The accuracy of in vitro inhibition parameters in scaling to in vivo drug-drug interactions (DDI) was examined for over 40 drugs using seven human P450-selective marker activities in pooled human liver microsomes. These data were combined with other parameters (systemic C max , estimated hepatic inlet C max , fraction unbound, and fraction of the probe drug cleared by the inhibited enzyme) to predict increases in exposure to probe drugs, and the predictions were compared with in vivo DDI gathered from clinical studies reported in the scientific literature. For drugs that had been tested as precipitants of drug interactions for more than one P450 in vivo, the order of inhibitory potencies in vitro generally aligned with the magnitude of the in vivo interactions. With the exception of many drugs known to be mechanism-based inactivators, the use of in vitro IC 50 , the fraction of the affected drug metabolized by the target enzyme [f m(CYP) ] and an estimate of free hepatic inlet C max , was generally successful in identifying those drugs that cause at least a 2-fold increase in the exposure to P450 marker substrate drugs. For CYP3A, incorporation of inhibition of both hepatic and intestinal metabolism was needed for the prediction of DDI. Many CYP3A inhibitors showed a different inhibitory potency for three different CYP3A marker activities; however, these differences generally did not alter the conclusions regarding whether a drug would cause a CYP3A DDI in vivo. Overall, these findings support the conclusion that P450 in vitro inhibition data are valuable in designing clinical DDI study strategies and can be used to predict the magnitudes of DDI.Drug-drug interactions remain an important issue in clinical practice and the discovery and development of new drugs. With our recently advanced knowledge of the human cytochrome P450 (P450) enzymes and their roles in drug metabolism, more systematic approaches to the study of drug interactions have evolved. Previous to this knowledge, studies of drug-drug interactions for new drugs were carried out empirically; combinations of drugs chosen for investigation of drug-drug interactions were selected based on the potential for alteration in the pharmacokinetics or dynamics of a narrow therapeutic index drug (e.g., digoxin, theophylline, warfarin, phenytoin, etc.) or whether there was a high likelihood that the new drug would be frequently coprescribed with another agent for a given condition. However, with an increased understanding of drug-metabolizing enzymes and their roles in the metabolism of specific drugs, a more mechanistic approach to assessing drug-drug interactions can be taken. The results of clinical drug-drug interaction studies with one drug can be extrapolated to other drugs that are cleared by the same enzyme.In vitro drug-drug interaction data are necessary for devising mechanistically based clinical drug-drug interaction study strategies. The effects of new drugs on well characterized drug metabolism reactions known to be specific for various huma...

ABSTRACT:The measurement of the effect of new chemical entities on human cytochrome P450 marker activities using in vitro experimentation represents an important experimental approach in drug development. In vitro drug interaction data can be used in guiding the design of clinical drug interaction studies, or, when no effect is observed in vitro, the data can be used in place of an in vivo study to claim that no interaction will occur in vivo. To make such a claim, it must be assured that the in vitro experiments are performed with absolute confidence in the methods used and data obtained. To meet this need, 12 semiautomated assays for human P450 marker substrate activities have been developed and validated using approaches described in the GLP (good laboratory practices) as per the code of U.S. Federal Regulations. The assays that were validated are: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), bupropion hydroxylase (CYP2B6), amodiaquine N-deethylase (CYP2C8), diclofenac 4-hydroxylase and tolbutamide methylhydroxylase (CYP2C9), (S)-mephenytoin 4-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), felodipine dehydrogenase, testosterone 6␤-hydroxylase, and midazolam 1-hydroxylase (CYP3A4 and CYP3A5). High-pressure liquid chromatography-tandem mass spectrometry, using stable isotope-labeled internal standards, has been applied as the analytical method. This analytical approach, through its high sensitivity and selectivity, has permitted the use of very low incubation concentrations of microsomal protein (0.01-0.2 mg/ml). Analytical assay accuracy and precision values were excellent. Enzyme kinetic and inhibition parameters obtained using these methods demonstrated high precision and were within the range of values previously reported in the scientific literature. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of cytochrome P450 activities.Drug-drug interactions are of great interest to scientists involved in drug research, regulatory authorities who are responsible for public safety, physicians, and their patients. Since "polypharmacy," or the practice of simultaneous prescription of more than one drug to treat one or more conditions in a single patient, has become a more common practice, drug interactions have been cited as one of the major reasons for hospitalization and even death (Lazarou et al., 1998). Thus, a great deal of effort is expended by researchers engaged in new drug research in avoiding the development of compounds that will cause drug-drug interactions.The most common mechanism underlying drug-drug interactions is the inhibition of cytochrome P450 activities. Several drugs in common use cause large increases in exposure to other drugs. Examples include ketoconazole, itraconazole, erythromycin, clarithromycin, diltiazem, and nefazodone (CYP3A); quinidine, paroxetine, and terbinafine (CYP2D6); ticlopidine (CYP2C19); ...

