R. Scott Obach

Mechanism-Based Inactivation of Human Cytochrome P450 Enzymes and the Prediction of Drug-Drug Interactions

The ability to use vitro inactivation kinetic parameters in scaling to in vivo drug-drug interactions (DDIs) for mechanism-based inactivators of human cytochrome P450 (P450) enzymes was examined using eight human P450-selective marker activities in pooled human liver microsomes. These data were combined with other parameters (systemic Cmax, estimated hepatic inlet Cmax, fraction unbound, in vivo P450 enzyme degradation rate constants estimated from clinical pharmacokinetic data, and fraction of the affected drug cleared by the inhibited enzyme) to predict increases in exposure to drugs, and the predictions were compared with in vivo DDIs gathered from clinical studies reported in the scientific literature. In general, the use of unbound systemic Cmax as the inactivator concentration in vivo yielded the most accurate predictions of DDI with a mean -fold error of 1.64. Abbreviated in vitro approaches to identifying mechanism-based inactivators were developed. Testing potential inactivators at a single concentration (IC25) in a 30-min preincubation with human liver microsomes in the absence and presence of NADPH followed by assessment of P450 marker activities readily identified those compounds known to be mechanism-based inactivators and represents an approach that can be used with greater throughput. Measurement of decreases in IC50 occurring with a 30-min preincubation with liver microsomes and NADPH was also useful in identifying mechanism-based inactivators, and the IC50 measured after such a preincubation was highly correlated with the kinact/KI ratio measured after a full characterization of inactivation. Overall, these findings support the conclusion that P450 in vitro inactivation data are valuable in predicting clinical DDIs that can occur via this mechanism.

Managing the challenge of chemically reactive metabolites in drug development

The normal metabolism of drugs can generate metabolites that have intrinsic chemical reactivity towards cellular molecules, and therefore have the potential to alter biological function and initiate serious adverse drug reactions. Here, we present an assessment of the current approaches used for the evaluation of chemically reactive metabolites. We also describe how these approaches are being used within the pharmaceutical industry to assess and minimize the potential of drug candidates to cause toxicity. At early stages of drug discovery, iteration between medicinal chemistry and drug metabolism can eliminate perceived reactive metabolite-mediated chemical liabilities without compromising pharmacological activity or the need for extensive safety evaluation beyond standard practices. In the future, reactive metabolite evaluation may also be useful during clinical development for improving clinical risk assessment and risk management. Currently, there remains a huge gap in our understanding of the basic mechanisms that underlie chemical stress-mediated adverse reactions in humans. This Review summarizes our views on this complex topic, and includes insights into practices considered by the pharmaceutical industry.

TREND ANALYSIS OF A DATABASE OF INTRAVENOUS PHARMACOKINETIC PARAMETERS IN HUMANS FOR 670 DRUG COMPOUNDS

We present herein a compilation and trend analysis of human i.v. pharmacokinetic data on 670 drugs representing, to our knowledge, the largest publicly available set of human clinical pharmacokinetic data. This data set provides the drug metabolism scientist with a robust and accurate resource suitable for a number of applications, including in silico modeling, in vitro-in vivo scaling, and physiologically based pharmacokinetic approaches. Clearance, volume of distribution at steady state, mean residence time, and terminal phase half-life were obtained or derived from original references exclusively from studies utilizing i.v. administration. Plasma protein binding data were collected from other sources to supplement these pharmacokinetic data. These parameters were analyzed concurrently with a range of simple physicochemical descriptors, and resultant trends and patterns within the data are presented. Our findings with this much expanded data set were consistent with earlier described notions of trends between physicochemical properties and pharmacokinetic behavior. These observations and analyses, along with the large database of human pharmacokinetic data, should enable future efforts aimed toward developing quantitative structure-pharmacokinetic relationships and improving our understanding of the relationship between fundamental chemical characteristics and drug disposition.

Aldehyde Oxidase: An Enzyme of Emerging Importance in Drug Discovery

David C. Pryde,* Deepak Dalvie, Qiyue Hu, Peter Jones, R. Scott Obach, ) and Thien-Duc Tran WorldWide Medicinal Chemistry, Pfizer Global Research and Development, Sandwich, Kent, CT13 9NJ, England, Pharmacokinetics, Dynamics andMetabolism, Pfizer Global Research andDevelopment, 10628 ScienceCenterDrive, La Jolla, California 92121, WorldWide Medicinal Chemistry, Pfizer Global Research and Development, 10628 Science Center Drive, La Jolla, California 92121, and ) Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340

Mechanism-based inactivation of cytochrome P450 enzymes : Chemical mechanisms, structure-activity relationships and relationship to clinical drug-drug interactions and idiosyncratic adverse drug reactions

