Absorption, Distribution, Metabolism, and Excretion of [14C]-Sotorasib in Rats and Dogs: Interspecies Differences in Absorption, Protein Conjugation and Metabolism.

Sotorasib is a first-in-class, targeted covalent inhibitor of Kirsten rat sarcoma viral oncogene homolog (KRAS)G12C approved by the FDA to treat patients with locally advanced or metastatic non-small cell lung cancer with the KRASG12C mutation. The mass balance, excretion, and metabolism of [14C]-sotorasib was characterized in rats and dogs after a single dose of 60 or 500 mg/kg, respectively. Mean recovery was >90% for both species. Excretion of unchanged sotorasib was a minor pathway in rats, accounting for <4% of administered dose in urine and <7% of administered dose in feces. Approximately 66% of administered dose was recovered in the bile from bile duct cannulated rats as metabolites. Excretion of unchanged sotorasib was the major excretion pathway in dogs, likely caused by solubility-limited absorption. Major pathways of sotorasib biotransformation included glutathione conjugation and oxidative metabolism. In vitro experiments demonstrated that nonenzymatic conjugation (Michael addition) was the primary mechanism of the reaction with glutathione. Extended radioactivity profiles in blood and plasma were observed in rats, but not dogs, after dosing with [14C]-sotorasib. In vitro experiments demonstrated that sotorasib-protein adducts were observed with both rat hemoglobin and serum albumin, explaining the extended radioactivity profile. SIGNIFICANCE STATEMENT: This study characterized the mass balance, excretion, and metabolism of [14C]-sotorasib, a covalent Kirsten rat sarcoma viral oncogene homolog G12C inhibitor, in rats and dogs. Rapid absorption and extensive metabolism of sotorasib was observed in rats, while sotorasib was primarily excreted unchanged in dog feces, likely due to solubility-limited absorption. Protein adducts with rat hemoglobin and serum albumin were characterized, explaining observed extended blood and plasma radioactivity profiles. The primary biotransformation pathway, glutathione conjugation, was mediated through nonenzymatic conjugation.

Strain-Dependent Variability of Early Discovery Small Molecule Pharmacokinetics in Mice: Does Strain Matter?

Drug discovery programs routinely perform pharmacokinetic (PK) studies in mice to prioritize lead compounds based on anticipated exposure-efficacy and exposure-toxicity relationships. Because of logistical and/or technical issues, the strain of mouse in early discovery PK studies may not always match the strain in toxicity or efficacy studies. This elicits the question do appreciable strain-dependent differences in PK parameters exist to an extent that would warrant conducting PK studies in a strain that matches efficacy and toxicity models? To understand the impact that strain may have on PK parameters, we selected eight marketed drugs with well characterized absorption, distribution, metabolism, and excretion properties and diverse structures to perform PK studies in three common mouse strains (Bagg Albino c, C57BL/6, and CD-1). Some statistical strain-dependent differences were observed; however, we found good general agreement of PK parameters between strains: 88%, 100%, 75%, 76%, 94%, and 88% of compounds were within twofold across strains for clearance, volume of distribution at steady state, t 1/2, C max, T max, and oral bioavailability, respectively. Overall, we recommend that an approach using a single strain of mouse is appropriate for discovery screening PK studies, provided that proper caution is exercised. SIGNIFICANCE STATEMENT: The mouse strain in discovery pharmacokinetic (PK) studies may not match the strain in efficacy and toxicology studies. Currently, there is a gap in the literature addressing whether differences in PK parameters across mouse strains exist such that multiple PK studies are warranted. The results from this study indicated that the PK properties of clinically used drugs between mouse strains are within an acceptable range such that single strain PK is appropriate.

Meta-analysis of hepatic cytochrome P450 ontogeny to underwrite the prediction of pediatric pharmacokinetics using physiologically based pharmacokinetic modeling.

