Efficacy and Improved Resistance Potential of a Cofactor-Independent InhA Inhibitor of Mycobacterium tuberculosis in the C3HeB/FeJ Mouse Model.

AN12855 is a direct, cofactor-independent inhibitor of InhA in Mycobacterium tuberculosis In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.

Novel Mechanism of Decyanation of GDC-0425 by Cytochrome P450.

GDC-0425 [5-((1-ethylpiperidin-4-yl)oxy)-9H-pyrrolo[2,3-b:5,4-c']dipyridine-6-carbonitrile] is an orally bioavailable small-molecule inhibitor of checkpoint kinase 1 that was investigated as a novel cotherapy to potentiate chemotherapeutic drugs, such as gemcitabine. In a radiolabeled absorption, distribution, metabolism, and excretion study in Sprague-Dawley rats, trace-level but long-lived 14C-labeled thiocyanate was observed in circulation. This thiocyanate originated from metabolic decyanation of GDC-0425 and rapid conversion of cyanide to thiocyanate. Excretion studies indicated decyanation was a minor metabolic pathway, but placing 14C at nitrile magnified its observation. Cytochrome P450s catalyzed the oxidative decyanation reaction in vitro when tested with liver microsomes, and in the presence of 18O2, one atom of 18O was incorporated into the decyanated product. To translate this finding to a clinical risk assessment, the total circulating levels of thiocyanate (endogenous plus drug-derived) were measured following repeated administration of GDC-0425 to rats and cynomolgus monkeys. No overt increases were observed with thiocyanate concentrations of 121-154 µM in rats and 71-110 µM in monkeys receiving vehicle and all tested doses of GDC-0425. These findings were consistent with results from the radiolabel rat study where decyanation accounted for conversion of <1% of the administered GDC-0425 and contributed less than 1 µM thiocyanate to systemic levels. Further, in vitro studies showed only trace oxidative decyanation for humans. These data indicated that, although cyanide was metabolically released from GDC-0425 and formed low levels of thiocyanate, this pathway was a minor route of metabolism, and GDC-0425-related increases in systemic thiocyanate were unlikely to pose safety concerns for subjects of clinical studies.

Inhibitory Effects of Trapping Agents of Sulfur Drug Reactive Intermediates against Major Human Cytochrome P450 Isoforms.

In some cases, the formation of reactive species from the metabolism of xenobiotics has been linked to toxicity and therefore it is imperative to detect potential bioactivation for candidate drugs during drug discovery. Reactive species can covalently bind to trapping agents in in vitro incubations of compound with human liver microsomes (HLM) fortified with ß-nicotinamide adenine dinucleotide phosphate (NADPH), resulting in a stable conjugate of trapping agent and reactive species, thereby facilitating analytical detection and providing evidence of short-lived reactive metabolites. Since reactive metabolites are typically generated by cytochrome P450 (CYP) oxidation, it is important to ensure high concentrations of trapping agents are not inhibiting the activities of CYP isoforms. Here we assessed the inhibitory properties of fourteen trapping agents against the major human CYP isoforms (CYP1A2, 2C9, 2C19, 2D6 and 3A). Based on our findings, eleven trapping agents displayed inhibition, three of which had IC50 values less than 1 mM (2-mercaptoethanol, N-methylmaleimide and N-ethylmaleimide (NEM)). Three trapping agents (dimedone, N-acetyl-lysine and arsenite) did not inhibit CYP isoforms at concentrations tested. To illustrate effects of CYP inhibition by trapping agents on reactive intermediate trapping, an example drug (ticlopidine) and trapping agent (NEM) were chosen for further studies. For the same amount of ticlopidine (1 µM), increasing concentrations of the trapping agent NEM (0.007-40 mM) resulted in a bell-shaped response curve of NEM-trapped ticlopidine S-oxide (TSO-NEM), due to CYP inhibition by NEM. Thus, trapping studies should be designed to include several concentrations of trapping agent to ensure optimal trapping of reactive metabolites.

