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In-vitro Drug Metabolism Studies DuringDevelopment of New Drugs

Anthony Y.H.Lu

Rutgers UniversityPiscataway, New Jersey, U.S.A.

Shiew-Mei Huang

Food and Drug AdministrationRockville, Maryland, U.S.A.

INTRODUCTION

Since late 1980s, the drug discovery and development process hasundergone significant changes, particularly in the preclinical stage involvingdrug candidate selection, drug metabolism and safety studies. These changesare directly related to the scientific progress in research areas ofcombinatorial chemistry, recombinant DNA technology, toxicology,metabolism, and analytical instrumentation. The increasing availability oftissues, cell cultures, and drug-metabolizing enzymes from human sourceshas led to the increased use of in vitro studies to select the most desirabledrug candidates. Well executed in vitro studies can provide valuableinformation regarding the metabolic fate of a new drug in humans, critical

Copyright 2004 by Marcel Dekker, Inc.

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factors contributing to the variability of pharmacokinetic parameters, andthe potential for drug-drug interactions. Consequently, in vitro study resultsare now being routinely included in New Drug Applications (NDA) by thesponsors.

What type of in vitro studies should be included in the NDA? Howshould these studies be conducted? In this chapter, we describe some of thecommonly used in vitro techniques used to study drug metabolism duringdrug development. However, as indicated in an FDA document on in vitrodrug metabolism studies [1], the assessment of drug metabolism in vitro is arapidly evolving area of drug development and regulation. Therefore, newmethods and additional studies will undoubtedly be added to this list. Sinceone of the guiding principles in drug development is to generate datautilizing up-to-date scientific technology and knowledge available in thefield, modification of currently used methods and approaches are expectedwith time. The goal of early in vitro studies conducted at the preclinicalstage is to obtain optimal information to maximize the possibility of successin developing a safe and effective drug for clinical use.

METHODS TO ASSESS DRUG-DRUG INTERACTION POTENTIAL

In vitro studies are useful for assessing the potential of metabolism-baseddrug-drug interaction [24], a major concern for the effective and safe use oftherapeutic agents and a critical factor contributing to the recentwithdrawal of various drugs from the United States market [56]. Sincecytochrome P450 plays a key role in the metabolism of numerous importantdrugs in clinical use, cytochrome P450-mediated drug-drug interactionshave attracted most attention, although the importance of transporter-based drug-drug interactions has also been recognized in the last few years.Central to the issue of metabolism-based drug-drug interactions is theidentification of the cytochrome P450(s) responsible for the metabolism ofthe interacting drugs. Major activity alterations of the involving cytochromeP450 species, due to either inhibition or induction, can result in potential,significant pharmacokinetic changes of interacting drugs in humans.

As described in the following sections, various in vitro methods can beused to assess the potential of drugs acting as inhibitors or inducers ofcytochrome P450. If the potential for interaction is great, in vivo studies inhuman should be considered to evaluate the clinical significance of the invitro findings. The in vivo approaches include specific pharmacokinetic andpharmacodynamic studies, population pharmacokinetic studies, andclinical safety and efficacy studies [79]. In vivo animal studies have limitedvalues in predicting human drug-drug interactions, particularly if the resultsin animals are negative. A single change in amino acid of the protein


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sequence can dramatically change the substrate specificity of cytochromeP450 [10, 11]. In addition, various researchers have described speciesdifferences in cytochrome P450 inhibition [12, 14] and induction [13].Thus, cytochrome P450s in the same gene family in animals and human maynot respond to inhibitors and inducers in similar manners.

GENERAL APPROACHES

In vitro Methodologies

Most of the in vitro metabolism studies involve the use of tissues or drug-metabolizing enzymes from the liver. The emphasis of metabolic researchhas been on the liver, as it is considered the major organ for drugmetabolism, and that we know the most about the properties andfunctions of liver drug-metabolizing enzymes, particularly cytochromeP450. In addition, human liver tissues and human recombinantcytochrome P450s are readily available. However, for some drugs,nonhepatic tissues, such as the gastrointestinal mucosa, may play a vitalrole in their metabolism. In these cases, in vitro metabolism studiesemploying tissues from the kidneys, intestines, or skin may be valuable.Similarly, although cytochrome P450s are the dominant enzymes for themetabolism of most drugs, other drug-metabolizing enzymes are alsopresent in the liver and extrahepatic tissues. These non-cytochrome P450enzymes are responsible for glucuronidation, sulfation, acetylation,glutathione conjugation, and other enzymatic reactions. In vitro studiesusing specific tissue fractions and cofactors are critical in characterizingthese metabolic reactions. In this chapter, unless specifically indicated, allin vitro studies refer to cytochrome P450-mediated hepatic metabolism ofnew drugs.

Many in vitro models are available to study hepatic drug metabolism,ranging from the simplest recombinant enzymes to subcellular fractions,hepatocytes, liver slices, to the more complicated isolated, perfused liver.The degree of physiological relevance of these models decreases as onechanges from the whole organ to the recombinant enzymes. It is importantto select in vitro systems that are most suitable to achieve specific goals ofthe study [2]. If the hepatic subcellular fractions are to be used formetabolism studies, it is important to recognize the distribution of theenzymes responsible for the metabolic events in various tissues and thespecific cofactors required for particular reactions.

One critical issue in conducting in vitro metabolism studies is theappropriateness of drug concentrations that are used in these studies. Sincethe drug concentration at the enzyme active site in the liver could not be


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easily measured and the plasma drug concentration is generally unknown atthe time of in vitro metabolism study, it is often difficult to define the in vitrodrug concentration of physiological relevance.

Despite this uncertainty, it is the general rule not to use unrealisticallyhigh drug concentrations (e.g., in the mM range) for in vitro metabolismstudies. Considering the assay sensitivity and the general plasma drugconcentrations in humans, drug concentrations in the low M rangerepresent a good range to study for most of the in vitro metabolism studies.A good practice is to use several drug concentrations (e.g., low, medium, andhigh, spanning two to three orders of magnitudes) in these studies. This isdesirable particularly for drugs that undergo metabolism via two or morepathways involving multiple enzymes (with different Km values). In thiscase, both high and low affinity metabolic pathways can be studied.

