Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
In Vitro Studies of Enzyme Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Choice of Drug Concentration Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Position in the Time Course of Drug Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

ROLE OF IN VIVO ANIMAL STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

STUDY OF METABOLIC DRUG INTERACTIONS IN VIVO IN CLINICAL TRIALS . . . . . . . 9

Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Choice of Potentially Interacting Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Route of Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Dose Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Statistical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

CORRELATION BETWEEN STUDIES IN VITRO AND IN VIVO . . . . . . . . . . . . . . . . . . . . . . . 14

FACTORS INFLUENCING DRUG INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

PRODUCT MONOGRAPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

LITERATURE REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

WEBSITE REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

APPENDIX A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

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 DRUG-DRUG INTERACTIONS:
STUDIES IN VITRO AND IN VIVO

INTRODUCTION

The Therapeutic Products Programme subscribes to the position that an adequate pre-marketing
investigation of the safety and efficacy of a new pharmaceutical or therapeutic biological (hereafter to be
referred to as drug) should include characterization of its metabolism and exploration of its interactions with
other drugs. This guidance document provides suggestions to industry scientists and Therapeutic Products
Programme evaluators concerning current approaches to the conduct and regulatory review of in vitro and
in vivo studies addressing drug interactions. The suggestions are not intended to be requirements, but
rather are offered as items of consideration for scientists and medical practitioners involved in the research
or regulatory assessment of drug interactions. The Therapeutic Products Programme recognizes that the
investigational approach used for a particular drug may have to be individualized depending on the
pharmacodynamic, pharmacokinetic, and safety characteristics of the product as well as its proposed
clinical application. As the methodological approaches to the study of drug metabolism and drug
interactions are undergoing rapid evolution, regular consultation of the scientific literature is recommended
to determine how the status of research in this field has changed since the issuance of this guidance
document.

Basic Considerations

Drug interactions are pharmacodynamic, pharmacokinetic, or clinical responses to the

administration of a drug combination that differ from the known effects of the individual drugs administered
alone. The clinical consequences of drug interactions may be antagonistic, additive, synergistic, or
idiosyncratic, resulting in treatment failure, increased pharmacologic effect, or toxic reactions, which may
be serious or fatal.

Pharmacodynamic drug interactions result in alteration of the response to one or both drugs without
affecting their plasma concentrations (e.g. displacement from receptor binding sites). Pharmacokinetic
drug interactions are a consequence of altered levels of exposure to the drug or its metabolites through one
or more of the following mechanisms (1, 2):

• altered absorption: Drug absorption may be altered by chelation or complex formation (e.g.
cholestyramine can form complexes with warfarin, nonsteroidal anti-inflammatory drugs, or
sulfonamides), prokinetic or antimotility effects (e.g. the absorption rate of acetaminophen is
increased by metoclopramide and delayed by propantheline), or pH effects (e.g. increased pH due
 to H2-blockers and antacids can reduce the absorption of ketoconazole) (3).

• altered distribution: Competition of two drugs for the same plasma protein-binding sites can
result in an increased free plasma concentration of the lower affinity drug. Significant interactions
are possible if an extensively protein-bound drug of low volume of distribution is co-administered
with a high affinity displacing drug that is present at a concentration approaching or exceeding the
molar concentration of the protein-binding sites (e.g. displacement of warfarin by trichloroacetic
acid, the major metabolite of chloral hydrate) (1).

• altered transport: Drug interactions can result from displacement of drugs from the ATP-binding
cassette (ABC) transporter proteins such as the MDR-1 gene-encoded P-glycoprotein, an energy-
dependent multidrug efflux pump located on the luminal surface of enterocytes and biliary
hepatocytes and the brush border of proximal renal tubule epithelium (e.g. the P-glycoprotein
inhibitor, quinidine, increases plasma levels of digoxin) (3, 4).

• induction: Induction or increased synthesis of one or more drug-metabolizing enzymes leads to

enhanced metabolism and hepatic clearance of all substrates for those particular pathways (5).
Numerous drugs have been identified as inducers of drug-metabolizing enzymes (1A).

• inhibition: Inhibition or decreased metabolism and hepatic clearance of substrates for a specific
drug-metabolizing enzyme can result from competition between drugs for the enzyme’s binding
sites, covalent modification of the enzyme by a reactive metabolite, or formation of a catalytically
inactive reversible complex between the enzyme and a drug or its metabolite (5). Inhibitory effects
on one or more drug-metabolizing enzymes have been demonstrated for a range of drugs (1A).

• altered excretion: Potential mechanisms for the altered renal excretion of drugs include
competition for renal anion or cation transport systems (e.g. probenecid inhibits the tubular
secretion of the penicillins), changes in urinary pH (e.g. sodium bicarbonate increases renal
elimination of phenobarbital), and inhibition of renal metabolism (6).

The objective of this guidance document is to address the issue of metabolic drug interactions.
Elimination of a drug may occur through metabolism to one or more active or inactive metabolites,
excretion of the parent drug by renal or biliary routes, or by a combination of excretory and metabolic
elimination pathways. If elimination occurs primarily through metabolism, identification of the principal
metabolic route may have important safety implications. Some drugs and dietary xenobiotics have the
ability to influence the metabolism of concomitantly administered drugs by acting as inhibitors or inducers
of drug-metabolizing enzymes. Thus, drug interactions may have the potential to result in substantial
increases or decreases in the blood and tissue concentrations of the drug under investigation or certain of
its metabolites.

The clinical significance of the interactions resulting from inhibition or induction will be dependent

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on the relative pharmacodynamic activity and toxicity of the parent drug and its metabolites. In the case of
a prodrug that is converted to an active metabolite (e.g. codeine), pharmacodynamic activity will be
decreased by inhibitors and increased by inducers. For drugs that are inactivated by the metabolic process,
inhibitors will tend to enhance pharmacodynamic activity, while inducers may result in therapeutic failure.
In situations where a drug interaction results in variations in the relative levels of a parent drug and
metabolite(s) that are approximately equipotent in terms of efficacy and safety considerations, inhibition and
induction may be of little therapeutic consequence.