The accurate prediction of pharmacokinetics (PK) is fundamental to underwriting safety and efficacy in pediatric clinical trials; age-dependent PK may be observed with pediatrics because of the growth and maturation processes that occur during development. Understanding the ontogeny of drug-metabolizing enzymes is a critical enabler for pediatric PK prediction, as enzyme expression or activity may change with age. Although ontogeny functions for the cytochrome P450s (CYPs) have been developed, disconnects between ontogeny functions for the same CYP may exist, depending on whether the functions were derived from in vitro or in vivo data. This report describes the development of ontogeny functions for all the major hepatic CYPs based on in vitro or in vivo data; these ontogeny functions were subsequently incorporated into a physiologically based pharmacokinetic model and evaluated. Pediatric PK predictions based on in vivo-derived ontogeny functions performed markedly better than those developed from in vitro data for intravenous (100% versus 51% within 2-fold, respectively) and oral (98% versus 67%, respectively) dosing. The verified models were then applied to complex pediatric scenarios involving active metabolites, CYP polymorphisms and physiological changes because of critical illness; the models reasonably explained the observed age-dependent changes in pediatric PK.

Predicting the drug interaction potential of AMG 853, a dual antagonist of the D-prostanoid and chemoattractant receptor-homologous molecule expressed on T helper 2 cells receptors.

2-(4-(4-(tert-Butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenylsulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid (AMG 853) is an orally bioavailable and potent dual antagonist of the D-prostanoid and chemoattractant receptor-homologous molecule expressed on T helper 2 cells receptors. The drug interaction potential of AMG 853, both as a victim and a perpetrator, was investigated using in vitro, in silico, and in vivo methodologies. Experiments in human liver microsomes (HLM) and recombinant enzymes identified CYP2C8, CYP2J2, and CYP3A as well as multiple UDP-glucuronosyltransferase isoforms as being responsible for the metabolic clearance of AMG 853. With use of HLM and selective probe substrates, both AMG 853 and its acyl glucuronide metabolite (M1) were shown to be inhibitors of CYP2C8. AMG 853 and M1 did not inhibit any of the other cytochrome P450 isoforms tested, and AMG 853 exhibited minimal enzyme induction properties in human hepatocytes cultures. In light of the in vitro findings, modeling and simulation approaches were used to examine the potential for ketoconazole (a CYP3A inhibitor) to inhibit the metabolism of AMG 853 as well as for AMG 853 to inhibit the metabolism of paclitaxel, rosiglitazone, and montelukast, commonly used substrates of CYP2C8. A weak and clinically insignificant drug interaction (area under the drug concentration-time curve (AUC)(i)/AUC <2) was predicted between ketoconazole and AMG 853. No drug interactions were predicted for AMG 853 and paclitaxel, rosiglitazone, or montelukast. Finally, administration of AMG 853 to healthy human subjects in clinical trials in the presence or absence of ketoconazole confirmed that AMG 853 is unlikely to be involved in clinically significant drug interactions.

Ligand-based design of a potent and selective inhibitor of cytochrome P450 2C19.

A series of omeprazole-based analogues was synthesized and assessed for inhibitory activity against CYP2C19. The data was used to build a CYP2C19 inhibition pharmacophore model for the series. The model was employed to design additional analogues with inhibitory potency against CYP2C19. Upon identifying inhibitors of CYP2C19, ligand-based design shifted to attenuating the rapid clearance observed for many of the inhibitors. While most analogues underwent metabolism on their aliphatic side chain, metabolite identification indicated that for analogues such as compound 30 which contain a heterocycle adjacent to the sulfur moiety, metabolism primarily occurred on the benzimidazole moiety. Compound 30 exhibited improved metabolic stability (Cl(int) = 12.4 mL/min/nmol) and was selective in regard to inhibition of CYP2C19-catalyzed (S)-mephenytoin hydroxylation in human liver microsomes. Finally, representative compounds were docked into a homology model of CYP2C19 in an effort to understand the enzyme-ligand interactions that may lead to favorable inhibition or metabolism properties.

Prediction of CYP2D6 drug interactions from in vitro data: evidence for substrate-dependent inhibition.