With the advancement in analytical methodologies and knowledge ofhuman drug-metabolizing enzymes, the major metabolic pathways of a newdrug in humans can be readily established and metabolites can be isolatedfrom in vitro models. If the metabolites are found to be pharmacologicallyactive, sensitive and specific assays could be developed to assess thepharmacokinetic profile of the metabolite(s) in subsequent clinical studies.

Animal toxicity studies are an important component of safety evaluationof new drugs. Comparative animal and human metabolic profiles generatedin vitro can help the selection of appropriate animal models for toxicityevaluation and may be useful in the interpretation or hypothesis-generatingof certain clinical findings.

The liver slices and hepatocyte suspensions from human and animalspecies are suitable for metabolic profiling, since these systems contain allthe necessary enzymes and cofactors for metabolism [2]. Hepatic subcellularfractions and recombinant drug-metabolizing enzymes can be used whenmetabolic profiles are relatively simple and only one or two well-recognizedenzymes are involved in the biotransformation of the new drug. Because ofthe known genetic polymorphism of many of the human drug-metabolizingenzymes and the well-recognized large inter-individual variability in drugmetabolism, it is desirable to use liver tissues derived from more than oneindividual (if possible) to generate metabolic profiles. In addition, as freshhuman livers are not always readily available, cryopreserved humanhepatocytes are now being increasingly used for drug metabolism studies[3]. Cryopreserved human hepatocytes retain most, if not all, of the majordrug-metabolizing enzyme activities.

In vitro/In vivo Correlation

Although significant progress has been made in recent years in theevaluation of drug-drug interaction potential based on in vitro data, a



complete understanding of the relationship between in vitro findings and invivo human results of metabolism-based drug-drug interaction studies isstill emerging. In some cases, excellent correlation of in vitro and in vivoresults has been demonstrated while in others, the in vitro and in vivocorrelation has been poor [15]. Because of the complexities of variousfactors impacting both in vitro and in vivo drug-drug interactions, accuratepredictions of the extent of in vivo drug interactions from in vitro metabolicstudies will require continued efforts in obtaining additional high qualitycorrelation data to permit rational evaluation of new drugs. At the presenttime, the feasibility of predicting in vivo drug interactions based on in vitrometabolic data is still under rigorous debate. Some investigators believe thata quantitative prediction of in vivo drug interaction is possible [1618]while others take the position that a qualitative prediction approach is morefeasible [19, 20]. In a recent commentary, Tucker et al. [21] used thequalitative terms low risk, medium risk, and high risk to describe theprojection of AUC changes based on the [I]/Ki ratio, where the Ki values aredetermined from in vitro studies.

Various factors contributing to the difficulty in predicting if a newmolecular entity (NME) is an inhibitor from in vitro data. Among them,the unusual cytochrome P450 property and the large number of drugsubstrates appear to be critical factors. In vitro drug-drug interactionpatterns (e.g., mutual inhibition, partial inhibition, activation, and lack ofreciprocal inhibition) for a given cytochrome P450, such as CYP3A4, areoften substrate-dependent. The Ki value of an inhibitor for a givencytochrome P450 is dependent on the probe substrates, enzyme sources,and experimental conditions such as protein concentration and incubationtime due to various degrees of inhibitor-protein binding, partition ofinhibitor to the lipid and aqueous layers, and inhibitor and substratedepletion.

One of the challenges in predicting the extent of in vivo drug-druginteraction from in vitro metabolism studies is the lack of information onthe inhibitor concentration in vivo in the active site of the enzyme or tissues.Since the plasma inhibitor concentration may be the only known parameter,both total inhibitor concentration and unbound inhibitor concentrationhave been used for in vitro-in vivo correlation evaluation. Claims of goodcorrelation with either of the parameters have been reported for differentdrugs. Other factors contributing to the lack of good in vitro-in vivocorrelation using either of the parameters may include the following: (1) theinhibiting drug may also act as an inducer; (2) other parallel eliminationpathways and/or extrahepatic metabolism of the drug may decrease theimportance of the in vitro-assessed pathway; (3) modulation of animportant cellular transport mechanism by the inhibitor may change theextent of in vivo drug-drug interaction, and (4) rapid elimination of


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inhibitor in vivo by noncytochrome P450 pathways may decrease the extentof in vivo drug-drug interaction.

Study Design Considerations

Cytochrome P450 Identification

Unequivocal identification of one or more specific cytochrome P450enzymes responsible for the metabolism of new therapeutic agents is thecornerstone of in vitro metabolism studies. This information is also criticalfor the follow-up cytochrome P450 inhibition and induction studies in theoverall evaluation of in vitro drug-drug interactions. For all these studies,the experimental conditions should be that the measured initial reactionrates (in terms of product formation) are linear with respect to enzymeconcentration and incubation time. It is preferable to use low enzymeconcentration (e.g., below 0.5mg human liver microsomal protein per mL)and short incubation time (less than 20 min) to minimize protein bindingand depletion of substrate and inhibitor (no more than 20% consumption,preferably less than 10%). If the analytical sensitivity is not an issue, lowerenzyme concentration and shorter incubation time are highly desirable. Incase of a slow substrate turnover, higher enzyme concentration and longerincubation time can be used as long as the initial metabolic rates are beingmeasured.

If the cytochrome P450-mediated metabolism represents a significantclearance mechanism for the NME, cytochrome P450 reaction phenotypingshould be carried out, generally, with human liver microsomes andrecombinant cytochrome P450s using a combination of several basicapproaches [22]. The NME concentrations used are generally at or belowthe Km values. Initial reaction rates are measured in the absence and thepresence of antibodies or chemical inhibitors, or with a panel of human livermicrosomes for correlation analysis with various cytochrome P450 probesubstrates. If there is an indication for the involvement of more than onecytochrome P450 in the metabolism of the drug, several drugconcentrations (e.g., low, medium, and high-spanning two to three orders ofmagnitude) should be used for inhibition studies.

Chemical Inhibitors and Inhibitory Antibodies. Specific and potentinhibitors are valuable for cytochrome P450 reaction phenotyping. In thisrespect, inhibitory antibodies (particularly monoclonal antibodies) withdemonstrated specificity and potency can be useful [23], as illustrated in arecent paper by Granvil et al. [24]. These investigators described that the 4-hydroxylation of debrisoquine, a well-recognized probe reaction ofCYP2D6, is mediated not only by CPY2D6 but also by human CYP1A1.Whereas quinidine, a recognized selective inhibitor of CYP2D6, inhibits the



4-hydroxylation of debrisoquine by both CYP2D6 and human CYP1A1,anti-CYP2D6 monoclonal anitbody inhibits specifically CYP2D6-medicated reaction, and not CYP1A1-dependent metabolism. To date,specific and potent monoclonal as well as polyclonal antibodies have notbeen widely used by the pharmaceutical industry possibly due to their highcost and limited availability from commercial sources.