Going Beyond Common Drug Metabolizing Enzymes: Case Studies of Biotransformation Involving Aldehyde Oxidase, γ-Glutamyl Transpeptidase, Cathepsin B, Flavin-Containing Monooxygenase, and ADP-Ribosyltransferase.

The significant roles that cytochrome P450 (P450) and UDP-glucuronosyl transferase (UGT) enzymes play in drug discovery cannot be ignored, and these enzyme systems are commonly examined during drug optimization using liver microsomes or hepatocytes. At the same time, other drug-metabolizing enzymes have a role in the metabolism of drugs and can lead to challenges in drug optimization that could be mitigated if the contributions of these enzymes were better understood. We present examples (mostly from Genentech) of five different non-P450 and non-UGT enzymes that contribute to the metabolic clearance or bioactivation of drugs and drug candidates. Aldehyde oxidase mediates a unique amide hydrolysis of GDC-0834 (N-[3-[6-[4-[(2R)-1,4-dimethyl-3-oxopiperazin-2-yl]anilino]-4-methyl-5-oxopyrazin-2-yl]-2-methylphenyl]-4,5,6,7-tetrahydro-1-benzothiophene-2-carboxamide), leading to high clearance of the drug. Likewise, the rodent-specific ribose conjugation by ADP-ribosyltransferase leads to high clearance of an interleukin-2-inducible T-cell kinase inhibitor. Metabolic reactions by flavin-containing monooxygenases (FMO) are easily mistaken for P450-mediated metabolism such as oxidative defluorination of 4-fluoro-N-methylaniline by FMO. Gamma-glutamyl transpeptidase is involved in the initial hydrolysis of glutathione metabolites, leading to formation of proximate toxins and nephrotoxicity, as is observed with cisplatin in the clinic, or renal toxicity, as is observed with efavirenz in rodents. Finally, cathepsin B is a lysosomal enzyme that is highly expressed in human tumors and has been targeted to release potent cytotoxins, as in the case of brentuximab vedotin. These examples of non-P450- and non-UGT-mediated metabolism show that a more complete understanding of drug metabolizing enzymes allows for better insight into the fate of drugs and improved design strategies of molecules in drug discovery.

Absorption, Metabolism, Excretion, and the Contribution of Intestinal Metabolism to the Oral Disposition of [14C]Cobimetinib, a MEK Inhibitor, in Humans.

The pharmacokinetics, metabolism, and excretion of cobimetinib, a MEK inhibitor, were characterized in healthy male subjects (n = 6) following a single 20 mg (200 µCi) oral dose. Unchanged cobimetinib and M16 (glycine conjugate of hydrolyzed cobimetinib) were the major circulating species, accounting for 20.5% and 18.3% of the drug-related material in plasma up to 48 hours postdose, respectively. Other circulating metabolites were minor, accounting for less than 10% of drug-related material in plasma. The total recovery of the administered radioactivity was 94.3% (±1.6%, S.D.) with 76.5% (±2.3%) in feces and 17.8% (±2.5%) in urine. Metabolite profiling indicated that cobimetinib had been extensively metabolized with only 1.6% and 6.6% of the dose remaining as unchanged drug in urine and feces, respectively. In vitro phenotyping experiments indicated that CYP3A4 was predominantly responsible for metabolizing cobimetinib. From this study, we concluded that cobimetinib had been well absorbed (fraction absorbed, Fa = 0.88). Given this good absorption and the previously determined low hepatic clearance, the systemic exposures were lower than expected (bioavailability, F = 0.28). We hypothesized that intestinal metabolism had strongly attenuated the oral bioavailability of cobimetinib. Supporting this hypothesis, the fraction escaping gut wall elimination (Fg) was estimated to be 0.37 based on F and Fa from this study and the fraction escaping hepatic elimination (Fh) from the absolute bioavailability study (F = Fa × Fh × Fg). Physiologically based pharmacokinetics modeling also showed that intestinal clearance had to be included to adequately describe the oral profile. These collective data suggested that cobimetinib was well absorbed following oral administration and extensively metabolized with intestinal first-pass metabolism contributing to its disposition.

