Preliminary concept paper

Table 3. Classification of CYP3A inhibitors(1)

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Table 3. Classification of CYP3A inhibitors(1)

Strong CYP3A inhibitors

Moderate CYP3A inhibitors

atanazavir, clarithromycin, indinavir, itraconazole,

ketoconazole, nefazodone, nelfinavir, ritonavir, saquinavir, telithromycin, voriconazole

Amprenavir, aprepitant, diltiazem, erythromycin, fluconazole, fosaprenavir, grapefruit juice(a), verapamil

(1) please note the following:

    • A “strong inhibitor” is one that caused a > 5-fold increase in the plasma AUC values of CYP3A substrates (not limited to midazolam) in clinical evaluations

    • A “moderate inhibitor” is one that caused a > 2- but < 5-fold increase in the AUC values of sensitive CYP3A substrates when the inhibitor was given at the highest approved dose and the shortest dosing interval in clinical evaluations

    • Note that this is not an extensive list; for an updated list, see URL???

(a) the effect of grapefruit juice varies widely

VI. Appendices- In vitro drug metabolism studies
Appendix A. Drug metabolism enzyme identification
Drug metabolizing enzyme identification studies, often referred to as reaction phenotyping studies, are a set of experiments that identify the specific enzymes responsible for metabolism of a drug. Oxidative and hydrolytic reactions involve cytochrome P450 (CYP) and non-CYP enzymes. For many drugs, transferase reactions are preceded by oxidation or hydrolysis of the drug. However, direct transferase reactions may represent a major metabolic pathway for compounds containing polar functional groups.
An efficient approach is to determine the metabolic profile (identify metabolites that are formed and their quantitative importance) of a drug and estimate the relative contribution of CYP enzymes to clearance before initiating studies to identify specific CYP enzymes that metabolize the drug. Identification of CYP enzymes is warranted if CYP enzymes contribute > 25% of a drug’s total clearance. The identification of drug metabolizing CYP enzymes in vitro helps predict the potential for in vivo drug-drug interactions and the impact of polymorphic enzyme activity on drug disposition and the formation of toxic or active metabolites. There are few documented cases of clinically significant drug-drug interactions related to non-CYP enzymes, but the identification of drug metabolizing enzymes in this class (i.e., glucuronosyltransferases, sulfotransferases, and N-acetyl transferases) is encouraged. Although classical biotransformation studies are not a general requirement for the evaluation of therapeutic biologics, certain protein therapeutics modify the metabolism of drugs that are metabolized by CYP enzymes. Given their unique nature, consultation with FDA is appropriate before initiating drug-drug interaction studies involving biologics.
1. Metabolic pathway identification experiments (Determination of metabolic profile)
a) Rationale and Goals- Data obtained from drug metabolic pathway identification experiments in vitro help determine whether experiments to identify drug metabolizing enzymes are warranted, and guide the appropriate design of any such experiments. The metabolic pathway identification experiments should identify the number and classes of metabolites produced by a drug and whether the metabolic pathways are parallel or sequential.
b) Tissue selection for metabolic pathway identification experiments
Freshly isolated hepatocytes are the preferred tissue for conducting metabolic pathway identification experiments. Hepatocytes provide cellular integrity with respect to enzyme architecture and contain the full complement of drug metabolizing enzymes. Alternative tissues include cyropreserved hepatocytes and freshly isolated liver slices. However, these tissues provide qualitative rather than quantitative information.
Subcellular liver tissue fractions or recombinant enzymes can be used in combination with the tissues mentioned above to identify the individual drug metabolites produced and classes of enzyme involved, but the methods do not provide quantitative information of fraction metabolized by a specific enzyme or pathway. Subcellular liver tissue fractions include microsomes, S9, and cytosol; appropriate co-factors are necessary.
c) Design of metabolic pathway identification experiments
The preferred first approach to metabolic pathway identification is to incubate the drug with hepatocytes or liver slices, followed by chromatographic analysis of the incubation medium by HPLC-MS/MS. This type of experiment leads to the direct identification of metabolites formed by oxidative, hydrolytic and transferase reactions and provides information concerning parallel vs. sequential pathways. An alternate approach is to analyze the incubation medium by HPLC using UV, fluorescent, or radiochemical detection.
In view of the known multiplicity and overlapping substrate specificity of drug metabolizing enzymes and the possibility of either parallel or sequential metabolic pathways, experiments should include several drug concentrations and incubation times. Expected steady-state in vivo plasma drug concentrations serve as a guide for the range of drug concentrations used for these experiments.
d) As indicated in the PhRMA position paper on drug-drug interactions (Bjornsson TD, et al, 2003), the methods listed in Table 1 can be used to identify CYP and non-CYP oxidative pathways responsible for the observed metabolites.
Table 1. Methods to identify pathways involved in the oxidative biotransformation of a drug

In vitro System



CYP, FMO vs other oxidases

microsomes, hepatocytes

+/- 1-aminobenzotriazole

broad specificity CYP inactivator


45oC pretreatment

inactivates FMO


+/- pargyline

broad MAO inactivator

S-9, cytosol

+/- menadione, allopurinol

Mo-CO (oxidase) inhibitors

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