A desirable antibody inhibition study can be conducted in two stages.Initially, metabolism of a drug by pooled human liver microsomes isexamined in the presence of antibodies against all major humancytochrome P450s at a single high concentration (known to give greaterthan 8095% inhibition with probe substrates) to determine whichantibodies significantly inhibit the metabolism. This study establishes thatone or more cytochrome P450 is involved in the metabolism of an NME.In subsequent studies, the effect of those inhibitory antibodies on themetabolism of the NME is studied in more detail using a series of antibodyconcentrations. A well-designed study should show that metabolism isinhibited strongly by the specific antibody in a concentration-dependentmanner at low antibody concentrations and then reaches maximuminhibition at higher antibody concentrations [25] as illustrated in Fig. 1(curves A and D). A steep inhibition slope indicates high potency of theantibody against specific cytochrome P450. The extent of the maximuminhibition indicates the extent (%) of the metabolism of the NME by thisparticular cytochrome P450 enzyme. No meaningful conclusion can bemade regarding the role of a specific cytochrome P450 in the metabolismof an NME when an antibody inhibition study showed a shallowinhibition slope (an indication of low antibody potency) and failed todemonstrate maximum inhibition (Fig. 1, curve B). Thus, a good antibodyinhibition study establishes not only the involvement but also thequantitative importance of a particular cytochrome P450 in themetabolism of the NME. When it is desirable to obtain informationregarding the variability of cytochrome P450 involvement, particularlywhen more than one cytochrome P450 enzymes are involved, similarstudies can be carried out with a panel of human liver microsomalpreparations. Frequently, one can demonstrate a wide range ofinvolvement of specific cytochrome P450 in the metabolism of a particulardrug with microsomes from different donors [23].

Although specific chemical inhibitors for individual human cytochromeP450 are rare, isoform-selective inhibitors are generally available at mostpharmaceutical laboratories and are valuable when properly used. Table 1lists preferred probe substrates and inhibitors for individual cytochromeP450 enzyme [21]. Similar to antibody inhibition studies, chemicalinhibition studies can be carried out first with a single inhibitorconcentration (known to give strong inhibition with probe substrates) to


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determine which probe inhibitors significantly inhibit the metabolism of theNME, followed by a more detailed study involving a series ofconcentrations of the inhibitors. As shown in Fig. 2 (curves A and B), a goodchemical inhibitor selective for a given cytochrome P450 isoform shouldgive strong inhibition (a steep inhibition slope) in the metabolism of anNME at low inhibitor concentrations and reach maximum inhibition athigher inhibitor concentrations so that the quantitative involvement of thiscytochrome P450 isoform in metabolism can be established. Gradualincrease in inhibition with a wide range of inhibitor concentrations (i.e., ashallow inhibition slope, Fig. 2, curve C) would suggest that the inhibitoreither has low potency toward the particular cytochrome P450 or it acts as apoor substrate of the enzyme. In this case inhibition results from the studyhave limited values. When studies are carried out using a panel of human

FIGURE 1 Inhibition of human liver microsomal drug metabolism by antibodiesagainst cytochrome P450. Curve A depicts the strong inhibition of compound Ametabolism by anti-CYP3A4 antibodies. The steep inhibition slope at low antibodyconcentrations indicates high potency of this antibody preparation. Maximuminhibition at higher antibody concentrations indicates that greater than 90% of themetabolism of compound A is mediated by CYP3A4 in this pooled human livermicrosomal sample. Curve B shows the inhibition of compound A metabolism inhuman liver microsome by a different anti-CYP3A4 antibody preparation. Theshallow inhibition slope indicates that either this antibody has a low potency againstCYP3A4 or it cross-reacts with another cytochrome P450. No conclusion can bemade regarding the role of CYP3A4 in the metabolism of compound A. Curve C isthe control experiment showing lack of inhibition of compound A metabolism bypre-immune IgG. Curve D depicts the inhibition of the metabolism of compound Bby anti-CYP3A4 antibodies. The steep inhibition slope is noted at lowconcentrations of this potent antibody. CYP3A4 is responsible for 50% of the


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TABLE 1 Recommended in vitro Probe Substrates and Inhibitors for CYPs (Ref. [21])


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liver microsomal preparations, different degrees of maximum inhibition inmetabolism provide information regarding the variability of specificcytochrome P450 involvement in the metabolism of the NME amongindividual subjects.

Recombinant Human Cytochrome P450 Enzymes. Microsomescontaining individually expressed human cytochrome P450s provide adifferent approach for cytochrome P450 reaction phenotyping. Thisapproach establishes the intrinsic capability of the individual cytochromeP450 in the metabolism of an NME, in the absence of other cytochromeP450 species. If one or more cytochrome P450 species are involved in anNMEs metabolism, it is important to examine the contribution of eachcytochrome P450 to human liver microsomal metabolism using inhibitoryantibodies or chemical inhibitors. Sometimes, a recombinant cytochromeP450 found to be involved in an NMEs metabolism, based on arecombinant enzyme study, may later be shown to play little or no role inliver microsomal metabolism of the drug in the presence of othercytochrome P450s, based on an inhibition study. Furthermore, for thesecytochrome enzymes for which activities are observed initially, adetermination of the enzyme kinetics (Km and Vmax) may be warranted sothat the intrinsic clearance and the relative importance of these different

FIGURE 2 Inhibition of human liver microsomal drug metabolism by a chemicalinhibitor of CYP3A4. Curve A depicts the strong inhibition of compound Ametabolism by this inhibitor. The steep inhibition slope at low inhibitorconcentrations indicates that this inhibitor of CYP3A4 is very potent. CYP3A4contributes to approximately 90% of the metabolism of compound A in this pooledmicrosomal preparation. Curve B shows that CYP3A4 contributes to 50% of themicrosomal metabolism of compound B. Curve C depicts the shallow inhibitionslope indicating poor inhibition of the metabolism of compound C even at highinhibitor concentrations. No conclusions can be made regarding the role ofCYP3A4 in the metabolism of compound C.



cytochrome P450 species contributing to the metabolism of the NME can beevaluated [2628].