Drugs that are substrates for metabolism by more than one drug-metabolizing enzyme generally
have a decreased likelihood of clinically significant drug interactions due to the availability of compensatory
metabolic pathways if one is inhibited. Drug interactions are likely to be particularly important in the
following situations (5, 7):

• when elimination of a drug occurs primarily through a single metabolic pathway

• when a drug is a potent inhibitor or inducer of a drug-metabolizing enzyme

• when one or both of the interacting drugs has a steep dose-response curve

• when one or both of the interacting drugs has a narrow therapeutic range

• when inhibition of the primary metabolic enzyme or induction of a secondary metabolic enzyme
results in diversion of the drug into an alternative pathway that generates a metabolite having toxic
or modified pharmacodynamic activity

• when a drug has non-linear pharmacokinetics or when the interaction results in conversion from
linear to non-linear pharmacokinetics

Drug Metabolism (5, 7, 8)

The biotransformation of drugs is generally a two phase process. Phase I reactions involve
oxidation, reduction, or hydrolysis of the parent compound, typically producing metabolites of increased
polarity, which may be subjected to excretion or further biotransformation. For metabolites undergoing
phase II of the biotransformation process, the polar groups present on the intermediate undergo conjugation
with glutathione, acetate, glucuronate, sulfate, or glycine to produce excretable compounds. Some drugs
undergo phase II conjugation reactions without prior phase I biotransformation.

The majority of phase I drug metabolism occurs through the cytochrome P450 system (CYP), a
superfamily of heme-containing isoenzymes located primarily in hepatocytes, within the membranes of the
smooth endoplasmic reticulum. The enterocytes of the small intestine are the principal extrahepatic source
of CYP isoforms, with smaller CYP quantities being present in the kidneys, lungs, and brain. The human

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CYP system has been subclassified into over 70 unique families based on amino acid sequence homology.

• The gene family name is denoted by an Arabic numeral (e.g. CYP3). CYPs within a given family
have greater than 40% sequence homology.

• CYP families are further differentiated into gene subfamilies denoted by an upper case letter (e.g.
CYP3A). CYPs within a particular subfamily have sequence homology in excess of 55%.

• Gene numbers of individual enzymes are denoted by a second Arabic numeral following the
subfamily letter (e.g. CYP3A4).

The isoforms of the CYP system are responsible for the oxidative metabolism of endogenous
substances such as steroid hormones, prostaglandins, lipids, and fatty acids and for the detoxification of
exogenous compounds such as drugs and dietary xenobiotics. The major CYP isoforms involved in human
drug metabolism are CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP1A2, and CYP2E1. CYP2A6 and
CYP2B6 likewise play a significant role in the metabolism of certain drugs.

Enzymes involved in phase II conjugation reactions include glutathione S-transferases, UDP-

glucuronosyl transferases, sulfotransferases, N-acetyltransferases, acyltransferases, and methyltransferases
(Appendix A).

STUDY OF METABOLIC DRUG INTERACTIONS IN VITRO

In order to investigate drug interactions in vitro, it is desirable first to identify all of the major
metabolic pathways involved in the biotransformation of the test drug, the specific enzymes responsible for
these biotransformation reactions, and the metabolites generated by the biotransformation process. While
many CYP inhibitors are also substrates for the affected enzyme (e.g. erythromycin is both a substrate and
inhibitor of CYP3A4), it should be recognized that a drug may be resistant to metabolism by a given CYP
isoform, yet still be capable of inhibiting the ability of the same isoenzyme to metabolize other drugs (e.g.
quinidine is a potent inhibitor of CYP2D6, but is metabolized by CYP3A4). Availability of this information
will enable the rational anticipation and exploration of drug interactions whereby the metabolism of the test
drug is affected by other agents or vice versa (2A, 5, 8).

In Vitro Studies of Drug Metabolism and Enzyme Inhibition

The following experimental model systems and probes have proved useful for the study of drug
interactions:

Subcellular Fractions of Human Liver Tissue

• Human liver microsomes (vesicles of endoplasmic reticulum) are a common system in which to

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 screen for the effects of a new drug on CYP pathways and to generate preliminary information on
potential drug-drug interactions (9, 10). Hepatic microsomes are a subcellular tissue fraction
obtained by differential high-speed centrifugation of homogenized liver. Important drug-
metabolizing enzymes present within the microsomal fraction include the cytochrome P450
superfamily, the flavin-containing mono-oxygenases, epoxide hydrolases, and a variety of
transferases (e.g. the UDP-glucuronosyltransferases). Microsomes from multiple donors should be
used, either as individual or pooled preparations, in order to avoid conclusions based on
microsomes that may exhibit one or more aberrant metabolic pathways. However, in certain
situations, determination of metabolism using aberrant microsomes may be a specific objective of
the study.

Microsomal preparations require the addition of exogenous cofactors, including a source of

NADPH. Transferase activity can be studied by supplementing microsomal preparations with
conjugating moieties. Predictions of drug interactions from cell-free systems such as microsomes
may be irrelevant if marked in vivo differences between plasma concentrations and intracellular
hepatocyte concentrations are not taken into account. Microsomes are generally not an
appropriate system in which to study sequential metabolic reactions (i.e. the coupling of phase I
and II reactions) owing to the disruption of the natural orientation between subcellular components.
However, advantages of the system include ease of preparation, commercial availability, and long-
term stability during cryopreservation.

• The S9 subcellular fraction (supernatant of liver homogenate resulting from precipitation of the
nuclei and mitochondria by centrifugation at 9,000-20,000g) is a useful preparation in which to
study drug metabolism involving both microsomal and cytosolic enzymes (10).

