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...

While it is generally recognized that misfolding of specific proteins can cause late-onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging in Caenorhabditis elegans. SDS-insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.

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 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...