Correlation Analysis. Using this approach, the drug is incubated with apanel of human liver microsomes (preferably more than 10 preparations)and the reaction rates of an NME determined in each preparation arecorrelated with the reaction rates of a cytochrome P450 probe substratemeasured in the same microsomal preparation. If a particular cytochromeP450 is responsible for the metabolism of the NME, a high correlationshould be observed between the metabolic rates of the drug and the markersubstrate. However, this type of correlation analysis appears to be lessreliable in identifying specific cytochrome P450 enzymes responsible for themetabolism of an NME. For example, Weaver et al. [29] reported that58C80 hydroxylation is catalyzed by CYP2C9 based on inhibition andrecombinant cytochrome P450 studies; however, there is no correlationbetween 58C80 hydroxylation and CYP2C9 probe substrate activity(r=0.023). In another study, Heyn et al. [30] reported that although highcorrelations between S-mephenytoin N-demethylation and CYP2B6(r=0.91), CYP2A6 (r=0.88), and CYP3A4 (r=0.74) were observed, otherapproaches showed CYP2B6 to be the major enzyme responsible for S-mephenytoin N-demethylation while CYP2A6 and CYP3A4 played nosignificant role in this reaction.

Cytochrome P450 Inhibition

It is important to examine if an NME is an inhibitor of cytochrome P450snot involved in the metabolism of the drug. For this type of study, the effectof NME on the metabolism of probe substrate for each of the individualcytochrome P450 (see Table 1) is evaluated, usually in human livermicrosomes, although individual recombinant human cytochrome P450enzymes have also been used. The incubation conditions should be such thatinitial rates could be measured. To determine the Ki value for any specificcytochrome P450, at least four to five probe substrate concentrations andtwo to three NME concentrations should be used in the assays. Substrateconcentrations should cover a wide range (preferably 1020-fold) with thenumber of concentrations evenly distributed below and above the Km value.The importance of proper selection of both substrate and inhibitorconcentrations in these studies is well illustrated in the paper by Madan etal. [22]. The rates of metabolite formation of probe substrate aredetermined in the presence and absence of the NME inhibitor and the dataare displayed in graphical representation to determine Ki and the type ofinhibition [22]. Substrate-dependent inhibition has been reported earlier forCYP3A [49, 51]. Two or more substrates may be needed when evaluatinginhibitors of CYP3A using in vitro methods [21, 47, 49]. Because of


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significant solvent effects (particularly when concentration >1%) reportedfor various CYP enzyme studies, low solvent concentrations should be usedin these in vitro studies [47].

In addition to reversible inhibition, time-dependent inhibition ofcytochrome P450 activity by a drug candidate may also be examined todetermine if the NME is a mechanism-based inhibitor. For this type of study,an NME, at various concentrations (covering a 1020-fold range), ispreincubated with human liver microsomes with and without NADPH forvarious lengths of time (e.g., 0, 10, 20, 30, 45, and 60min) to allow thegeneration of reactive metabolites that inhibit cytochrome P450 activityirreversibly or quasi-irreversibly [22]. At various incubation time points, analiquot of the samples is removed and diluted several folds with fresh assaybuffer. The activity of the remaining cytochrome P450 is determined by thereaction rates of a probe substrate, and the data are displayed in graphicalrepresentation to determine the Ki and Kinact values [22, 31].

If an NME and clinically co-administered drugs are metabolized by thesame cytochrome P450 isoform, inhibition of this cytochrome P450 canlead to the accumulation of either of the drugs and thereby cause potentialserious drug-drug interactions. This potential can be evaluated using an invitro system of human liver microsomes in the presence of both the drugs.The importance in the proper use of concentrations of either of the drugs isas described in the preceding section. The Ki value for either of the drugs canbe determined and the potential of drug-drug interaction of co administereddrugs can be evaluated.

Cytochrome P450 Induction

Cytochrome P450 induction represents another mechanism formetabolismbased drug-drug interactions, although it is much less commonthan inhibition-mediated interaction events. Drug treatment can result inthe induction of cytochrome P450 responsible for its own metabolism (i.e.,auto-induction) or other cytochrome P450s responsible for the metabolismof co-administered drugs. The major effect of cytochrome P450 induction isthe alteration of drug efficacy and safety over time due to increasedclearance of therapeutic agents resulting in decreased parent drugconcentrations and increased metabolite levels.

To determine if an NME is a cytochrome P450 inducer, the compound, atseveral concentrations, is incubated with primary human hepatocytes fortwo to five days, and the metabolic rates for probe substrates of individualcytochrome P450 (generally CYP1A2, 2C9, 2C19, and 3A) are measured[32, 33]. The NME concentrations should be relevant to its therapeuticrange or, if the theoretical range is not known, a pilot study covering two tothree orders of magnitude may be appropriate. The enzyme activity is



considered to be the most relevant measure while mRNA and Western blotanalyses are useful primarily for mechanistic interpretation [21, 50]. In viewof the individual variability in cytochrome P450 induction, primary humanhepatocytes prepared from at least three individual donor livers should beused to obtain reliable results. Appropriate positive controls (e.g.,omeprazole for CYP1A2 induction, rifampicin for 2C9, 2C19, and 3A4induction) should be included in the study.

In addition to primary human hepatocytes, other in vitro methods suchas receptor ligand assay and reporter gene assay have also been used toevaluate the intrinsic induction potential of drug candidates [13, 32, 34]. Apositive result of the in vitro induction study can help design clinical trials todetermine if induction is likely to occur at clinical doses and if the extent ofinduction may result in significant drug-drug interactions.

Transferases

If an NME is primarily metabolized by a noncytochrome P450 enzyme, itmay become necessary to identify the specific enzyme form responsible forthe metabolism of the compound, particularly if a co-administered drug isalso biotransformed by a similar metabolic pathway and the same enzyme.However, for enzymes such as flavin-containing monooxygenases,monoamine oxidases, epoxide hydrolases, glucuronosyl transferases (UGT),sulfotransferases, methyltransferases, acetyltransferases, and glutathione-S-transferases, analytical tools are generally not available for carrying outreaction phenotyping experiments. For example, specific or highly selectiveprobe substrates and inhibitors are still not available for most of theseenzymes. In addition, antibodies against many of these enzymes are oftennoninhibitory so that antibody inhibition experiments can not be performedto identify the specific enzyme form(s) involved in the metabolism of anNME. For some of the enzymes, recombinant isoforms remain the only toolfor reaction phenotyping.