Whole Cell Models

• Biopsy samples and harvested livers not used for transplantation represent sources of human liver
tissue for experimental purposes. The characteristics of these preparations will vary depending
upon the age, health, and genotypic status of the donor, as well as factors such as diet, alcohol or
tobacco consumption, and medication usage. Isolated hepatocytes in suspension or primary culture
(11, 12) and precision-cut liver slices (13) offer numerous advantages over subcellular fractions,
including a full complement of hepatic drug- metabolizing enzymes, endogenous cofactors,
preservation of the natural orientation for linked enzymes, and a more accurate reproduction of
potential differences between extracellular and intracellular drug concentrations across the plasma
membrane barrier. They also afford the opportunity to study the role of alternative metabolic
routes for a substrate in the presence of drugs that inhibit the principal drug-metabolizing pathway.

Liver slices have the additional advantages of preserving the tissue cytoarchitecture and cell- to-
cell communications. However, clearance predictions from liver slices may be lower than those
from hepatocytes or microsomes if, due to slice thickness, equilibration is not achieved between

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 the cells of the slice and the incubation medium (5).

Short-term stability of enzymatic activities represents the major problem with hepatocytes and liver
slices (typically <3-4 h for suspensions, <24 h for cultures or slices). The viability of these
preparations varies depending on culture conditions and the relative stability of the isoforms under
investigation.

• Caco-2 cell monolayers serve as a surrogate model for absorption and metabolism at the level of
the human intestine (14). Caco-2 cells are derived from human colon cancer cells. When cultured
on porous membranes, they differentiate spontaneously into monolayers of polarized cells. Caco-2
cell monolayers are a useful system in which to investigate drug interactions resulting from inhibition
of the P-glycoprotein efflux mechanism. Disadvantages of Caco-2 cell monolayers include the
underexpression of metabolizing enzymes and a time-dependent loss of enzyme activity in culture.

Heterologous Expressed and Purified Human Drug-Metabolizing Enzymes

• The cDNAs for common CYPs have been cloned and the recombinant human enzymatic proteins
expressed in a variety of cells with low intrinsic cytochrome P450 enzyme activity including
bacteria, yeast, insect cells, mammalian cells, and human lymphoblastoid cells or HepG2 human
hepatoma cells (15, 16). Lysates of these transgenic cells are subjected to subcellular fractionation.
The activity of the heterologously expressed enzyme can be studied either in microsomes prepared
from the transgenic cell lysates or in reconstitution systems containing electrophoretically purified
enzyme. Because of the low level of endogenous CYPs in these cell types, adequate protein yields
with high specificity for the heterologously expressed enzyme are possible. In contrast, purification
of CYPs from human liver microsomes is a complicated process in which it is often difficult to
achieve acceptable resolution of CYPs of the same subfamily. cDNA-expressed enzymes
generally exhibit affinity properties (Km, Ki) that are representative of native enzymes, though rates
of metabolism (Vmax) can be more variable.

Recombinant models have the advantage of offering a single enzyme system in which to investigate
isoform-specific metabolism of a drug or the ability of the drug to inhibit substrate metabolism
through that isoform. However, the absence of competing pathways prevents these systems from
supplying information on the relative contribution of the isoform in question to the overall
metabolism of the drug in vivo. Nevertheless, cDNA-expressed and purified enzymes can be a
useful system in which to confirm results obtained with native human liver tissue or microsomes.
Purified preparations of the following heterologously expressed human drug-metabolizing enzymes
are now commercially available: CYPs, flavin-containing mono-oxygenase, epoxide hydrolase, and
glutathione S-transferase.

The study of metabolically competent recombinant enzymes in transfected cells is an emerging

technology.

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Pharmacological Probes

• The metabolic pathways for the test drug in subcellular fractions or whole cell models can be
demonstrated through the use of selective chemical inhibitors of specific CYP enzymes (5, 8, 17,
18). Inhibitory activity can be expressed either as an inhibition constant (Ki) or the 50% inhibitory
concentration (IC 50). The IC50 is an estimate of the concentration of the drug inhibiting the
maximum rate of metabolism of a fixed concentration of substrate by 50% and, as such, is a
measure of the inhibitory potency. IC50 determinations have the advantage of being independent
of the biochemical mechanism of enzyme inhibition. However, the IC50 is applicable only to the
substrate concentration studied. Therefore, extrapolation to in vivo situations may be unreliable
if plasma concentrations of the substrate differ markedly from the substrate concentration studied
in vitro. For competitive inhibitors, the IC 50 will approximate the Ki only if the substrate
concentration studied is considerably below the K m (the substrate concentration at which the rate
of metabolism is half-maximal).

Knowledge of the in vitro inhibition constant of a drug (K i) for a particular CYP isoform is,
therefore, considered more useful in assessing the probability of a drug interaction. The Ki is a
measure of the affinity of the inhibitor for the enzyme. Ki determinations require the study of
inhibition at a range of concentrations for both the inhibitor and substrate. While Ki values are
theoretically dependent on the binding characteristics of the inhibitor, different Ki values may be
obtained using different substrates. It is, therefore, desirable to perform independent K i estimations
using multiple substrates. Positive controls should be used to establish assay sensitivity. The effect
of concentration on the selectivity of inhibition should be considered.

A rationale should be provided for the choice of incubation time over which the test system is
exposed to the investigational drug in combination with the pharmacological probe. The linearity
of metabolic rate versus incubation time should have been confirmed in preliminary studies (18).

Immunochemical Probes

• Alternatively, selective inhibition of certain CYP enzymes in microsome preparations can be

achieved through use of polyclonal or monoclonal antibodies to specific isoforms (19). Inhibition
of metabolite formation by an antibody specific to a particular cytochrome implicates that isoform
in the metabolism of the test drug. Current problems with this approach include a lack of wide
commercial availability of these antibodies, deficient antibody selectivity between subfamily
members, failure to achieve maximal inhibition if the molar concentration of the antibody is not
equivalent to the molar concentration of the enzyme, inability to achieve 100% inhibition (probably
because the large size of the antibody molecule does not permit access to all isoform molecules),
and a high degree of inter-laboratory variation in the degree of inhibition achieved.