ABSTRACT: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 C max , estimated hepatic inlet C max , 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 C max 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 (IC 25 ) 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 IC 50 occurring with a 30-min preincubation with liver microsomes and NADPH was also useful in identifying mechanismbased inactivators, and the IC 50 measured after such a preincubation was highly correlated with the k inact /K I 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.The prediction of drug-drug interactions (DDIs) using in vitro enzyme kinetic data has been an area of increasing advances and sophistication. This has proven to be a valuable endeavor because DDIs remain an important issue in clinical practice and the discovery and development of new drugs. The earlier that the potential for DDIs can be identified in new compounds being studied as potential drugs, the greater the likelihood that this deleterious property can be removed through improved design of the molecule. Also, for those compounds already undergoing clinical trials, in vitro DDI data can be leveraged in the design of adequate and appropriate clinical DDI studies. With our increased understanding of drug-metabolizing enzymes and their roles in the metabolism of specific drugs, a mechanistic approach to assessing DDIs can be taken. The results of clinical DDI studies with one drug can be extrapolated to other drugs that are cleared by the same enzyme.The alteration of drug-metabolizing enzyme activities can occur by three main mechanisms: reversible inhibition, mechanism-based inactivation, and induction. Confidence in quantitatively extrapolating in vitro results to in vivo varies with these mechanisms. For reversible inhibitio...

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 muM 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 microM) 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 microM) 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.

ABSTRACT:The measurement of the effect of new chemical entities on human UDP-glucuronosyltransferase (UGT) marker activities using in vitro experimentation represents an important experimental approach in drug development to guide clinical drug-interaction study designs or support claims that no in vivo interaction will occur. Selective high-performance liquid chromatography-tandem mass spectrometry functional assays of authentic glucuronides for five major hepatic UGT probe substrates were developed: ␤-estradiol-3-glucuronide (UGT1A1), trifluoperazine-N-glucuronide (UGT1A4), 5-hydroxytryptophol-O-glucuronide (UGT1A6), propofol-O-glucuronide (UGT1A9), and zidovudine-5-glucuronide (UGT2B7). High analytical sensitivity permitted characterization of enzyme kinetic parameters at low human liver microsomal and recombinant UGT protein concentration (0.025 mg/ml), which led to a new recommended optimal universal alamethicin activation concentration of 10 g/ml for microsomes. Alamethicin was not required for recombinant UGT incubations. Apparent enzyme kinetic parameters, particularly for UGT1A1 and UGT1A4, were affected by nonspecific binding. Unbound intrinsic clearance for UGT1A9 and UGT2B7 increased significantly after addition of 2% bovine serum albumin, with minimal changes for UGT1A1, UGT1A4, and UGT1A6. Eleven potential UGT and cytochrome P450 inhibitors were evaluated as UGT inhibitors, resulting in observation of nonselective UGT inhibition by chrysin, mefenamic acid, silibinin, tangeretin, ketoconazole, itraconazole, ritonavir, and verapamil. The pan-cytochrome P450 inhibitor, 1-aminobenzotriazole, minimally inhibited UGT activities and may be useful in reaction phenotyping of mixed UGT and cytochrome P450 substrates. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of human UGT activities and the identification of UGT enzymeselective chemical inhibitors.

C]metformin by PF-04971729 also were very weak (IC 50 ‫؍‬ ϳ900 M). Single-species allometric scaling of rat pharmacokinetics of PF-04971729 was used to predict human clearance, distribution volume, and oral bioavailability. Human pharmacokinetic predictions were consistent with the potential for a low daily dose. First-in-human studies after oral administration indicated that the human pharmacokinetics/dose predictions for PF-04971729 were in the range that is likely to yield a favorable pharmacodynamic response.

