ABSTRACT: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

Raloxifene is a selective estrogen receptor modulator which is effective in the treatment of osteoporosis in postmenopausal women. We report herein that cytochrome P450 (P450)3A4 is inhibited by raloxifene in human liver microsomal incubations. The nature of the inhibition was irreversible and was NADPH- and preincubation time-dependent, with K(I) and k(inact) values estimated at 9.9 microM and 0.16 min(-1), respectively. The observed loss of P450 3A4 activity was attenuated partially by glutathione (GSH), implying the involvement of a reactive metabolite(s) in the inactivation process. Subsequently, GSH adducts of raloxifene were identified in incubations with human liver microsomes; substitution with GSH occurred at the 5- or 7-position of the benzothiophene moiety or at the 3'-position of the phenol ring, with the 7-glutathionyl derivative being most abundant based on LC/MS and NMR analyses. These adducts are postulated to derive from addition of GSH to raloxifene arene oxides followed by dehydration and aromatization. Alternatively, raloxifene may be oxidized to an extended quinone intermediate, which then is trapped by GSH conjugation. The bioactivation of raloxifene most likely is catalyzed by P450 3A4, since the formation of GSH adducts was almost abolished when liver microsomes were pretreated with ketoconazole or with an inhibitory anti-P450 3A4 IgG. The GSH adducts also were detected in incubations of raloxifene with rat or human hepatocytes, while the corresponding N-acetylcysteine adducts were identified in the bile and urine from rats treated orally with the drug at 5 mg/kg. Taken together, these data indicate that P450 3A4-mediated bioactivation of raloxifene in vitro is accompanied by loss of enzyme activity. The significance of these findings with respect to the clinical use of raloxifene remains to be determined.

We have expressed in Escherichia coli functionally active proteins encoded by two human cDNAs that were isolated previously by using rat 3 alpha-hydroxysteroid dehydrogenase cDNA as the probe. The expressed proteins catalyzed the interconversion between 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol. Therefore, we name these two enzymes type I and type II 3 alpha-hydroxysteroid dehydrogenases. The type I enzyme has a high affinity for dihydrotestosterone, whereas the type II enzyme has a low affinity for the substrate. The tissue-specific distribution of these two enzymes was determined by reverse transcription polymerase chain reaction using gene-specific oligonucleotide primers. The mRNA transcript of the type I enzyme was found only in the liver, whereas that of the type II enzyme appeared in the brain, kidney, liver, lung, placenta, and testis. The structure and sequence of the genes encoding these two 3 alpha-hydroxysteroid dehydrogenases were determined by analysis of genomic clones that were isolated from a lambda EMBL3 SP6/T7 library. The genes coding for the type I and type II enzymes were found to span approximately 20 and 16 kilobase pairs, respectively, and to consist of 9 exons of the same sizes and boundaries. The exons range in size from 77 to 223 base pairs (bp), whereas the introns range in size from 375 bp to approximately 6 kilobase pairs. The type I gene contains a TATA box that is located 27 bp upstream of multiple transcription start sites. In contrast, the type II gene contains two tandem AP2 sequences juxtaposed to a single transcription start site.

The multidrug resistance protein Mrp2 is an ATP-binding cassette (ABC) transporter mainly expressed in liver, kidney, and intestine. One of the physiological roles of Mrp2 is to transport bilirubin glucuronides from the liver into the bile. Current in vivo models to study Mrp2 are the transporter-deficient and Eisai hyperbilirubinemic rat strains. Previous reports showed hyperbilirubinemia and induction of Mrp3 in the hepatocyte sinusoidal membrane in the mutant rats. In addition, differences in liver cytochrome P450 and UGT1a levels between wild-type and mutant rats were detected. To study whether these compensatory mechanisms were specific to rats, we characterized Mrp2 Ϫ/Ϫ mice. Functional absence of Mrp2 in the knockout mice was demonstrated by showing increased levels of bilirubin and bilirubin glucuronides in serum and urine, a reduction in biliary excretion of bilirubin glucuronides and total glutathione, and a reduction in the biliary excretion of the Mrp2 substrate dibromosulfophthalein. To identify possible compensatory mechanisms in Mrp2 Ϫ/Ϫ mice, the expression levels of 98 phase I, phase II, and transporter genes were compared in liver, kidney, and intestine of male and female Mrp2 Ϫ/Ϫ and control mice. Unlike in Mrp2 mutant rats, no induction of Mrp3 in Mrp2 Ϫ/Ϫ mice was detected. However, Mrp4 mRNA and protein in liver and kidney were increased ϳ6-and 2-fold, respectively. Phenotypic analysis of major cytochrome P450-mediated activities in liver microsomes did not show differences between wild-type and Mrp2 Ϫ/Ϫ mice. In conclusion, Mrp2Ϫ/Ϫ mice are a new valuable tool to study the role of Mrp2 in drug disposition.

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.