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In Vitro Studies of Enzyme Induction

Primary human hepatocyte cultures and hepatocyte cell lines (e.g. HepG2 human hepatoma cell
line) have sometimes proved useful for the study of induction phenomena (11). Artifacts are common,
however, as cultured hepatocytes typically undergo a time-dependent loss of CYP expression and may also
exhibit a diminished capacity for induction. The predictive value of hepatocyte cell lines is often limited by
substantial phenotypic differences between these cells and the tissues from which they were derived.
Enzyme induction can be measured by assaying for the activity of specific isoforms, immunodetection of
isoform protein, or quantification of mRNA. The use of known inducers as positive control agents is
necessary to verify the sensitivity of these systems.

Choice of Drug Concentration Range

In vitro drug interaction studies should employ clinically relevant concentrations of both the
substrate and the inhibitor or inducer. Studies conducted at supratherapeutic drug concentrations may
produce in vitro drug interactions that are not representative of the in vivo situation. Moreover, the major
metabolic pathway for a given drug may be concentration-dependent. For instance, N-demethylation is
the principal in vivo metabolic pathway for diazepam at therapeutic doses, while at high in vitro drug
concentrations (100 µM) 3-hydroxylation predominates.

Controversy exists as to whether the free (unbound) or total (unbound + bound) in vivo plasma
concentration of the drug represents the most appropriate concentration for prediction of drug interactions
from in vitro studies. Some compounds have high liver to plasma partition ratios despite extensive protein
binding. Use of free fraction plasma concentrations for the prediction of metabolic drug interactions for
such agents has resulted in an underestimation of the clinical outcome. In some instances, hepatic drug
levels may even exceed the total plasma concentration by several fold (5, 17, 20).

If the drug is to be developed as a resolved enantiomer, in vitro preclinical metabolism studies

should be conducted with that enantiomer, rather than the racemate, as the metabolic pathway may be
stereoselective (e.g. S-warfarin is metabolized by CYP2C9, while R-warfarin is metabolized by CYP3A4
and CYP1A2).

Position in the Time Course of Drug Development

The availability of in vitro metabolic studies prior to phase II clinical investigations should be
encouraged. Appropriatein vitro studies of metabolism and drug interactions should have been completed
prior to the initiation of phase III clinical trials.

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ROLE OF IN VIVO ANIMAL STUDIES

Whenever feasible, drug metabolism should be investigated in human tissues, cells, or subcellular
fractions as interspecies and intraspecies differences in drug-metabolizing enzymes limit the ability to
extrapolate between experimental animals and humans. While animal cell lines and liver microsomal
fractions may be used to supply preliminary data, such information should always be confirmed in human
liver preparations.

However, animal species may provide useful models in which to determine whether a new chemical
species generated in vitro by human liver microsomes produces pharmacological or toxicological effects
in vivo and how these compare with the effects of the parent compound. An appreciation of the
pharmacodynamics and toxicology of the human metabolites of a drug is helpful in anticipating the clinical
implications of drug interactions.

A knowledge of comparative drug metabolism in humans and animals can play a useful role in the
rational selection of animal models for toxicology studies. Major discrepancies in the metabolism of a drug
between laboratory test species and humans diminish the predictive value of toxicology data obtained from
these animals.

The development of transgenic animal models for heterologous expression of human drug-
metabolizing enzymes or for gene inactivation by homologous recombination represents an in vivo
approach to the investigation of the role of specific enzymes involved in drug metabolism (21, 22).
However, owing to interspecies differences, the endpoints of such studies may not reflect the human
situation (21).

STUDY OF METABOLIC DRUG INTERACTIONS IN VIVO IN CLINICAL TRIALS (3A,

6, 7, 23)

The clinical investigation of potential drug-drug interactions should be a systematic process of well
designed trials performed upon appropriately selected cohorts of subjects or patients, receiving both
interacting drugs under conditions of dosage and administration that are intended to reproduce the
proposed or approved therapeutic dosing recommendations. Such studies should address relevant
pharmacokinetic and pharmacodynamic endpoints and employ suitable statistical analyses.

Design

• 0pen label (unblinded) design is generally acceptable for studies of pharmacokinetic interactions,
unless the interaction assessment includes pharmacodynamic measurements.

• Possible designs include randomized crossover (randomization to A followed by A+B or A+B

followed by A); one sequence crossover (A followed by A+B), or parallel group dosing

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 (randomization to A or A+B). Through use of a “within individual” design, crossover studies offer
greater statistical power for a given number of subjects than do parallel group studies. Parallel
group studies require more subjects for a shorter duration of time, whereas crossover studies
require fewer subjects for a longer time period. Crossover studies have the advantage of allowing
individual subjects to serve as their own controls. Randomized crossover studies represent an
improvement over one sequence crossover studies in that they are sensitive to sequence and carry-
over effects as well as to period effects. Parallel group studies may be preferred for drugs with
long elimination half-lives for which lengthy time intervals are required to achieve steady-state or
to complete washout.

• The choice of single or multiple dose regimens for each of the drugs in an interaction study should
be based upon (1) whether these agents are intended for acute or chronic use in their target patient
populations, (2) the presence or absence of safety considerations precluding multiple dose use, and
(3) pharmacodynamic/pharmacokinetic characteristics of the drugs (e.g. elimination half-life, lag
time to pharmacodynamic response of interest, enzyme-inducing properties, etc.). On the whole,
multiple dose regimens result in smaller confidence intervals at steady state. However, for drugs
with low accumulation ratios, confidence intervals may be similar for single dose and multiple dose
study situations.

• The timing of drug co-administration may be an important variable in drug interaction studies (e.g.
concurrent administration versus administration separated by 1, 2, 3, or more hours). The
sequence of administration of the interacting drugs may likewise affect the magnitude of the
interaction.