Predicting the magnitude of potential drug-drug interactions is important for underwriting patient safety in the clinical setting. Substrate-dependent inhibition of cytochrome P450 enzymes may confound extrapolation of in vitro results to the in vivo situation. However, the potential for substrate-dependent inhibition with CYP2D6 has not been well characterized. The inhibition profiles of 20 known inhibitors of CYP2D6 were characterized in vitro against four clinically relevant CYP2D6 substrates (desipramine, dextromethorphan, metoprolol, and thioridazine) and bufuralol. Dextromethorphan exhibited the highest sensitivity to in vitro inhibition, whereas metoprolol was the least sensitive. In addition, when metoprolol was the substrate, inhibitors with structurally constrained amino moieties (clozapine, debrisoquine, harmine, quinidine, and yohimbine) exhibited at least a 5-fold decrease in inhibition potency when results were compared with those for dextromethorphan. Atypical inhibition kinetics were observed for these and other inhibitor-substrate pairings. In silico docking studies suggested that interactions with Glu216 and an adjacent hydrophobic binding pocket may influence substrate sensitivity and inhibition potency for CYP2D6. The in vivo sensitivities of the clinically relevant CYP2D6 substrates desipramine, dextromethorphan, and metoprolol were determined on the basis of literature drug-drug interaction (DDI) outcomes. Similar to the in vitro results, dextromethorphan exhibited the highest sensitivity to CYP2D6 inhibition in vivo. Finally, the magnitude of in vivo CYP2D6 DDIs caused by quinidine was predicted using desipramine, dextromethorphan, and metoprolol. Comparisons of the predictions with literature results indicated that the marked decrease in inhibition potency observed for the metoprolol-quinidine interaction in vitro translated to the in vivo situation.

Evaluation of CYP2C8 inhibition in vitro: utility of montelukast as a selective CYP2C8 probe substrate.

Understanding the potential for cytochrome P450 (P450)-mediated drug-drug interactions is a critical step in the drug discovery process. Although in vitro studies with CYP3A4, CYP2C9, and CYP2C19 have suggested the presence of multiple binding regions within the P450 active site based on probe substrate-dependent inhibition profiles, similar studies have not been performed with CYP2C8. The ability to understand CYP2C8 probe substrate sensitivity will enable appropriate in vitro and in vivo probe selection. To characterize the potential for probe substrate-dependent inhibition with CYP2C8, the inhibition potency of 22 known inhibitors of CYP2C8 were measured in vitro using four clinically relevant CYP2C8 probe substrates (montelukast, paclitaxel, repaglinide, and rosiglitazone) and amodiaquine. Repaglinide exhibited the highest sensitivity to inhibition in vitro. In vitro phenotyping indicated that montelukast is an appropriate probe for CYP2C8 inhibition studies. The in vivo sensitivities of the CYP2C8 probe substrates cerivastatin, fluvastatin, montelukast, pioglitazone, and rosiglitazone were determined in relation to repaglinide on the basis of clinical drug-drug interaction (DDI) data. Repaglinide exhibited the highest sensitivity in vivo, followed by cerivastatin, montelukast, and pioglitazone. Finally, the magnitude of in vivo CYP2C8 DDI caused by gemfibrozil-1-O-ß-glucuronide was predicted. Comparisons of the predictions with clinical data coupled with the potential liabilities of other CYP2C8 probes suggest that montelukast is an appropriate CYP2C8 probe substrate to use for the in vivo situation.

Mechanism-based inactivation of cytochrome P450 3A4 by mibefradil through heme destruction.

Mibefradil (Posicor) was developed as a calcium channel blocker for the treatment of chronic hypertension. The compound was withdrawn from the market in 1998 because of the potential for rhabdomyolysis, renal failure, or bradycardia when it was coadministered with other drugs. Mibefradil has previously been shown to be a potent reversible (IC(50) = 0.3-2 µM) and mechanism-based (K(i) = 2.3 µM; k(inact) = 0.4 min(-1)) inhibitor of CYP3A4-catalyzed statin metabolism. At present, the mechanism of CYP3A4 inactivation by mibefradil is not known. Mechanism-based inactivation experiments and spectral studies were used to examine the mechanism of CYP3A4 inactivation by mibefradil and its major metabolite, des-methoxyacetyl mibefradil (Ro 40-5966), in vitro. Both mibefradil and Ro 40-5966 were shown to exhibit type I binding characteristics (K(s) = 0.69 ± 0.06 and 1.39 ± 0.04 µM, respectively) toward CYP3A4. Complete K(i)/k(inact) experiments were performed, revealing a rapid and irreversible decrease in CYP3A4-catalyzed 1'-hydroxymidazolam formation. Approximately 70% of CYP3A4 activity was lost in the first minute of incubation with mibefradil, and inactivation was nonlinear after 2 min. Ro 40-5966 also resulted in time-dependent inhibition of CYP3A4, albeit to a lesser extent than mibefradil. The decrease in CYP3A4 activity in the presence of mibefradil and NADPH was subsequently shown to have a good correlation with the time-dependent loss of CO binding, which, coupled with the lack of stable heme and/or apoprotein adducts, suggests heme destruction as the mechanism of inactivation of CYP3A4 by mibefradil.