When a drug molecule contains functional groups such asOH,NH2,SH orCOOH, glucuronidation often represents the mostimportant pathway for its clearance. Therefore, considerable attention hasbeen paid to UGT reaction phenotyping and its role in drug-druginteractions [35, 39]. At the present time, highly selective chemicalinhibitors and inhibitory antibodies for individual UGT isoforms are notavailable. The only method available to identify the specific isoformresponsible for the metabolism of a drug is to conduct a study withrecombinant UGT enzymes. In addition, a study using a combination ofdrugs in human liver microsomes or recombinant system may be valuable inorder to determine if one drug inhibits the metabolism of the other drug or ifmutual inhibition occurs.


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In the literature, there are limited clinical data on UGT-dependent drug-drug interaction [35], either because of the generally high Km and Ki forUGTs (therefore low intrinsic clearance and low interaction potential) ordue to the lack of clinical studies designed to address UGT-dependent drug-drug interactions. Further studies are needed to evaluate the clinicalsignificance of UGT-dependent drug-drug interactions.

Transporters

It has become increasingly evident that drug transporters, such as P-glycoprotein, play an important role in the absorption, distribution, andexcretion of many drugs [3638, 40]. Many substrates, inhibitors, andinducers of CYP3A4 are also substrates, inhibitors, and inducers of P-gp[4045]. Drug-Drug interactions involving transporters, particularly P-glycoprotein, have become the new focuses in drug discovery anddevelopment. When drugs compete for the same binding sites on the P-glycoprotein molecule, drug-drug interactions can occur.

To determine if an NME is a substrate of P-glycoprotein and whether thecompound acts as an inhibitor of P-glycoprotein, various in vitro systems,such as Caco-2 cells, cDNA-transfected Madine-Darby canine kidney cellsand LLC-PK1 pig kidney cells, and derivative cells containing MDR1 (L-MDR1) can be used. Many studies use digoxin and vinblastine as in vitroprobes and fexofenadine and digoxin as in vivo probe substrates of P-glycoprotein. The experiments are usually carried out under linearcondition, and the substrate concentrations are at or below their Km values.Although ATPase and calcein-AM assays have been used, it appears that theefflux assay (also known as the bi-directional permeability assay) is themethod of choice for evaluating compounds [38, 41].

At the present time, the in vitro methodologies have not beenstandardized for the identification of substrates and inhibitors for P-glycoprotein and other transporters. Prediction of the in vivo drug-druginteractions from in vitro studies is still problematic. It is expected that moreselective probe substrates and inhibitors will be available for P-glycoproteinand other transporters (e.g., OATP, MRP, BCRP) in the future, and that ourability to predict drug-drug interactions in vivo at the transporters level willbe greatly improved.

REGULATORY CONSIDERATIONS

Evaluation of an NMEs drug-drug interaction potential is an integral partof the regulatory review prior to its market approval [1, 7]. The clinicalpharmacology and biopharmaceutic review of an NDA focuses on keyquestions relevant to the review and integrates information across various



studies [46]. For example, in addition to questions addressing how thefollowing intrinsic factors (age, gender, race, weight, height, disease,genetic polymorphism, pregnancy, and organ dysfunction) may influenceexposure and/or response, the reviewers also ask questions related toextrinsic factors:

What extrinsic factors (co-administered drugs, herbal products,diet, smoking, and alcohol use) influence exposure and/orresponse and what is the impact of differences, if any, inexposure on pharmacodynamics of an NME?

Based upon what is known about exposure-responserelationships and their variability, what dosage regimenadjustments, if any, do you recommend for each of these factors?

Among drug-drug interaction questions, the following may be addressed viain vitro studies:

Is there an in vitro basis to suspect in vivo drug-drug interaction? Is the drug a substrate of CYP enzymes? Is the drug an inhibitor and/or an inducer of CYP enzymes? Is the drug a substrate and/or an inhibitor of P-glycoprotein

transport processes? Are there other metabolic/transporter pathways that may be

important?

Depending on the answers to the above questions, additional studies may beconducted to fully assess the interaction potential of an NME with otherdrugs, herbal products, and/or food/juices. Figure 3 illustrates onealgorithm in the evaluation of CYP enzyme-based drug-drug interactions ofan NME; starting with in vitro evaluations of the metabolic profile and theCYP enzyme-modulating effects of the NME using human enzymes. Basedon the outcomes of these in vitro evaluations, which are reviewed along withadditional in vivo clearance information, further clinical studies may beconducted (Fig. 3).

The appropriate use of in vitro metabolism and drug interactioninformation can provide the basis for the design of subsequent in vivostudies, or obviate the need for further in vivo studies, as illustrated in thefollowing two cases. For example, Drug As effects on various cytochromeP450 enzyme activities have been evaluated with the following probereactions (phenacetin O-deethylation for CYP1A2; tolbutamide 4'-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation forCYP2C19, bufuralol 1'-hydroxylation for CYP2D6 and testosterone 6-hydroxylation for CYP3A) using human liver microsomes. The data show


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that Drug A does not inhibit CYP1A2, CYP2C9, CYP2C19, and CYP2D6at concentrations 100-fold the mean steady state Cmax level achievableafter the administration of the highest proposed clinical dose. Based on thisinformation, no further in vivo studies on Drug As inhibitory effects onCYP1A2, 2D6, 2C9, and 2C19 will be needed. Drug A inhibits CYP3A.Further analysis indicates the Ki value to be 1/100 of the Cmax level;suggesting Drug A to be a strong CYP3A inhibitor. A follow-up clinicalstudy with oral midazolam administration confirmed its effect on substratesof CYP3A. The focus of the clinical evaluation on CYP3A has provided datauseful for risk/benefit evaluation of Drug A and subsequent productlabeling. Similarly, Drug B has been evaluated using in vitro methods andshown to have Ki values in the following rank order:CYP1A2=CYP2C9>CYP3A>CYP2C19>CYP2D6. As many of these I/Kiratios fall within the gray area between low risk and high risk (21), anin vivo study focused on CYP2D6 was performed. By focusing on the CYPenzyme that appeared to be affected most by Drug B, the lack of interactionfrom this latter in vivo study would eliminate the need to study Drug Bseffects on the other CYP enzymes.