Cytochrome P450 constitute a superfamily of heme-containing enzymes that catalyze the oxidative biotransformation of structurally diverse xenobiotics including drugs. Inhibition of P450 enzymes is by far the most common mechanism which can lead to DDIs. P450 inhibition can be categorized as reversible (competitive or non-competitive) or irreversible (mechanism-based inactivation). Mechanism-based P450 inactivation usually involves bioactivation of the xenobiotic to a reactive intermediate, which covalently modifies an active site amino acid residue and/or coordinates to the heme prosthetic group. Covalent modification of P450 enzymes can also lead to hapten formation and can in some cases trigger an autoimmune response resulting in toxicological consequences. Compared to reversible inhibition, irreversible inhibition more frequently results in unfavorable DDIs as the inactivated P450 enzyme has to be replaced by newly synthesized protein. For these reasons, most drug metabolism groups within pharmaceutical companies have well-established screening paradigms to assess mechanism-based inactivation of major human P450 enzymes by new chemical entities followed by in-depth mechanistic studies to elucidate the mechanism of P450 inactivation when a positive finding is discerned. A deeper understanding of the process leading to enzyme inactivation by drug candidates can lead to rational chemical intervention strategies to circumvent the P450 inactivation/bioactivation liability. Apart from structure-activity relationship studies, methodology to predict the magnitude of in vivo metabolic DDIs using in vitro P450 inactivation data and predicted human pharmacokinetics of the candidate drug also exists and can be utilized to project the extent of clinical DDIs against P450 enzyme-specific substrates. In this review, a comprehensive analysis of the biochemical basis and known structure-activity relationships for P450 inactivation by xenobiotics is described. In addition, the current state-of-the-art of the methodology used in predicting the magnitude of DDIs using in vitro P450 inactivation data and human pharmacokinetic parameters is discussed in detail.

METABOLISM AND DISPOSITION OF VARENICLINE, A SELECTIVE α4β2 ACETYLCHOLINE RECEPTOR PARTIAL AGONIST, IN VIVO AND IN VITRO

The metabolism and disposition of varenicline (7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine), a partial agonist of the nicotinic acetylcholine receptor for the treatment of tobacco addiction, was examined in rats, mice, monkeys, and humans after oral administration of [14C]varenicline. In the circulation of all species, the majority of drug-related material was composed of unchanged varenicline. In all four species, drug-related material was primarily excreted in the urine. A large percentage was excreted as unchanged parent drug (90, 84, 75, and 81% of the dose in mouse, rat, monkey, and human, respectively). Metabolites observed in excreta arose via N-carbamoyl glucuronidation and oxidation. These metabolites were also observed in the circulation, in addition to metabolites that arose via N-formylation and formation of a novel hexose conjugate. Experiments were conducted using in vitro systems to gain an understanding of the enzymes involved in the formation of the N-carbamoylglucuronide metabolite in humans. N-Carbamoyl glucuronidation was catalyzed by UGT2B7 in human liver microsomes when incubations were conducted under a CO2 atmosphere. The straightforward dispositional profile of varenicline should simplify its use in the clinic as an aid in smoking cessation.

Comparison of Different Algorithms for Predicting Clinical Drug-Drug Interactions, Based on the Use of CYP3A4 in Vitro Data: Predictions of Compounds as Precipitants of Interaction

Cytochrome P450 3A4 (CYP3A4) is the most important enzyme in drug metabolism and because it is the most frequent target for pharmacokinetic drug-drug interactions (DDIs) it is highly desirable to be able to predict CYP3A4-based DDIs from in vitro data. In this study, the prediction of clinical DDIs for 30 drugs on the pharmacokinetics of midazolam, a probe substrate for CYP3A4, was done using in vitro inhibition, inactivation, and induction data. Two DDI prediction approaches were used, which account for effects at both the liver and intestine. The first was a model that simultaneously combines reversible inhibition, time-dependent inactivation, and induction data with static estimates of relevant in vivo concentrations of the precipitant drug to provide point estimates of the average magnitude of change in midazolam exposure. This model yielded a success rate of 88% in discerning DDIs with a mean -fold error of 1.74. The second model was a computational physiologically based pharmacokinetic model that uses dynamic estimates of in vivo concentrations of the precipitant drug and accounts for interindividual variability among the population (Simcyp). This model yielded success rates of 88 and 90% (for “steady-state” and “time-based” approaches, respectively) and mean -fold errors of 1.59 and 1.47. From these findings it can be concluded that in vivo DDIs for CYP3A4 can be predicted from in vitro data, even when more than one biochemical phenomenon occurs simultaneously.

Can in vitro metabolism-dependent covalent binding data in liver microsomes distinguish hepatotoxic from nonhepatotoxic drugs? An analysis of 18 drugs with consideration of intrinsic clearance and daily dose.

In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the approach still would falsely identify some agents as potentially hepatotoxic.