Selection of alternative CYP3A4 probe substrates for clinical drug interaction studies using in vitro data and in vivo simulation.

Understanding the potential for cytochrome P450-mediated drug-drug interactions (DDIs) is a critical step in the drug discovery process. DDIs of CYP3A4 are of particular importance because of the number of marketed drugs that are cleared by this enzyme. In response to studies that suggested the presence of several binding regions within the CYP3A4 active site, multiple probe substrates are often used for in vitro CYP3A4 DDI studies, including midazolam (the clinical standard), felodipine/nifedipine, and testosterone. However, the design of clinical CYP3A4 DDI studies may be confounded for cases such as 1-(2-hydroxy-2-methylpropyl)-N-[5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl]-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458), with which testosterone is predicted to exhibit a clinically relevant DDI whereas midazolam and felodipine/nifedipine are not. To develop an appropriate path forward for such clinical DDI studies, the inhibition potency of 20 known inhibitors of CYP3A4 were measured in vitro using 8 clinically relevant CYP3A4 probe substrates and testosterone. Hierarchical clustering suggested four probe substrate clusters: testosterone; felodipine; midazolam, buspirone, quinidine, and sildenafil; and simvastatin, budesonide, and fluticasone. The in vivo sensitivities of six clinically relevant CYP3A4 probe substrates (buspirone, cyclosporine, nifedipine, quinidine, sildenafil, and simvastatin) were determined in relation to midazolam from literature DDI data. Buspirone, sildenafil, and simvastatin exhibited similar or greater sensitivity than midazolam to CYP3A4 inhibition in vivo. Finally, Simcyp was used to predict the in vivo magnitude of CYP3A4 DDIs caused by AMG 458 using midazolam, sildenafil, simvastatin, and testosterone as probe substrates.

Application of cytochrome P450 drug interaction screening in drug discovery.

Advances in drug interaction screening have resulted in reduced compound attrition rates due to unfavorable CYP-mediated drug interactions in clinical trials and improved patient safety. A major driver for the success in predicting drug interactions is a better understanding of the biological, chemical or mechanical factors that can impact the prediction of drug interactions in vitro. The enzyme source, probe substrate, accessory proteins and pharmacogenetics can all have profound effects upon the robustness and relevance of data generated with in vitro drug-drug interaction assays. Furthermore, the use of in silico techniques can potentially afford a priori knowledge of drug interaction potential, thus reducing the time and cost associated with drug interaction screening. This review will focus on recent advances in in vitro, in silico and bioanalytical techniques and demonstrate how these tools are currently used to provide effective CYP drug interaction screening in a discovery setting.

In vitro inhibition of multiple cytochrome P450 isoforms by xanthone derivatives from mangosteen extract.

Mangosteen is a xanthone-containing fruit found in Southeast Asia for which health claims include maintaining healthy immune and gastrointestinal systems to slowing the progression of tumor growth and neurodegenerative diseases. Previous studies have identified multiple xanthones in the pericarp of the mangosteen fruit. The aim of the current study was to assess the drug inhibition potential of mangosteen in vitro as well as the cytochrome P450 (P450) enzymes responsible for the metabolism of its individual components. The various xanthone derivatives were found to be both substrates and inhibitors for multiple P450 isoforms. Aqueous extracts of the mangosteen pericarp were analyzed for xanthone content as well as inhibition potency. Finally, in vivo plasma concentrations of alpha-mangostin, the most abundant xanthone derivative found in mangosteen, were predicted using Simcyp and found to be well above their respective in vitro K(i) values for CYP2C8 and CYP2C9.