FIGURE 3 An algorithm for evaluating drug-drug interactions [21].



LABELING

In a proposed revision of physician labeling format and content, significant(or evidence of no) drug-drug interactions would appear in the Highlightssection, in addition to having this information in the main body of the labeling[48]. In vitro and in vivo information on the metabolic pathways andmetabolites, including contribution of specific enzymes, and known or expectedeffects of inducers or inhibitors of the pathway, is described in the clinicalpharmacology section of the labeling. Any information on pathways orinteractions that have been ruled out by in vitro data is also included in thissection. Important clinical consequences of this information would be placedin drug interactions, warnings, precautions, boxed warning, contraindications,and dosage and administration sections of the main labeling, as appropriate.Examples of appropriate labeling language are provided in italic below:

[Case 1] In vitro interaction has been studied for the new drugand no interactions have been demonstrated; no in vivo studieshave been conducted to confirm or refute the in vitro finding.

In vitro drug interaction studies reveal no inhibition of themetabolism of the new drug by the CYP3A4 inhibitorketoconazole. No clinical studies have been performed to evaluatethis finding. However, based on the in vitro findings, a metabolicinteraction with ketoconazole, itraconazole, and other CYP3A4inhibitors is not anticipated.

Recent examples, such as rosiglitazone (inhibitory effect on CYP enzymes),and sildenafil (inhibitory effects on CYP1A2, 2C9, 2C19, 2D6, 2E1, and3A4), are listed in Table 2.

[Case 2] Through in vitro investigations, specific enzymes havebeen identified as metabolizing the test drug, but no in vivo or invitro drug interaction studies have been conducted.

In vitro drug metabolism studies reveal that the new drug is asubstrate of the CYP ____ enzyme. No in vitro or clinical druginteraction studies have been performed. However, based on thein vitro data, blood concentrations of the new drug are expectedto increase in the presence of inhibitors of the CYP ____ enzymesuch as _____, _____, or.

Recent examples, such as pimozide (substrate of CYP3A, ventriculararrhythmia observed in patients also taking CYP3A inhibitors, macrolideantibiotics) and Ketoconazole are listed in Table 2.

Recently approved product labels have reflected the increasedunderstanding of metabolic pathways and consequences of drug


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TABLE 2 Labeling Examples of Metabolism and Drug-Drug Interaction Information


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TABLE 2 Continued.


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interactions by health care practitioners. Newer labels frequently includein vitro parameters evaluating the drugs effect on specific cytochromeP450 metabolism and the clinical consequences of the changes in theseenzyme activities have on co-administered drugs. In addition, the labelsalso include the influence of concomitantly administered drugs on thedrug itself. Table 2 lists some examples of the labeling language based onin vitro information. Less frequently included in the labels today aretransporter information and metabolic interactions based on othernoncytochrome P450 enzymes. As the science progresses and technologiesin the evaluation become standard, future labeling should include theseother types of information.

SUMMARY

As many of the new drugs are to be indicated for patients who receive otherdrugs or biologies, it is necessary to know the drug interaction potentialearly on in the development. For compounds eliminated by a singlepathway, there is a high probability of drug interaction. The appropriate useof in vitro metabolism (including isozyme characterization) and druginteraction information can provide the basis for the design of confirmatoryin vivo studies or obviate the need for further in vivo studies. Furtherimprovement in the in vitro methodologies evaluating other,noncytochrome P450-based metabolilsm/drug interactions andtransporterbased interactions should improve our abilities to assess drug-drug interactions for risk/benefit evaluation during drug development andregulatory review.

REFERENCES

1. Guidance for Industry: Drug Metabolism/Drug Interactions in the DrugDevelopment Process: Studies in vitro. Internet: http://www.fda.gov/cder April1997.

2. Lin, J.H.; Rodrigues, A.D. In vitro Model, for Early Studies of DrugMetabolism. In Pharmacokinetic Optimization in Drug Research: Biological,Physicochemical and Computational Strategies, Testa, B., VanderWaterbeemed, H., Folkes, G., Guy, R., Eds.; Wiley-Verlag, 2001, 217243.

3. Li, A.P.; Gerycki, P.D.; Hengstler, J.G.; Kedderis, G.L.; Keebe, H.G.; Rahman,R.; de Sousas, G.; Silva, J.M.; Skett, P. Present Status of the Application ofCryopreseved Hepatocytes in the Evaluation of Xenobiotic: Consensus of anInternational Expert Panel. Chem. Biol. Interact. 1999, 121, 117123.

4. Drug-Drug Interactions, Rodrigues, A.D., Ed.; Marcel Dekker: New York,2001.



5. Huang, S.-M.; Booth, B.; Fadiran, E.; Uppoor, R.S.; Doddapaneni, S.; Chen, M.;Ajayi, F.; Martin, T.; Lesko, L.J. What Have We Learned from the RecentMarket Withdrawal Of Terfenadine and Mibefradil? Presentation at the 101Annual Meeting of American Society of Clinical Pharmacology andTherapeutics. March 1517, 2000, Beverly Hills, CA, abstract in ClinPharmacol Ther.

6. Huang, S.-M.; Miller, M.; Toigo, T.; Chen, M.; Sahajwalla, C; Lesko, L.J.;Temple, R. Evaluation of Drugs in Women: Regulatory Perspectivein Section11, Drug Metabolism/Clinical Pharmacology (section editor: Schwartz, J). InPrinciples of Gender-Specific Medicine; Legato, M., Ed.; Academic Press, inpress.

7. CDER MPCC/CPS In vivo Drug-Drug Interaction Working Group. Guidancefor industry: in vivo metabolism/drug interactions: study design, data analysisand recommendation for dosing and labeling, Internet: http://www.fda.gov/cder, December 1999.

8. CDER MPCC/CPS Population PK Working Group. Guidance for industry:population pharmacokinetic. Internet: http://www.fda.gov/cder, February1999.