Examination of 209 drugs for inhibition of cytochrome P450 2C8.

Cytochrome P450 2C8 is involved in the metabolism of drugs such as paclitaxel, repaglinide, rosiglitazone, and cerivastatin, among others. An in vitro assessment of 209 frequently prescribed drugs and related xenobiotics was carried out to examine their potential to inhibit CYP2C8. A validated sensitive, moderate‐throughput high‐performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay was used to detect N‐desethylamodiaquine, the CYP2C8‐derived major metabolite of amodiaquine metabolism, using heterologously expressed recombinant CYP2C8 (rhCYP2C8) and pooled human liver microsomes. The 209 drugs were first tested at 30 μM for their ability to inhibit rhCYP2C8. Forty‐eight compounds exhibited greater than 50% inhibition and were further evaluated for measurement of IC50. The six most potent inhibitors (IC50 <1 μM) from this set were measured for IC50 in pooled human liver microsomes, and the most potent inhibitor identified was the leukotriene receptor antagonist, montelukast (IC50 = 19.6 nM). Inhibitors of CYP2C8 were identified from a wide variety of therapeutic classes, with no single class predominating. Other potent inhibitors included candesartan cilexetil (cyclohexylcarbonate ester prodrug of candesartan), zafirlukast, clotrimazole, felodipine, and mometasone furoate. Seventeen moderate inhibitors of rhCYP2C8 (1 < IC50 < 10 μM) included salmeterol, raloxifene, fenofibrate, ritonavir, levothyroxine, tamoxifen, loratadine, quercetin, oxybutynin, medroxyprogesterone, simvastatin, ketoconazole, ethinyl estradiol, spironolactone, lovastatin, nifedipine, and irbesartan. These in vitro data were used along with clinical pharmacokinetic information in predicting potential drug‐drug interactions that could occur by inhibition of CYP2C8. Although almost all drugs tested are not expected to cause drug interactions via inhibition of CYP2C8, montelukast was identified as being of concern as a potential inhibitor of clinical relevance. These findings are discussed in context to potential drug interactions that could be observed between these agents and drugs for which CYP2C8 is involved in metabolism and warrant investigation of the possibility of clinical drug interactions mediated by inhibition of this enzyme.

WHAT IS THE OBJECTIVE OF THE MASS BALANCE STUDY? A RETROSPECTIVE ANALYSIS OF DATA IN ANIMAL AND HUMAN EXCRETION STUDIES EMPLOYING RADIOLABELED DRUGS

Mass balance excretion studies in laboratory animals and humans using radiolabeled compounds represent a standard part of the development process for new drugs. From these studies, the total fate of drug-related material is obtained: mass balance, routes of excretion, and, with additional analyses, metabolic pathways. However, rarely does the mass balance in radiolabeled excretion studies truly achieve 100% recovery. Many definitions of cutoff criteria for mass balance that identify acceptable versus unacceptable recovery have been presented as ad hoc statements without a strong rationale. To address this, a retrospective analysis was undertaken to explore the overall performance of mass balance studies in both laboratory animal species and humans using data for 27 proprietary compounds within Pfizer and extensive review of published studies. The review has examined variation in recovery and the question of whether low recovery was a cause for concern in terms of drug safety. Overall, mean recovery was greater in rats and dogs than in humans. When the circulating half-life of total radioactivity is greater than 50 h, the recovery tends to be lower. Excretion data from the literature were queried as to whether drugs linked with toxicities associated with sequestration in tissues or covalent binding exhibit low mass balance. This was not the case, unless the sequestration led to a long elimination half-life of drug-related material. In the vast majority of cases, sequestration or concentration of drug-related material in an organ or tissue was without deleterious effect and, in some cases, was related to the pharmacological mechanism of action. Overall, from these data, recovery of radiolabel would normally be equal to or greater than 90%, 85%, and 80% in rat, dog, and human, respectively. Since several technical limitations can underlie a lack of mass balance and since mass balance data are not sensitive indicators of the potential for toxicity arising via tissue sequestration, absolute recovery in humans should not be used as a major decision criteria as to whether a radiolabeled study has met its objectives. Instead, the study should be seen as an integral part of drug development answering four principal questions: 1) Is the proposed clearance mechanism sufficiently supported by the identities of the drug-related materials in excreta, so as to provide a complete understanding of clearance and potential contributors to interpatient variability and drug-drug interactions? 2) What are the drug-related entities present in circulation that are the active principals contributing to primary and secondary pharmacology? 3) Are there findings (low extraction recovery of radiolabel from plasma, metabolite structures indicative of chemically reactive intermediates) that suggest potential safety issues requiring further risk assessment? 4) Do questions 2 and 3 have appropriate preclinical support in terms of pharmacology, safety pharmacology, and toxicology? Only if one or more of these four questions remain unanswered should additional mass balance studies be considered.