• In the case of a drug that competitively inhibits the metabolism of a substrate, inhibition is usually
maximal at the time when the inhibitor reaches its steady state level (4 or 5 half-lives) and when
the inhibited drug reaches steady state at its new, longer half-life. For enzyme inducers, the time
to maximal induction may extend beyond the attainment of steady state plasma levels, therefore,
proving more difficult to predict. If the drugs are to be studied at steady-state, attainment of
steady-state plasma concentrations should be documented for the parent drugs and their
metabolites of interest by the analysis of samples collected over several days prior to the period
at which the interactive effects are to be studied. It should be recognized that time-dependent
changes in drug pharmacokinetics can occur over the course of chronic therapy, such that plasma
exposure and metabolic profile may not stabilize until several months after the initiation of treatment.

• Investigation of drug levels during the washout phase following discontinuation of the inhibitor or
inducer may be necessary in order to determine the time period post-discontinuation during which
the patient remains susceptible to drug interactions. The rate-limiting factor in the offset of induction
may be the half-life of CYP protein turnover, rather than the half-life of the inducer.

• Useful information on drug interactions can sometimes be obtained from plasma concentration

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 measurements performed upon blood samples collected according to sparse or dense sampling
schedules during phase II and III clinical trials (24). These population pharmacokinetic studies
have the advantage of providing data from the intended patient population. This approach allows
identification of correlations between pharmacokinetic inter- and intra-individual variability and
factors such as demographics, co-morbidity, or concomitant medication. Quantitative estimates
of the magnitude of this variability can be generated. Such data may be of value in confirming an
interaction suspected on the basis of in vitro findings or in the identification of unsuspected
interactions. However, as the power of population pharmacokinetic screens to detect drug
interactions is not well established, such data should not be used to dismiss the possibility of a
suspected drug interaction, especially if positive in vitro or in vivo data are available from
systematic drug interaction studies. False negative findings may be an issue if the study contains
an insufficient number of patients taking the drug combination of interest. Furthermore,
interpretation of population pharmacokinetic data may be difficult if the sequence and timing of co-
administration were not standardized.

Population

The following subject or patient groups may represent appropriate populations for drug interaction
studies:

• healthy subjects who meet restrictive eligibility criteria that increase the homogeneity of the study
cohort

• subjects drawn from the general population, meeting expanded eligibility criteria that permit
heterogeneity of the study cohort

• patients for whom the investigational drug is intended

• subjects who have been selected on the basis of phenotyping and/or genotyping for metabolic
polymorphisms, predisposition to adverse drug reactions, or therapeutic outcome

Healthy volunteers as a subject group for drug interaction studies have the advantage of minimizing
the confounding effects introduced by disease states or concomitant medications that may affect drug
absorption, metabolism, distribution, or excretion. However, use of subjects drawn from the general
population or patients for whom the investigational drug is intended may result in data that correlate better
with the clinical situation. Phenotyping and/or genotyping during screening may be used for inclusion,
exclusion, or stratification of subjects. Such approaches are useful in characterizing the effect of metabolic
status (e.g. poor, extensive, ultra-rapid metabolizer) on the magnitude of the drug interaction, but introduce
a bias which limits the generalizability of the results. Alternatively, the availability of phenotype and/or
genotype information following study completion may be useful in the interpretation of pharmacokinetic
outliers as well as inter-individual variability in pharmacodynamics and adverse events.

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Choice of Potentially Interacting Drugs

The selection of potentially interacting drugs for drug interaction investigations should be based on
a knowledge of the enzymes subject to inhibition or induction by the test drug or responsible for its
metabolism, as identified in in vitro studies.

• Selected drugs should be potent and specific inhibitors or inducers of the principal drug-
metabolizing enzymes responsible for the biotransformation of the investigational drug.

• Selected drugs should be sensitive substrates of the principal drug-metabolizing enzymes subject
to inhibition or induction by the investigational drug or involved in its biotransformation.

• Selection may favour approved drugs for which co-administration is likely given the indication and
target population characteristics of the investigational drug.

• Selection may be based on known interactions affecting chemically related drugs of the same
therapeutic class.

• Selection may favour drugs having low therapeutic indices for which interactions would be
expected to have clinically significant consequences (e.g. digoxin, warfarin).

• Selection of substances ingested socially (e.g. alcohol) or through the diet (e.g. grapefruit juice) may
be advisable where these are anticipated to affect the metabolism of the test drug.

Route of Administration

• The routes of administration used in the clinical investigation of drug interactions should correspond
to those recommended in the proposed or approved Product Monographs for the drugs in
question.

• If multiple routes of administration are to be recommended in the Product Monograph,

positive/negative drug interaction findings for one route should not necessarily be extrapolated to
the others (e.g. an intravenous drug interaction study would not reveal an effect on intestinal
CYP3A4 activity that might influence bioavailability).

Dose Selection

• The highest proposed or approved doses of the test drugs and the shortest dosing intervals should
be used in order to maximize the possibility of finding an interaction.

• On occasion lower doses may be acceptable for safety reasons.

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• Exploration of differential effects over a range of doses may be useful to characterize the dose-
dependency of a drug interaction.

Endpoints

1. Pharmacokinetic

• Parameters that serve as useful endpoints in the analysis of pharmacokinetic drug interaction data
include the area under the blood/plasma/serum concentration time curve (AUC), the peak
concentration (C max), the time to peak concentration ( tmax), the trough concentration (C min), the
clearance (CL), the volume of distribution (Vd), and the elimination half-life (t1/2).

• The fraction of drug existing unbound in the plasma (fu) may be a useful parameter in the
assessment of drug interactions for certain highly protein-bound agents that are distributed primarily
in the plasma rather than in tissues (i.e. low Vd).

• Pharmacokinetic parameters should be determined for the parent drug and important active or toxic
metabolites.

• The availability of pharmacokinetic data for both interacting drugs may be desirable, especially in
situations where interactive effects are complex (e.g. concomitant use of an inhibitor and an inducer,
co-administration of two drugs having reciprocal effects on each other’s primary or secondary
metabolic pathways through inhibitory activity on different drug-metabolizing enzymes, or use of
an inhibitor having active or toxic metabolites that are substrates for enzymes affected by elevated
levels of the interacting drug). In some instances, two independent studies may be required to
adequately characterize the effects on an interaction on each of the drugs involved, while in other
cases such information may be obtained from a single study (e.g. three period crossover approach).