Prediction of CYP-mediated drug interactions in vivo using in vitro data.

Over the past 15 years, a concerted effort has been undertaken by the pharmaceutical industry to reduce the attrition of clinical candidates resulting from undesirable ADME characteristics. Increasing regulatory and competitive pressures demand that pharmaceutical products brought to the market possess pristine safety and drug co-administration profiles for most therapeutic areas. The high-profile withdrawal of drugs such as mibefradil from the market because of unfavorable drug-drug interaction profiles has focused efforts on screening for cytochrome P450 (CYP)-mediated drug interactions early in the discovery paradigm and on predicting the impact of inhibition on the in vivo situation. This paper discusses current practices used to screen for CYP-mediated drug-drug interactions in vitro (inhibition and induction) and how these data are being used to predict whether a clinically relevant drug-drug interaction is likely to occur in vivo.

Oral delivery of 1,3-dicyclohexylurea nanosuspension enhances exposure and lowers blood pressure in hypertensive rats.

Cytochrome P450-derived epoxyeicosatrienoic acids (EET) are biologically active metabolites of arachidonic acid that have potent effects on renal vascular reactivity and tubular ion transport and have been implicated in the control of blood pressure. EETs are hydrolyzed to their less active diols, dihydroxyeicosatrienoic acids (DHET), by the enzyme soluble epoxide hydrolase (sEH). 1,3-dicyclohexylurea (DCU), a potent sEH inhibitor, lowers systemic blood pressure in spontaneously hypertensive rats when dosed intraperitoneally. However, DCU has poor aqueous solubility, posing a challenge for in vivo oral delivery. To overcome this limitation, we formulated DCU in a nanosuspension using wet milling. Milling reduced particle size, increasing the total surface area by approximately 40-fold. In rats chronically infused with angiotensin II, the DCU nanosuspension administered orally twice daily for 4 days produced plasma exposures an order of magnitude greater than unmilled DCU and lowered blood pressure by nearly 30 mmHg. Consistent with the mechanism of sEH inhibition, DCU increased plasma 14,15-EET and decreased plasma 14,15-DHET levels. These data confirm the antihypertensive effect of sEH inhibition and demonstrate that greatly enhanced exposure of a low-solubility compound is achievable by oral delivery using a nanoparticle drug delivery system.

CYP2C19 inhibition: the impact of substrate probe selection on in vitro inhibition profiles.

Understanding the potential for cytochrome P450 (P450)-mediated drug-drug interactions is a critical part of the drug discovery process. Factors such as nonspecific binding, atypical kinetics, poor effector solubility, and varying ratios of accessory proteins may alter the kinetic behavior of an enzyme and subsequently confound the extrapolation of in vitro data to the human situation. The architecture of the P450 active site and the presence of multiple binding regions within the active site may also confound in vitro-in vivo extrapolation, as inhibition profiles may be dependent on a specific inhibitor-substrate interaction. In these studies, the inhibition profiles of a set of 24 inhibitors were paneled against the CYP2C19 substrate probes (S)-mephenytoin, (R)-omeprazole, (S)-omeprazole, and (S)-fluoxetine, on the basis of their inclusion in recent U.S. Food and Drug Administration guidance for in vitro drug-drug interactions with CYP2C19. (S)-Mephenytoin was inhibited an average of 5.6-fold more potently than (R)- or (S)-omeprazole and 9.2-fold more potently than (S)-fluoxetine. Hierarchical clustering of the inhibition data suggested three substrate probe groupings, with (S)-mephenytoin exhibiting the largest difference from the rest of the substrate probes, (S)-fluoxetine exhibiting less difference from (S)-mephenytoin and the omeprazoles and (R)- and (S)-omeprazole exhibiting minimal differences from each other. Predictions of in vivo inhibition potency based on the in vitro data suggest that most drug-drug interactions will be identified by either (S)-mephenytoin or omeprazole, although the expected magnitude of the interaction may vary depending on the chosen substrate probe.

Differential time-dependent inactivation of P450 3A4 and P450 3A5 by raloxifene: a key role for C239 in quenching reactive intermediates.