Cytochrome P450 2B6 (CYP2B6) is involved in the metabolism of drugs such as bupropion, efavirenz, propofol, and selegiline, among others. More than 200 commonly prescribed drugs and other xenobiotics were examined for their ability to inhibit CYP2B6-mediated bupropion hydroxylase activity. Thirty compounds were found exhibiting greater than 50% inhibition at 30 microM. Inhibitors of CYP2B6 were identified from a wide variety of therapeutic classes. The 2 platelet aggregation inhibitors, clopidogrel and ticlopidine, were both identified as potent inhibitors (IC50 = 0.0206 and 0.149 microM, respectively). Other inhibitors (IC50 < 1 microM) included clotrimazole, itraconazole, sertraline, and raloxifene. These in vitro data were used along with clinical pharmacokinetic information in the prediction of potential drug-drug interactions that could occur by inhibition of CYP2B6. Although few drugs tested are expected to cause drug interactions, clopidogrel and ticlopidine were identified as being of concern as potential inhibitors of clinical relevance. These findings are discussed in context to potential drug interactions that could be observed between these agents and drugs for which CYP2B6 is involved in metabolism.

ABSTRACT:The leukotriene receptor antagonist montelukast was examined for its inhibition of the human drug-metabolizing enzyme cytochrome P4502C8 (CYP2C8). Montelukast was demonstrated to be a potent inhibitor of CYP2C8-catalyzed amodiaquine N-deethylase, rosiglitazone N-demethylase, and paclitaxel 6␣-hydroxylase in human liver microsomes. Inhibition was also observed when the reaction was catalyzed by recombinant heterologously expressed CYP2C8. The mechanism of inhibition was competitive, with K i values ranging from 0.0092 to 0.15 M. Inhibition potency was highly dependent on the microsomal protein concentration. Increasing the microsomal protein concentration by 80-fold yielded a 100-fold decrease in inhibition potency. Preincubation of montelukast with human liver microsomes and NADPH did not alter the inhibition potency, suggesting that montelukast is not a mechanism-based inactivator. Montelukast was a selective inhibitor for human CYP2C8; inhibition of other human cytochrome P450 enzymes was substantially less. These in vitro data support the use of montelukast as a selective CYP2C8 inhibitor that could be used to determine the contribution of this enzyme to drug metabolism reactions. These data also raise the possibility that montelukast could have an effect on the metabolic clearance of drugs possessing CYP2C8-catalyzed metabolism as a major clearance pathway, thereby eliciting pharmacokinetic drug-drug interactions.Cytochrome P450 (P450) enzymes represent a family of proteins that contribute predominantly in the metabolism of drugs and other xenobiotics. Alteration in the activity of these enzymes in vivo represents the major underlying mechanism behind pharmacokinetic drug-drug interactions (Clarke and Jones, 2002). In humans, some P450 enzymes have been designated as more predominant in the metabolism of drugs than others. These include CYP1A2, 2C9, 2C19, 2D6, and 3A. These five enzymes have been studied extensively with regard to their role in drug metabolic clearance as well as drug-drug interactions. However, recent investigations have uncovered roles for other cytochrome P450 enzymes, such as CYP2C8, in the metabolism of some drugs. Thus, CYP2C8 can also be the target of drug-drug interactions for those drugs in which it plays a predominant role in the clearance. Drugs for which CYP2C8 appears to play a major role in metabolic clearance include repaglinide, rosiglitazone, and cerivastatin. Drug interactions that arise by inhibition of CYP2C8 have been described recently, including interactions between cerivastatin and gemfibrozil (Backman et al., 2002), repaglinide, and gemfibrozil (Niemi et al., 2003b), and repaglinide and trimethoprim (Niemi et al., 2004).In previous work, a vast number of drugs have been tested for their potential to inhibit CYP2C8 (Bun et al., 2003, Ong et al., 2000Walsky et al., 2005). In a study conducted in our laboratories, we found that montelukast (Fig. 1), a leukotriene receptor antagonist used in the treatment of asthma, potently inhibited CYP2C8-catalyzed amodiaquin...