2. Pharmacodynamic

• Pharmacodynamic parameters may serve as useful endpoints when interactions are not wholly
accounted for by pharmacokinetic effects (e.g. additive haemodynamic or anticoagulant effects).

Adverse Events

• In clinical pharmacology studies addressing drug interactions, attention should be directed to

adverse events occurring with greater frequency or severity during combination treatment than
during treatment with either agent alone.

• Clinical trial protocols should contain directions for the collection of blood samples from patients

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 experiencing serious, severe, or unexpected adverse events. Plasma level determinations for
concomitant medications should likewise be encouraged in such cases.

Statistical Considerations

• The 90% confidence intervals should be provided for the mean ratios of the pharmacokinetic
exposure measures of the drug in the presence and absence of the interacting agent.

• Significance tests are of limited value as small, consistent differences in exposure may result in p-
values that are statistically significant, but of doubtful clinical relevance.

• Knowledge of the width of the equivalence interval beyond which a dosage adjustment is necessary
is useful in determining sample size.

• In the absence of other information to determine an equivalence interval, a standard interval of 80-
125% can be employed for investigational and approved drugs in drug interaction studies.

• Sample size should be sufficient such that the study has statistical power to detect a clinically
important difference between the treatment groups. Sample size requirements will be increased
if inter- or intrasubject variability in pharmacokinetic measurements is high.

CORRELATION BETWEEN STUDIES IN VITRO AND IN VIVO

In assessing the correlation between in vitro and in vivo drug interaction studies, attention should
be directed to the following considerations:

• In vitro drug interaction studies should be conducted at concentrations similar to those attained in
vivo.

• The clinical importance of positive in vitro results should always be confirmed in vivo. Depending
on the role of the investigational drug in the suspected interaction, the in vivo studies should be
performed using one or more sensitive substrates or potent and specific inhibitors/inducers of the
drug-metabolizing enzyme implicated. Results from such studies may be used to extrapolate
qualitatively to other inhibitors, inducers, or substrates of the enzyme in question.

• In vitro drug interaction studies may not accurately predict in vivo interactions if alternative
metabolic or excretory routes play a major role in the clearance of the drug in vivo.

• In the event of conflicting results between in vitro and in vivo studies, the in vivo studies should
receive precedence, provided that these have been conducted under clinically relevant conditions.

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 Confounding factors that may result in failure to make accurate correlations between in vitro and
in vivo studies include, but are not limited to, the following (20):

• Free (unbound) in vivo drug concentrations in the plasma may be lower than the drug
concentrations in the hepatic biophase as the hepatic uptake of many lipophilic compounds is not
necessarily limited by protein binding.

• When a drug interaction involves elements of both induction and inhibition, the predominant effect
on drug clearance can be either positive or negative and may be time-dependent (e.g. metabolic
inhibition in single dose studies and induction at steady-state conditions).

• In vitro drug interaction results may underestimate the in vivo interactions for agents that inhibit
of the intestinal P-glycoprotein transporter, thus increasing the bioavailability of concomitantly
administered drugs which are substrates for extrusion by this mechanism.

• High microsomal protein concentrations may result in overestimation of Ki values if the inhibitor
becomes depleted due to microsomal metabolism or nonspecific binding to microsomal proteins
(5).

FACTORS INFLUENCING DRUG INTERACTIONS

Inter-individual susceptibility to drug interactions may be influenced by a wide range of factors

including, but not limited to, the following:

• genetic: Many drug-metabolizing enzymes, for example CYP2D6, CYP2C19, CYP2A6,

CYP2C9, and N-acetyltransferase, are subject to genetic polymorphism such that large inter-
individual differences exist in the ability to metabolize substrates for these enzymes (25, 26). Some
genetic polymorphisms show an increased incidence in certain ethnic groups. For example, poor
metabolizers of CYP2D6 substrates represent 5 to 10% of the Caucasian population, but only 1
to 2% of the Asian population. Conversely, the incidence of poor metabolizers of CYP2C19
substrates is 18-22% in Asian populations, but only 2-6% in Caucasian populations (5, 25, 26).

• age: The magnitude and clinical consequences of a pharmacokinetic drug interaction may be age-
dependent. Neonates exhibit reduced hepatic metabolism and renal excretion of drugs due to
immaturity of liver and kidney function. The susceptibility of elderly individuals to drug interactions
may be affected by age-related alterations in absorption, hepatic metabolism, renal clearance, or
volume of distribution (27).

• gender: Studies of certain drugs have shown inconsistencies in pharmacokinetic parameters or

pharmacodynamic endpoints between male and female subjects that are suggestive of gender-
dependent differences in bioavailability, volume of distribution, drug-metabolizing enzyme activity,

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 renal clearance, or physiological characteristics. Normalization of pharmacokinetic parameters for
body weight may be advisable in assessing apparent differences between male and female patients
in drug interaction studies (28).

• disease states: The effects of hepatic impairment on the magnitude and clinical consequences of
drug interactions may be complex and difficult to predict. Patients with renal impairment may be
at an increased risk for metabolic drug interactions, due to a diminished contribution of the
excretory component of the drug elimination process.

• social: Tobacco smoke is an inducer of CYP1A1, CYP1A2, and possibly CYP2E1 (29).
Chronic alcohol ingestion results in the induction of CYP2E1 (7).