9. Huang, S.-M.; Honig, P.; Lesko, L.J.; Temple, R.; Williams, R. An IntegratedApproach to Assessing Drug-Drug Interactions: A Regulatory Perspective. InDrug-Drug Interactions, Rodrigues, A.D., Ed.; Marcel Dekker: New York,2001, 605632.

10. Matsunaga, E.; Zeugin, T.; Zanger, U.M.; Aoyama, T.; Meyer, U.A.; Gonzalez,E.F. Sequence Requirements for Cytochrome P450IID1 Catalytic Activity: ASingle Amino Acid Change (ILD380PHE) Specifically Decreases Vm of theEnzyme for Bufuralol but not Debrisoquine Hydroxylation. J. Biol. Chem.1990, 265, 1719717201.

11. Crespi, C.L.; Steimel, D.T.; Peuman, B.W.; Korzekwa, K.R.; FernandezSalguero,P.; Buters, J.T.M.; Gelboin, H.V.; Gonazelez, E.J.; Idle, J.R.; Doly, A.K.Comparison of Substrate Metabolism by Wild Type CYP2D6 Protein and aVariant Containing Methionine, but not Valine at Position 374.Pharmacogenetics 1995, 5, 234243.

12. Boobis, A.R.; Davies, D.S. Human Cytochrome P450s. Xenobiotica 1984, 14,151185.

13. Goodwing, B.; Redinbo, M.R.; Kliewer, S.A. Regulation of CYP3A GeneTranscription by the Pregnane X Receptor. Annu. Rev. Pharmacol. Toxicol.2002, 42, 123.

14. Sesardic, R.; Boobis, A.R.; Murray, B.P.; Murray, S.; Sequra, J.; De La Torre, R.;Davies, D.S. Furafylline is a Potent and Selective Inhibitor of CytochromeP4501A2 in Man. Br. J. Clin. Pharmac. 1990, 29, 651663.

15. Davit, B.; Reynolds, K.; Yuan, R.; Ajayi, F.; Conner, D.; Fadiran, E.;Gillespie, B.; Sahajwalla, C.; Huang, S.-M.; Lesko, L.J. FDA Evaluationusing in vitro Metabolism to Predict and Interpret in vivo Metabolic Drug-Drug Interactions: Impact on Labeling. J. Clin. Pharmacol. 1999, 39, 899910.


Lu and Huang108

16. Sugiyama, Y.; Iwatsudo, Y.; Ueda, K.; Ito, K. Strategic Proposals for AvoidingToxic Interactions with Drugs for Clinical use During Development and AfterMarketing of a New Drug: Pharmacokinetic Consideration. J. Toxicol. Sci.1996, 21, 309316.

17. Von Moltke, L.L.; Greenblatt, D.J.; Schmider, J.; Duan, S.X.; Wrigh, C.E.;Harmatz, J.S.; Shader, R.I. Midazolam Hydroxylation by Human LiverMicrosomes in vitro: Inhibition by Fluoxetine, Norfluoxetine and by AzoleAntifungal Agents. J. Clin. Pharmacol. 36, 783791.

18. Ito, K.; Iwatsubo, T.; Kanamitsu, S.; Ueda, K.; Suzuki, H.; Sugiyama, Y.;Prediction of Pharmacokinetic Alterations Caused by Drug-Drug Interactions:Metabolic Interactions in the Liver. Pharmacol. Rev. 1998, 50, 387411.

19. Lin, J.H. Sense and Nonsense in the Prediction of Drug-Drug Interactions.Current Drug Metabolism 2000, 1, 305331.

20. Lin, J.H.; Pearson, P.G. Prediction of Metabolic Drug Interactions: Quantitativeor Qualitative? In Drug-Drug Interactions; Rodrigues, A.D., Ed.; MarcelDekker: New York, 2001, 415438.

21. Tucker, G.T.; Houston, J.B.; Huang, S.M. Optimizing Drug Development:Strategies to Assess Drug Metabolism/Transporter Interactions PotentialToward a Consensus. Clin. Pharmacol. Ther. 2001, 70, 103114; Br. J. Clin.Pharmacol. 2001, July 52 (1), 107117; Eur. J. Pharm. Sci. July 2001, 13 (4),417428; Pharm. Res. Aug. 2001, 18 (8), 10711180.

22. Madam, A.; Usuki, E.; Burton, L.A.; Ogilive, B.W.; Parkinson, A. In vitroApproaches for Studying the Inhibition of Drug-metabolizing Enzymes andIdentifying the Drug-metabolizing Enzymes Responsible for the Metabolism ofDrugs. In Drug-Drug Interaction, Rodrigues, A.D., Ed.; Marcel Dekker: New

23. Gelboin, H.V.; Krausz, K.W.; Gonzalez, F.J.; Yang, T.J. Inhibitory MonoclonalAntibodies to Human Cytochrome P450 Enzymes: A New Avenue for DrugDiscovery. TIPS 1999, 20, 432438.

24. Granvil, C.P.; Krausz, K.W.; Gelboin, H.V.; Idle, J.R.; Gonzalez, F.J. 4-Hydroxylation of Debrisoquine by Human CYP1A1 and its Inhibition byQuinidine and Quinine. J. Pharmacol. Exp. Ther. 2002, 301, 10251032.

25. Wang, R.W.; Lu, A.Y.H. Inhibitory Anti-peptide Antibody Against HumanCYP3A4. Drug Metab. Dispos. 1997, 25, 762767.

26. Rodrigues, A.D. Integrated Cytochrome P450 Reaction Phenotyping:Attempting to Bridge the Gap Between CDNA-expressed Cytochrome P450and Native Human Liver Microsomes. Biochem. Pharmacol. 1999, 57, 465480.

27. Crespi, C.L.; Miller, V.P. The Use of Heterologously ExpressedDrugMetabolizing Enzymes-state of the Art and Prospects for the Future.Pharmacol. Ther. 1999, 84, 121131.

28. Venkatakrishnan, K.; Von Moltke, L.L.; Greenblatt, D.J. Application ofthe Relative Activity Factor Approach in Scaling from HeterologouslyExpressed Cytochrome P450 to Human Liver Microsomes: Studies onAmitriptyline as Model Substrate. J. Pharmcol. Exp. Ther. 2001, 297, 326337.


York, 2001, 217294 (Figures 3, 4).


29. Weaver, R.J.; Dickins, M.; Burke, M.D. Hydroxylation of the Antimalarial Drug58C80 by CYP2C9 in Human Liver Microsomes: Comparison withMephenytoin and Tolbutamide Hydroxylations. Biochem. Pharmacol. 1995,49, 9971004.