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ABSTRACT:The contribution of human cytochrome P450 (P450) isoforms to the metabolism of aprepitant in humans was investigated using recombinant P450s and inhibition studies. In addition, aprepitant was evaluated as an inhibitor of human P450s. Metabolism of aprepitant by microsomes prepared from baculovirus-expressed human P450s was observed only when CYP1A2, CYP2C19, or CYP3A4 was present in the expression system. Incubation with CYP1A2 and CYP2C19 yielded only products of O-dealkylation, whereas (Navari et al., 1999;Campos et al., 2001). A dosing regimen of aprepitant consists of a combination therapy with a 5-hydroxytryptamine 3 receptor antagonist, such as ondansetron, and a corticosteroid (e.g., dexamethasone, methylprednisolone) (Roila et al., 1998;Gralla et al., 1999). Thus, the use of this novel drug presents a potential for interactions with chemotherapeutic agents as well as adjuvant therapies.In drug-drug interaction studies, it was observed that coadministration with aprepitant resulted in increases in the area under the plasma concentration versus time curve (AUC) for dexamethasone and methylprednisolone . When the standard dexamethasone regimen (20 mg on day 1 and 12 mg on days 2-5) was given concomitantly with aprepitant, dexamethasone AUC 0 -24 h increased ϳ2-fold on both day 1 and day 5 compared with the standard dexamethasone regimen alone. When a modified dexamethasone regimen (12 mg on day 1 and 4 mg on days 2 and 5) was given concomitantly with aprepitant, the dexamethasone AUC 0 -24 h also increased, making it similar to that of the standard regimen alone. When 125 mg of methylprednisolone i.v. on day 1 and 40 mg p.o. on days 2 and 3 was given with aprepitant, the AUC of methylprednisolone increased approximately 30% on day 1 (after i.v. administration) and 2-fold on day 3 (after oral administration). Aprepitant had no effect on either ondansetron (i.v.) or granisetron (p.o.) pharmacokinetics (Blum et al., 2003). Dexamethasone, methylprednisolone, and granisetron are metabolized by CYP3A4 (Glynn et al., 1986;Bloomer et al., 1994;Gentile et al., 1996;Varis et al., 1998), whereas ondansetron is metabolized by multiple P450 isoforms, including CYP1A2, CYP2D6, and CYP3A4 (Fischer et al., 1994;Dixon et al., 1995).Furthermore, clinical drug interaction studies indicated that administration of aprepitant at two different dosing regimens for 5 days altered the pharmacokinetics of the CYP3A4 probe substrate midazolam, when administered on days 1 and 5 of aprepitant therapy. The pharmacokinetic changes included 2.3-and 1.5-fold increases in midazolam AUC and maximum observed plasma concentration, respectively . Collectively, these results suggested that aprepitant is a moderate inhibitor of CYP3A4.

ABSTRACT:Marker substrates, chemical inhibitors, and inhibitory antibodies are important tools for the identification of cytochrome P450 (P450) isoform responsible for the metabolism of therapeutic agents in vitro. In view of the versatile and nonspecific nature of P450 enzymes, many of the marker substrates and chemical inhibitors used for P450 in vitro reaction phenotyping are isoform selective but not specific. Recently, the use of marker substrate and chemical inhibitors in CYP2D6 in vitro reaction phenotyping was questioned by Granvil et al. (2002). In comparison of a panel of 15 recombinant P450 enzymes, they found that in addition to CYP2D6, CYP1A1 is also capable of catalyzing the formation of 4-hydroxylated metabolite of debrisoquine and that the intrinsic clearance of debrisoquine by CYP2D6-mediated 4-hydroxylation is only about twice that by CYP1A1. In their study, they have also demonstrated that quinidine inhibits both CYP2D6-and CYP1A1-mediated debrisoquine 4-hydroxylation. In view of these important findings, we have reevaluated various approaches used to identify P450 isoform(s) responsible for the metabolism of therapeutic agents. While acknowledging the value of inhibitory antibodies in P450-phenotyping studies, it is our opinion that in well conducted in vitro experiments, isoform-selective chemical inhibitors can also provide valuable and reliable information. Hopefully, future efforts may produce even better P450 isoform-selective marker substrates and inhibitors.For most drugs, biotransformation is the major route of elimination, and oxidative metabolism by P450 1 enzymes is a common metabolic pathway (Rendic, 2002). Therefore, it is important to assess the relative contribution of metabolic pathways to the overall elimination processes and to identify the P450 isoforms responsible for oxidative reactions. In vitro P450 phenotyping has proven to be very successful in predicting the potential of drug interactions and the polymorphic impact on drug disposition (Lin and Lu, 1998;Dahl, 2002). As a result, pharmaceutical companies routinely include in vitro reaction P450 phenotyping in the drug candidate selection process.Ever since the pioneering work of Smith and coworkers (Mahgoub et al., 1977;Sloan et al., 1978), the 4-hydroxylation of debrisoquine has been recognized as a marker reaction of human CYP2D6. Urinary metabolic ratio defined as debrisoquine to 4-hydroxydebrisoquine measured following an oral dose of debrisoquine is generally considered as an accurate assessment of an individual's metabolic capacity of CYP2D6. Based on the metabolic ratios, individuals are phenotypically classified as being poor metabolizer (PM), extensive metabolizer (EM), or ultra-rapid metabolizer (UM) phenotypes, each displaying characteristic pharmacokinetic patterns and clinical outcomes (Eichelbaum and Gross, 1990;Dahl et al., 1995;Meyer and Zanger, 1997). The debrisoquine pharmacokinetic profile of an individual with EM phenotype can be changed to a pattern similar to that of an individual with PM phenotyp...