• dietary: Grapefruit juice contains chemicals that are potent inhibitors of CYP3A4 in the intestinal
wall mucosa (30). Cruciferous vegetables (e.g. brussels sprouts, cabbage, cauliflower) and
hydrocarbons present in charcoal-broiled meat can induce CYP1A2 (7). The calcium present in
dairy products has the potential to chelate drugs such as tetracyclines and fluoroquinolones (3).
Irreversible, non-selective monoamine oxidase inhibitors reduce the metabolism of endogenous
norepinephrine and exogenous tyramine ingested in foods and beverages that have undergone
protein breakdown as a result of aging, fermentation, pickling, smoking, or bacterial contamination
(e.g. beer, wine, certain cheeses and sausages). The increased bioavailability of tyramine in
combination with the augmented endogenous norepinephrine stores can lead to an exaggeration
of the indirect sympathomimetic effect of tyramine, resulting in hypertensive crises (31).

PRODUCT MONOGRAPH

The choice of whether to deal with a given drug interaction as a contraindication, warning, or
precaution should be based on an assessment of the seriousness and severity of the clinical consequences
of the established or suspected drug interaction. All documented and anticipated drug interactions should
be included in the “Drug Interactions” sub-section of the “Precautions” section with appropriate cross-
references to other sections in which they may also appear. Drug interactions should be presented as
contraindications if they have the capacity to be life-threatening, cause permanent damage, or elicit other
reactions that would prohibit concomitant administration. Interactions having the potential to cause serious
or severe consequences that are reversible or not life-threatening should normally be included in the
“Warnings” section together with recommendations for appropriate risk management measures. Drug
interactions of unknown clinical significance or resulting in adverse effects that are merely bothersome can
generally be adequately dealt with in the “Drug Interactions” sub-section of the “Precautions” section.

When describing the results of in vivo clinical drug interaction studies, the monograph should
indicate the number of individuals studied, whether the subjects were healthy volunteers or patients, the
dose and duration of treatment with the drugs in question, and the magnitude of the observed effect on
important pharmacokinetic parameters (e.g. % increase or multiple of control value). Drug interactions

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identified through population pharmacokinetic approaches, clinical trial case reports, or spontaneous post-
marketing adverse event reports should be identified as such. When sufficient data are available, comments
should be provided on the mechanism of the interaction, the clinical manifestations, and appropriate actions
to prevent or respond to an interaction (e.g. adjustments of dosage and/or dosing interval, timing and
sequence of co-administration, recommended washout periods between administration of interacting drugs,
plasma level monitoring, pharmacodynamic monitoring).

For drugs known to be potent and selective inhibitors or inducers or sensitive substrates of specific
drug-metabolizing enzymes, standard labelling concerning drug interactions may be appropriate, even if
specific data are not available concerning the interactions in question. For example, potent inhibitors of
CYP3A4 and CYP2D6 are likely to decrease the metabolism of all substrates for these isoforms.
However, drug interactions that are anticipated from in vitro studies should be identified as such and
distinguished from drug interactions that have been demonstrated under in vivo conditions.

Warnings or precautionary statements based upon a documented interaction with a particular drug
need not necessarily be extrapolated to other members of that drug’s therapeutic class if a reasonable basis
exists for believing that these drugs do not share a common metabolic pathway. For example, the
benzodiazepines, triazolam and alprazolam, are substrates for CYP3A4 and undergo clinically significant
interactions with inhibitors of this isoform, whereas lorazepam, a drug metabolized primarily by
glucuronidation, is not subject to interactions with CYP3A4 inhibitors. Manufacturers wishing to gain
exemption from therapeutic class labelling for drug interactions should convincingly demonstrate that the
possibility of such interactions with their products has been adequately investigated and dismissed.

The “Pharmacokinetics” sub-section of the “Actions and Clinical Pharmacology” section of the
Product Monograph should contain information on the major metabolic pathways involved in the
biotransformation of the drug, the specific enzymes responsible for these reactions, and the principal
metabolites generated. A comprehensive list of the metabolic enzymes tested should be provided.

When approval is granted to Product Monographs that contain information on clinically important
new drug interactions, the manufacturers of the interacting drugs should be notified in order to ensure that
consistent information appears in the Product Monographs for all agents involved in the interactions.

Prepared: C. F. Strnad, Ph.D.

Reviewed: W. Casley, Ph.D.

B. Foster, Ph.D.
I. Hynie, M.D., Ph.D.
S. Robertson, M.D.

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 LITERATURE REFERENCES

1. Kedderis, G.L. Pharmacokinetics of drug interactions. Adv. Pharmacol. 1997; 43:189-203.

2. Hooper, W.D. Metabolic drug interactions. In “Handbook of Drug Metabolism” (T.F. Woolf
ed., 1999), pp. 229-238. Marcel Dekker, Inc., New York, NY.

3. Fleisher, D. et al. Drug, meal and formulation interactions influencing drug absorption after oral
administration. Clin. Pharmacokinet. 1999; 36(3):233-254.

4. Yu, D. K. The contribution of P-glycoprotein to pharmacokinetic drug-drug interactions. J. Clin.

Pharmacol. 1999; 39:1203-1211.

5. Lin, J.H., Lu, A.Y.H. Inhibition and induction of cytochrome P450 and the clinical implications.
Clin. Pharmacokinet. 1998; 5(5):361-390.

6. Bonate, P.L. et al. Drug interactions at the renal level. Clin. Pharmacokinet. 1998; 34(5):375-
404.

7. Johnson, M. D. et al. Clinically significant drug interactions. Postgraduate Medicine 1999;

105(2):193-222.

8. Bertz, R.J., Granneman, G.R. Use of in vitro and in vivo data to estimate the likelihood of
metabolic pharmacokinetic interactions. Clin. Pharmacokinet. 1997; 32(3):210-258.

9. Rodrigues, A.D., Wong, S.L. Application of human liver microsomes in metabolism-based drug-
drug interactions. Adv. Pharmacol. 1997; 43:65-101.

10. Ekins, S. et al. In vitro metabolism: Subcellular fractions. In “Handbook of Drug Metabolism”
(T.F. Woolf ed., 1999), pp. 363-399. Marcel Dekker, Inc. New York, NY.

11. Li, A.P. Primary hepatocyte cultures as an in vitro experimental system for the evaluation of
pharmacokinetic drug-drug interactions. Adv. Pharmacol. 1997; 43:103-130.

