Perspective The Conduct of in Vitro Studies to Address Time-Dependent Inhibition of Drug-Metabolizing Enzymes: A Perspective of the Pharmaceutical Research and Manufacturers of America

Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes caused by new molecular entities (NMEs) is of concern because such compounds can be responsible for clinically relevant drug-drug interactions (DDI). Although the biochemistry underlying mechanism-based inactivation (MBI) of P450 enzymes has been generally understood for several years, significant advances have been made only in the past few years regarding how in vitro time-dependent inhibition data can be used to understand and predict clinical DDI. In this article, a team of scientists from 16 pharmaceutical research organizations that are member companies of the Pharmaceutical Research and Manufacturers of America offer a discussion of the phenomenon of TDI with emphasis on the laboratory methods used in its measurement. Results of an anonymous survey regarding pharmaceutical industry practices and strategies around TDI are reported. Specific topics that still possess a high degree of uncertainty are raised, such as parameter estimates needed to make predictions of DDI magnitude from in vitro inactivation parameters. A description of follow-up mechanistic experiments that can be done to characterize TDI are described. A consensus recommendation regarding common practices to address TDI is included, the salient points of which include the use of a tiered approach wherein abbreviated assays are first used to determine whether NMEs demonstrate TDI or not, followed by more thorough inactivation studies for those that do to define the parameters needed for prediction of DDI. Pharmacokinetic drug-drug interactions (DDIs) can occur when one drug alters the metabolism of a coadministered drug. The outcome is an increase or decrease in the systemic clearance and/or bioavailability, and a corresponding change in the exposure to a coadministered drug. The clinical consequences of DDIs range from lack of therapeutic efficacy of a life saving drug to severe adverse drug reactions, including fatalities. Significant drug-drug interactions can lead to termination of development of otherwise promising new therapies, withdrawal of a drug from the market, or severe restrictions/limitations on its use (Wienkers and Heath, 2005). Because of the impact on patient health and safety, DDI was the subject of a position paper in 2003 by scientists from member companies of the Pharmaceutical Research and Manufacturers of America (PhRMA) that focused on Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.109.026716. ABBREVIATIONS: DDI, drug-drug interactions; PhRMA, Pharmaceutical Research and Manufacturers of America; FDA, Food and Drug Administration; P450, cytochrome P450; TDI, time-dependent inhibition; MBI; mechanism-based inactivation; MIC, metabolite-intermediate complex; NME, new molecular entity; IVIVE, in vitro-in vivo extrapolation; AUC, area under the curve; ADME, absorption, distribution, metabolism, and excretion; PK, pharmacokinetic; PBPK, physiologically based pharmacokinetics; MDMA, 3,4-methylenedioxymethamphetamine; SAR, structure activity relationships; HLM, human liver microsome. 0090-9556/09/3707-1355–1370$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 7 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 26716/3483234 DMD 37:1355–1370, 2009 Printed in U.S.A. 1355 at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from the in vitro and in vivo assessment and implications of reversible mechanisms of enzyme inhibition (Bjornsson et al., 2003). The U.S. Food and Drug Administration (FDA) has issued a revised draft guidance for the conduct of in vitro and in vivo drug-drug interaction studies (FDA Guidance for Industry, 2006; http://www.fda.gov/cder/ guidance/index.htm). This draft guidance was broadly written with the intention of assisting in the harmonization of approaches and study designs for better comparison between different drugs and data from various laboratories. Oxidation reactions catalyzed by the cytochrome P450 (P450) enzymes are the most prevalent biotransformations of administered drugs, as well as other xenobiotics and some endogenous compounds (Soars et al., 2007). P450 inhibition has been implicated in the majority of reported clinically relevant drug-drug interactions (Thummel et al., 2000; Bachmann et al., 2003). In addition, the inhibited metabolic pathway could lead to decreased formation of an active metabolite (or formation of a drug from a prodrug) resulting in decreased efficacy. The increase of plasma concentrations caused by DDIs can be substantial, as reported for the interaction between ketoconazole or itraconazole (strong CYP3A4 inhibitors) and triazolam (a CYP3A4 substrate), in which