The role of C239 as the active-site residue responsible for forming the covalent linkage with raloxifene during P450 3A4 time-dependent inactivation (TDI) was recently identified. The corresponding residue in CYP3A5 is S239, and when the potential for TDI in P450 3A5 was investigated, only reversible inhibition was observed against midazolam and testosterone, with median inhibitory concentration (IC50) values of 2.4 and 2.9 microM, respectively. In a similar fashion, when C239 was replaced with alanine in P450 3A4, TDI was successfully engineered out, and the reversible inhibition was characterized by IC50 values of 3.7 and 3.5 microM against midazolam and testosterone, respectively. Metabolism studies confirmed that the reactive diquinone methide intermediate required for P450 3A4 inactivation formed in all of the P450 3A enzymes investigated. Furthermore, the absence of TDI in P450 3A5 led to an increase in the formation of GSH-related adducts of raloxifene compared with that for P450 3A4. Consequently, the absence of the nucleophilic cysteine leads to differential TDI and generation of reactive metabolites in the P450 3A enzyme, providing the foundation for pharmacogenetics that contributes to individual differences in susceptibility to adverse drug reactions.

The in vitro drug interaction potential of dietary supplements containing multiple herbal components.

Herbal-based remedies are widely used as alternative treatments for a number of ailments. In addition, the use of products that contain both single and multiple herbal constituents is becoming increasingly common. The work described in this report examined the in vitro drug interaction potential for a commonly used herbal cold remedy reported to contain a mixture of eight herbal components. Experiments conducted in human liver microsomes exhibited significant inhibition (<10% of control activity remaining) of multiple cytochrome P450 (P450) isoforms, including CYP2B6, CYP2C9, and CYP2D6, by the herbal mixture. In an attempt to explain the observed P450 inhibition by the herbal mixture, individual active components were obtained and tested for inhibitory potency. Inhibition of multiple P450 activities by a single constituent, luteolin, was observed. Conversely, inhibition of a single isoform by several herbal components was noted for CYP2B6. Based on the data presented, it is concluded that mixtures of herbal components may exhibit multiple modes of P450 inhibition, indicating the potential for complex herbal-drug interaction scenarios to occur.

CYP2C9 inhibition: impact of probe selection and pharmacogenetics on in vitro inhibition profiles.

Drug-drug interactions may cause serious adverse events in the clinical setting, and the cytochromes P450 are the enzyme system most often implicated in these interactions. Cytochrome P450 2C is the second most abundant subfamily of cytochrome P450 enzymes and is responsible for metabolism of almost 20% of currently marketed drugs. The most abundant isoform of this subfamily is CYP2C9, which is the major clearance pathway for the low therapeutic index drugs warfarin and phenytoin. Considering the importance of CYP2C9 to drug-drug interactions, the in vitro-in vivo extrapolation of drug-drug interactions for CYP2C9 may be confounded by the presence of polymorphic variants and the possibility of multiple binding regions within the CYP2C9 active site, leading to the potential for genotype- and substrate-dependent inhibition. To address the issues of genotype-dependent enzyme inhibition as well as probe substrate correlations, the inhibitory potency (Ki) of 28 effector molecules was assessed with five commonly used probes of CYP2C9 in both the CYP2C9.1 and CYP2C9.3 proteins. The inhibition of CYP2C9.1 and CYP2C9.3 by the battery of inhibitors with five substrate probes demonstrated differential inhibition potency not only between the two genotypes but also across substrate probes. Furthermore, the substrate probes fell into three distinct classes depending on genotype, suggesting that multiple probes may be needed to fully assess inhibition of CYP2C9 in vitro. Thus, both genotype and choice of probe substrate must be considered when attempting to predict potential CYP2C9 drug-drug interactions from in vitro data.

Enzyme source effects on CYP2C9 kinetics and inhibition.