12. Sinz, M. W. In vitro metabolism: Hepatocytes. In “Handbook of Drug Metabolism” (T.F.

Woolf ed., 1999), pp. 401-424. Marcel Dekker, Inc. New York, NY.

13. Ferrero, J.L., Brendel, K. Liver slices as a model in drug metabolism. Adv. Pharmacol. 1997;
43:131-169.

14. Yee, S., Day, W.W. Application of Caco-2 cells in drug discovery and development. In

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 “Handbook of Drug Metabolism” (T.F. Woolf ed., 1999), pp. 507-522. Marcel Dekker, Inc.
New York, NY.

15. Crespi, C.L., Penman, B.W. Use of cDNA-expressed human cytochrome P450 enzymes to study
potential drug-drug interactions. Adv. Pharmacol. 1997; 43:171-188.

16. Rodrigues, A.D. Applications of heterologous expressed and purified human drug-metabolizing
enzymes: An industrial perspective. In “Handbook of Drug Metabolism” (T.F. Woolf ed.,
1999), pp. 279-320. Marcel Dekker Inc., New York, NY.

17. von Moltke, L. L. et al. In vitro approaches to predicting drug interactions in vivo. Biochem.
Pharmacol. 1998; 55(2):113-122.

18. Yuan, R. et al. In vitro metabolic interaction studies: Experience of the Food and Drug
Administration. Clin. Pharmacol. Ther. 1999; 66(1):9-15.

19. Gonzalez, F.J. Overview of experimental approaches for study of drug metabolism and drug-drug
interactions. Adv. Pharmacol. 1997; 43:255-277.

20. Davit, B. et al. FDA evaluations using in vitro metabolism to predict and interpret in vivo
metabolic drug-drug interactions: impact on labeling. J. Clin. Pharmacol. 1999; 39:899-910.

21. Friedberg, T. et al. In vivo and in vitro recombinant DNA technology as a powerful tool in drug
development. In: “Handbook of Drug Metabolism” (T. F. Woolf ed., 1999) pp. 321-362.
Marcel Dekker Inc., New York, NY.

22. McKinnon, R.A. & Nebert, D.W. Cytochrome P450 knockout mice: New toxicological models.
Clin. Exp. Pharmacol. Physiol. 1998; 25:783-787.

23. Huang, S.-M. et al. Assessment of the quality and quantity of drug-drug interaction studies in
recent NDA submissions: Study design and data analysis issues. J. Clin. Pharmacol. 1999;
39:1006-1014.

24. Sun, H. et al. Population pharmacokinetics. Clin. Pharmacokinet. 1999, 37(1):41-58.

25. Ingelman-Sundberg, M. et al. Polymorphic human cytochrome P450 enzymes: an opportunity for
individualized treatment. TIPS 1999; 20(8):342-349.

26. Daly, A. K. Pharmacogenetics. In: “Handbook of Drug Metabolism” (T. F. Woolf ed., 1999)
pp. 175-202. Marcel Dekker Inc., New York, NY.

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27. Hammerlein, A. et al. Pharmacokinetic and pharmacodynamic changes in the elderly. Clin.
Pharmacokinet. 1998; 35(1):49-64.

28. Beierle, I. et al. Gender differences in pharmacokinetics and pharmacodynamics. Int. J. Clin.
Pharmacol. Ther. 1999; 37 (11):529-547.

29. Zevin, S., Benowitz, N.L. Drug interactions with tobacco smoking. Clin. Pharmacokinet. 1999;
36 (6): 425-438.

30. Ameer, B., Weintraub, R.A. Drug interactions with grapefruit juice. Clin. Pharmacokinet. 1997;
33(2): 103-121.

31. Stockley, I. H. Monoamine oxidase inhibitor drug interactions. In “Drug Interactions” (Stockley
ed., 1999) pp. 594-615. Pharmaceutical Press, London, UK.

WEBSITE REFERENCES

1A. Clinically Used Drugs Metabolized by Cytochrome P450.

http://www.dml.georgetown.edu/depts/pharmacology/clinlist.html

2A. Guidance for Industry: Drug Metabolism/Drug Interaction Studies in the Drug Development
Process: Studies In Vitro (1997)
Food and Drug Administration
http://www.fda.gov/cder/guidance/clin3.pdf

3A. Guidance for Industry: In Vivo Drug Metabolism/Drug Interaction Studies–Study Design, Data
Analysis, and Recommendations for Dosing and Labelling (1999)
Food and Drug Administration
http://www.fda.gov/cder/guidance/2635fnl.htm
http://www.fda.gov/cder/guidance/2635fnl.pdf

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 APPENDIX A

Phase I Enzymes

Cytochrome P450 Superfamily

CYP1
CYP2
CYP3
CYP4
CYP5
CYP7
CYP11
CYP17
CYP19
CYP21
CYP27

Flavin-containing mono-oxygenases
Alcohol dehydrogenase
Aldehyde dehydrogenase
Dihydropyrimidine dehydrogenase
Butyrylcholinesterases
Cholinesterase
Hydrolases
Monoamine-diamine oxidase
Polyamine oxidase
Xanthine oxidase
Alkylhydrazine oxidase
Paroxonase
Prostaglandin synthetase-lipoxygenase
Aromatases
Azo and nitro reductases
Carbonyl reductase
Epoxide hydrolases

Phase II Enzymes

Glutathione S-transferases
GST A1-1
GST A2-2
GST M1a-1a

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 GST M1b-1b
GST M2-2
GST M3-3
GST M4-4
GST M5-5
GST P1-1
GST T1-1
GST T2-2
GST microsomal

N-Acetyltransferases
NAT1
NAT2

Uridine diphosphate-glucuronyltransferases
UGT1
UGT2

Methyltransferases
thiol methyltransferase
catechol-O-methyltransferase
thiopurine methyltransferase

Sulfotransferases
ST1A2
ST1A3
ST1A5
ST2A3

Acyltransferase

Acyl-CoA synthetases

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