exposure to triazolam increased by 22or 27-fold after coadministration with ketoconazole or itraconazole, respectively (Varhe et al., 1994). For drugs with narrow therapeutic indices, increases in plasma concentrations can lead to adverse drug reactions. Inhibition of P450 enzymes can be classified as either reversible or time-dependent inhibition (TDI) (White, 2000). Reversible inhibition involves rapid association and dissociation of drugs and enzymes and may be competitive, noncompetitive, or uncompetitive. In general, TDI results from irreversible covalent binding or quasi-irreversible noncovalent tight binding of a chemically reactive intermediate to the enzyme that catalyzes its formation, resulting in loss of enzyme function. In some cases, TDI could result from reversible inhibition from a metabolite(s) generated in situ. The distinction between the terminology TDI and mechanism-based inactivation (MBI) must be appreciated. A TDI is defined as a compound that demonstrates an increase in the extent of inhibition it causes when it is incubated with the enzyme before addition of the substrate. As such, this is a kinetic definition only. MBI refers to a subset of TDI in which specific biochemical experiments are conducted that show that the enzyme acts upon the substrate to form a chemically reactive metabolite that subsequently inactivates the enzyme (Silverman, 1996). These two terms will be used throughout this document and are not interchangeable. Metabolic drug-drug interactions resulting from TDI can display a delayed onset due to the time dependence in inhibition and can persist even after the inhibitor has been eliminated because enzymatic activity is only restored by de novo protein synthesis. Time-dependent inhibitors of various human P450 enzymes have been well documented from in vitro studies, and many have been shown to cause DDI (Table 1). Several comprehensive reviews on TDI have been published recently (Zhou et al., 2005, 2007; Venkatakrishnan and Obach, 2007; Johnson, 2008). Drug metabolism sciences have advanced significantly since the publication of the PhRMA position paper (Bjornsson et al., 2003), the 1997 FDA in vitro drug interactions guidance (Drug Metabolism/ Drug Interaction Studies in the Drug Development Process: Studies In Vitro), and even since the release of the updated FDA draft drug interactions guidance in 2006, with reports on drug-drug interactions involving TDI steadily increasing (Ghanbari et al., 2006). Thus, the Drug Metabolism Technical Group of PhRMA assembled a working group of pharmaceutical industry researchers to address the topic of TDI, which builds upon the treatment of reversible inhibition offered in the earlier publication (Bjornsson et al., 2003). This group devised an anonymous survey to collect data on current practices on TDI within pharmaceutical industry research organizations and developed recommendations regarding the conduct of in vitro TDI studies. The outcome of these efforts is presented in this article. The Science of Time-Dependent Inhibition and Predicting DDI Bioorganic Chemistry of P450 Inactivation. The P450 catalytic cycle is illustrated in Fig. 1, and the reader is referred to authoritative accounts of the biochemistry of these enzymes (Guengerich, 2001; Ortiz de Montellano and De Voss, 2002). Several steps will be sensitive to inhibition or inactivation including substrate and oxygen binding or electron transfer. The final products of the catalytic cycle can be stable metabolites or reactive intermediates. The reactivity and nature of a formed metabolite will determine further interactions with P450. The biochemical mechanisms by which compounds function as mechanism-based P450 inactivators can be divided into three categories: quasi-irreversible or metabolite-intermediate complex (MIC) formation, heme alkylation, and protein alkylation. In some cases, inactivation can be caused by protein alkylation by heme fragments (He et al., 1998). Comprehensive reviews of the chemistry and biochemistry of these processes have previously been provided and are beyond the scope of the present discussion (Correia and Ortiz de Montellano, 2005; Fontana et al., 2005; Kalgutkar et al., 2007; Hollenberg et al., 2008). In brief, MICs generally form from molecules possessing amine or methylenedioxyphenyl functional groups. Oxidative bioactivation of TABLE 1 Drugs and other compounds known to cause time-dependent inhibition of drugmetabolizing human cytochrome P450 enzymes 1-Aminobenzotriazole (several) Methoxsalen (CYP2A6) Amiodarone (CYP3A) 3,4-Methylenedioxymethamphetamine (CYP2D6) Amprenavir (CYP3A) 3-Methylindole (CYP2F) Azamulin (CYP3A) Mibefradil (CYP3A) Azithromycin (CYP3A) Midazolam (CYP3A) Bergamottin (CYP3A) Mifepristone (CYP3A) Cannabidiol (CYP3A) Nefazodone (CYP3A) Carbamazepine (CYP1A2) Nelfinavir (CYP3A) Chlorgyline (CYP1A2) Nicardipine (CYP3A) Cimetidine (CYP2D6) Paroxetine (CYP2D6) Clarithromycin (CYP3A) Phencyclidine (CYP2B6) Clopidogrel (CYP2B6) Phenelzine (several) Delavirdine (CYP3A) Pioglitazone (CYP3A) Diclofenac (CYP3A) n-Propylxanthate (CYP2B6) Dihydralazine (CYP1A2) Raloxifene (CYP3A) 6,7-Dihydroxybergamottin (CYP3A) Resveratrol (CYP3A) Diltiazem (CYP3A) Rhapontigenin (CYP1A1) Disulfiram (CYP2E1) Ritonavir (CYP3A) Efavirenz (CYP2B6) Rofecoxib (CYP1A2) EMTPP (CYP2D6) Rosiglitazone (CYP3A) Enoxacin (CYP1A2) Roxithromycin (CYP3A) Erythromycin (CYP3A) Rutaecarpine (CYP1A1, 1B1) Ethinyl estradiol (CYP3A) Saquinavir (CYP3A) 2-Ethynylnaphthalene (CYP1A) Silybin (CYP3A) Fluoxetine (CYP3A) Suprofen (CYP2C9) Furafylline (CYP1A2) Tabimorelin (CYP3A) Gemfibrozil glucuronide (CYP2C8) Tamoxifen (CYP3A) Gestodene (CYP3A) ThioTEPA (CYP2B6) Glabridin (CYP2B6) Ticlopidine (CYP2B6, 2C19) Hydrastine (several) Tienilic acid (CYP2C9) 4-Ipomeanol (CYP3A) Troglitazone (CYP3A, 2C8, 2C9) Irinotecan (CYP3A) Troleandomycin (CYP3A) Isoniazid (several) Verapamil (CYP3A) Lopinavir (CYP3A) Zafirlukast (CYP3A) Menthofuran (CYP2A6) Zileuton (CYP1A2) EMTPP, (1(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl -44-(trifluoromethyl)-2-pyridinyl piperazine. 1356 GRIMM ET AL. at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from these functional groups can lead to intermediate species (e.g., nitroso, carbene, etc.) that form very tight complexes with the heme iron of the P450. The complex probably disrupts oxygen binding and subsequent catalysis, and it produces a characteristic absorption spectrum with a Soret maximum of 448 to 455 nm. Although chemically reversible, MICs are stable under physiological conditions. Several examples of compounds that inactivate P450s by this mechanism include the macrolide antibiotics (Franklin, 1991), calcium channel blockers such as verapamil and diltiazem (Ma et al., 2000), and the selective serotonin reuptake inhibitor paroxetine (Bertelsen et al., 2003). Another P450 inactivation mechanism involves covalent attachment of a reactive species to either the prosthetic heme of the P450 or to protein itself. Although in some instances heme adducts are not stable, the inactivation is generally considered to be irreversible (Correia and Ortiz de Montellano, 2005). Perhaps one of the best characterized compounds that functions by covalently modifying the heme is the relatively nonselective P450 inactivator 1-aminobenzotriazole (Ortiz de Montellano and Mathews, 1981). Other functional groups that can be activated to MBI by P450 metabolism are listed in Table 2. The reader is referred to reviews of the chemistry of P450 inactivators (Fontana et al., 2005; Kalgutkar et al., 2007; Riley et al., 2007; Hollenberg et al., 2008). Enzyme Kinetic Aspects of P450 Inactivation. P450-catalyzed reactions are generally rapid equilibrium interactions between P450 and its substrate, followed by electron transfers (from oxidoreductase/ NADPH and cytochrome b5/NADH) and oxygen incorporation (Guengerich, 2001), leading to oxidized product (metabolite) formation. As illustrated in Scheme 1, a P450 enzyme catalyzes the conversion of a mechanism-based inactivator to its reactive form EI* with the rate constant k2. The reactive metabolite can either be released as a stable product (k3) or it can react with the enzyme (k4). The interaction of a reactive metabolite with a P450 enzyme results in inactivation of the enzyme (E-X) through covalent protein binding, heme modification, or formation of MIC (Silverman, 1995; Correia and Ortiz de Montellano, 2005) as discussed above. The rate of E-X formation is related to k2, k3, and k4. The rate of P450 enzyme inactivation is proportional to inactivator concentration and can be saturated at high concentration (eq. 1): kobs kinact I KI I (1) where the kobs is the pseudo first-order rate constant of inactivation at inactivator concentration [I], kinact is the maximum inactivation rate (a theoretical value that cannot be experimentally observed), and KI is the inactivator concentration when the rate of inactivation reaches half of kinact. Further details of the relationships among the micro rate constants and these parameters are beyond the scope of this discussion and can be found elsewhere (Silverman, 1996). Experimental conditions to determine these kinetic parameters typically involve an “inactivation” incubation (termed in this article as the “preincubation”) for specific time periods in the presence/absence of a putative inactivator and NADPH followed by P450 activity measurement by dilution into a secondary incubation. The percentage of P450 enzyme activity loss due to inactivation can be calculated using eq. 2 (Obach et al., 2007): % activity loss 100 Ainactivator Avehicle noNADPH Ainactivator Avehicle NADPH