When choosing a recombinant cytochrome P450 (P450) enzyme system for in vitro studies, it is critical to understand the strengths, limitations, and applicability of the enzyme system to the study design. Although literature kinetic data may be available to assist in enzyme system selection, comparison of data from separate laboratories is often confounded by differences in experimental conditions and bioanalytical techniques. We measured the Michaelis-Menten kinetic parameters for four CYP2C9 substrates (diclofenac, (S)-warfarin, tolbutamide, and (S)-flurbiprofen) using four recombinant CYP2C9 enzyme systems (Supersomes, Baculosomes, RECO system, and in-house purified, reconstituted enzyme) to determine whether the enzyme systems exhibited kinetic differences in metabolic product formation rates under uniform experimental conditions. The purified, reconstituted enzyme systems exhibited higher K(m) values, reduced substrate affinity, and lower calculated intrinsic clearance values compared with baculovirus microsomal preparations. Six- to 25-fold differences in predicted intrinsic clearance values were calculated for each substrate depending on the enzyme system-substrate combination. Results suggest that P450 reductase interactions with the CYP2C9 protein and varying ratios of CYP2C9/P450 reductase in the enzyme preparations may play a role in these observed differences. In addition, when (S)-flurbiprofen was used as a substrate probe to determine CYP2C9 inhibition with a set of 12 inhibitors, decreased inhibition potency was observed across 11 of those inhibitors in the RECO purified, reconstituted enzyme compared with the Supersomes baculovirus microsomal preparation and pooled human liver microsomes. Considering these differences, consistent use of an enzyme source is an important component in producing comparable and reproducible kinetics and inhibition data with CYP2C9.

Mechanism of inactivation of human cytochrome P450 2B6 by phencyclidine.

The mechanism behind the observed inactivation of human P450 2B6 by phencyclidine (PCP) has been evaluated over the past 2 decades. The scope of the current investigation was to contribute to the fundamental knowledge of PCP oxidation and perhaps the mechanism behind P450 inactivation. To study the chemistry of PCP oxidation, we subjected PCP to the Fenton reagent. Under Fenton chemistry conditions, oxidation on all three PCP rings was observed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). When PCP was incubated with the Fenton system in the presence of glutathione (GSH), three GSH-PCP conjugates were identified. Subsequent LC-MS/MS analysis of these conjugates revealed two species that had GSH attached to the cyclohexane ring of PCP and a third conjugate in which GSH was adducted to the piperidine ring. When PCP was incubated across a panel of P450 enzymes, several enzymes, including P450s 2D6 and 3A4, were able to catalyze the formation of the PCP iminium ion, whereas P450s 2B6 and 2C19 were exclusively able to hydroxylate secondary carbons on the cyclohexane ring of PCP. Subsequent mechanistic experiments revealed that only P450s 2B6 and 2C19 demonstrated loss of catalytic activity after preincubation with 10 microM PCP. Finally, investigation of P450 2B6 inactivation using structural analogs of PCP revealed that blocking the para-carbon atom on the cyclohexane ring of PCP from oxidation protected the P450 2B6 from inactivation, which suggests that a reactive intermediate generated during the hydroxylation of the cyclohexane ring may be linked to the mechanism of inactivation of P450 2B6 by PCP.

Influence of fluorescent probe size and cytochrome b5 on drug-drug interactions in CYP2C9.

7-Methoxy-4-trifluoromethylcoumarin (MFC) has been used extensively in high-throughput screens for the identification of potential CYP2C9 interactions. More recently, additional probes from Invitrogen have been used. Vivid 2C9 Green is the largest of the probes and has had limited prior characterization. The new series of probes differ significantly from MFC and were examined for their ability to identify interactions with 19 CYP2C9 substrates/inhibitors. The inhibition profiles depend largely on the physical differences between the fluorescent probe substrates. Cytochrome b5 (cyt b5) was also investigated for the ability to alter the inhibition profile of a given compound. The stoichiometric addition of cyt b5 caused an increase in V max of MFC and Vivid 2C9 Green 4.4 and 1.7 times, respectively. Furthermore, cyt b5 imposes a steric component to the active site as the inhibition profiles were significantly affected in incubations with MFC. The addition of cyt b5 had limited impact on the inhibition profiles generated with Vivid 2C9 Green. The K(m) of Vivid 2C9 Green increased from 1.2+/-0.2 micro M to 4.8+/-0.3 micro Mas a result of cyt b5 addition. These results illustrate that multiple substrate probes may be necessary for screening drug-drug interaction in CYP2C9 and that cyt b5 effects can impart steric restraints on the CYP2C9 active site.