30. Heyn, H.; White, R.B.; Stevens, J.C. Catalytic Role of Cytochrome P4502B6 inthe N-demethylation of S-mephenytoin. Drug Metab. Dispos. 1996, 24, 948954.

31. Jones, D.R.; Hall, S.D. Mechanism-based Inhibition of Human CytochromeP450: in vitro Kinetics and in vitro-in vivo Correlations. In Drug-DrugInteractions, Rodrigues, A.D., Ed.; Marcel Dekker: New York, 2001, 387413.

32. Silva, J.M.; Nicoll-Griffith, D.A. In vitro Models for Studying Induction ofCytochrome P450 Enzymes. In Drug-Drug Interactions, Rodrigues, A.D., Ed.;Marcel Dekker: New York, 2001, 189216.

33. Li, A.P.; Reith, M.K.; Rasmussen, A.; Gorski, J.C.; Hall, S.D.; Xu, L.; Kaminski,D.L.; Cheng, K.L. Primary Human Hepatocytes as a Tool for the Evaluation ofStructure-Activity Relationship in Cytochrome P450 Induction Potential ofXenobiotics: Evaluation of Rifampin, Rifapentine and Rifabutin. Chem. Biol.Interact. 1997, 107, 1730.

34. Rodrigues, A.D.; Lin, J.H. Screening of Drug Candidates for their Drug-DrugInteraction Potential. Current Opinion in Chemical Biology 2001, 5, 396401.

35. Remmel, R.P. Review of Human UDP-glucuronosyltransferases and their Rolein Drug-Drug Interactions. In Drug-Drug Interactions, Rodrigues, A.D., Ed.;Marcel Dekker: New York, 2001, 89114.

36. Troutman, M.D.; Luo, G.; Gan, L.S.; Thakker, D.R. The Role of P-glycoprotein in Drug Disposition: Significance to Drug Development. In Drug-Drug Interactions, Rodrigues, A.D., Ed.; Marcel Dekker: New York, 2001,295357.

37. Kusuhara, H.; Sugiyana, Y. Drug-Drug Interactions Involving the MembraneTransport Process. In Drug-Drug Interactions, Rodrigues, A.D., Ed.; MarcelDekker: New York, 2001, 123188.

38. Polli, J.W.; Wring, S.A.; Humpreys, J.E.; Huang, L.; Morgan, J.B.; Webster,L.O.; Serabjit-Singh, C.S. Rational use of in vitro P-glycoprotein Assays in DrugDiscovery. J. Pharmacol. Exp. Ther. 2001, 299, 620628.

39. Green, M.D.; Tephly, T.R. Glucuronidation of Amine Substrates by Purified andExpressed UDP-glucuronosyltransferase Proteins. Drug Metab. Dispos. 1998,26, 860867.

40. Cvetkovic, M.; Leake, B.; Fromm, M.F.; Wilkinson, G.R.; Kim, R.B. OATP andP-glycoprotein Transporters Mediate the Cellular Uptake and Excretion ofFexofenadine. Drug Metab. Dispos. 1999, 27 (8), 866871.

41. Hochman, J.H.; Yamazaki, M.; Ohe, T.; Lin, J.H.; Evaluation of DrugInteractions with P-glycoprotein in Drug Discovery: In vitro Assessment of thePotential for Drug-Drug Interactions with P-glycoprotein. Current DrugMetabolism 2002, 3, 257273.


Lu and Huang110

42. Kim, R.B. Drugs as P-glycoprotein Substrates, Inhibitors, and Inducers. DrugMetabolism Reviews 2002, 34, 4754.

43. Kim, R.B., et al. Interrelationship between Substrates and Inhibitors of HumanCYP3A4 and P-glycoprotein. Pharm Res 1999, 16 (3), 39443948.

44. Cummins, C.L.; Jacobsen, W.; Benet, L.Z.; Unmasking the Dynamic Interplaybetween Intestinal P-Glycoprotein and CYP3A4. J. Pharmacol Exp. Ther. 2002,300 (3), 10361045.

45. Yasuda, K.; Lan, L.B.; Sanglard, D.; Furuya, K.; Schuetz, J.D.; Schuetz, E. G.Interaction of Cytochrome P450 3A Inhibitors with P-Glycoprotein. J.Pharmacol. Exp. Ther. 2002, 303 (1), 323332.

46. Lesko, L.J.; Williams, R.L. The Question based ReviewA ConceptualFramework for Good Review Practices. Appl. Clin. Trials 1999, 8 (6), 5662.

47. Yuan, R.; Madani, S.; Wei, S.; Reynolds, K.; Huang, S.-M. Evaluation ofCytochrome P450 Probe Substrates Commonly used by the PharmaceuticalIndustry to Study in vitro Drug Interactions. Drug Metab. Disp. 2002, 30 (12),in press.

48. FR notice. Labeling Guideline (Federal Register 65, 247; 8108281131;December 22, 2000).

49. Kentworthy, K.E.; Bloomer, J.C.; Clarke, S.E.; Houston, J.B. CYP3A4 DrugInteractions: Correlation of 10 in vitro Probe Substrates. Br. J. Clin. Pharmacol.1999, 48, 716727.

50. Lecluyse, E. Human Hepatocyte Culture Systems for the in vitro Evaluation ofCytochrome P450 Expression and Regulation. Eur. J. Pharm. Sci. 2001, 4, 343368.

51. Wang, R.W.; Newton, D.J.; Lin, N.; Atkins, W.M.; Lu, A.Y.H. HumanCytochrome P450 3A4: In vitro Drug-Drug Interaction Patterns areSubstrateDependent. Drug Metab. Disp. 2000, 28, 360366.


Table of ContentsChapter 5: In-vitro Drug Metabolism Studies During Development of New DrugsINTRODUCTIONMETHODS TO ASSESS DRUG-DRUG INTERACTION POTENTIALGENERAL APPROACHESIn vitro MethodologiesIn vitro/In vivo CorrelationStudy Design ConsiderationsCytochrome P450 IdentificationCytochrome P450 InhibitionCytochrome P450 InductionTransferasesTransporters

REGULATORY CONSIDERATIONSLABELINGSUMMARYREFERENCES

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