Computational predictions of the site of metabolism of cytochrome P450 2D6 substrates: comparative analysis, molecular docking, bioactivation and toxicological implications.

Cytochrome P450 2D6 (CYP2D6) is a polymorphic enzyme responsible for metabolizing approximately 25% of all drugs. CYP2D6 is highly expressed in the brain and plays a role as the major CYP in the metabolism of numerous brain-penetrant drugs, including antipsychotics and antidepressants. CYP2D6 activity and inhibition have been associated with numerous undesirable effects in patients, such as bioactivation, drug-associated suicidality and prolongation of the QTc interval. Several in silico tools have been developed in recent years to assist safety assessment scientists in predicting the structural identity of CYP2D6-derived metabolites. The first goal of this study was to perform a comparative evaluation on the ability of four commonly used in silico tools (MetaSite, StarDrop, SMARTCyp and RS-WebPredictor) to correctly predict the CYP2D6-derived site of metabolism (SOM) for 141 compounds, including 10 derived from the Genentech small molecule library. The second goal was to evaluate if a bioactivation prediction model, based on an indicator of chemical reactivity (ELUMO-EHOMO) and electrostatic potential, could correctly predict five representative compounds known to be bioactivated by CYP2D6. Such a model would be of great utility in safety assessment since unforeseen toxicities of CYP2D6 substrates may in part be due to bioactivation mechanisms. The third and final goal was to investigate whether molecular docking, using the crystal structure of human CYP2D6, had the potential to compliment or improve the results obtained from the four SOM in silico programs.

A novel reaction mediated by human aldehyde oxidase: amide hydrolysis of GDC-0834.

GDC-0834, a Bruton's tyrosine kinase inhibitor investigated as a potential treatment of rheumatoid arthritis, was previously reported to be extensively metabolized by amide hydrolysis such that no measurable levels of this compound were detected in human circulation after oral administration. In vitro studies in human liver cytosol determined that GDC-0834 (R)-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo- 4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b] thiophene-2-carboxamide) was rapidly hydrolyzed with a CLint of 0.511 ml/min per milligram of protein. Aldehyde oxidase (AO) and carboxylesterase (CES) were putatively identified as the enzymes responsible after cytosolic fractionation and mass spectrometry-proteomics analysis of the enzymatically active fractions. Results were confirmed by a series of kinetic experiments with inhibitors of AO, CES, and xanthine oxidase (XO), which implicated AO and CES, but not XO, as mediating GDC-0834 amide hydrolysis. Further supporting the interaction between GDC-0834 and AO, GDC-0834 was shown to be a potent reversible inhibitor of six known AO substrates with IC50 values ranging from 0.86 to 1.87 µM. Additionally, in silico modeling studies suggest that GDC-0834 is capable of binding in the active site of AO with the amide bond of GDC-0834 near the molybdenum cofactor (MoCo), orientated in such a way to enable potential nucleophilic attack on the carbonyl of the amide bond by the hydroxyl of MoCo. Together, the in vitro and in silico results suggest the involvement of AO in the amide hydrolysis of GDC-0834.

1-Aminobenzotriazole coincubated with (S)-warfarin results in potent inactivation of CYP2C9.

1-Aminobenzotriazole (ABT) is a nonselective, mechanism-based inactivator of cytochrome P450 (P450) and a useful tool compound to discern P450- from non-P450-mediated metabolism. ABT effectively inactivates major human P450 isoforms, with the notable exception of CYP2C9. Here we propose that ABT preferentially binds to the warfarin-binding pocket in the CYP2C9 active-site cavity; thus, ABT bioactivation and subsequent inactivation is not favored. Therefore, coincubation with (S)-warfarin would result in displacement of ABT from the warfarin-binding pocket and subsequent binding to the active site, converting ABT into a potent inactivator of CYP2C9. To test this hypothesis, in vitro studies were conducted using various coincubation combinations of ABT and (S)-warfarin or diclofenac to modulate the effectiveness of CYP2C9 inactivation by ABT. Coincubation of ABT with (S)-warfarin (diclofenac probe substrate) resulted in potent inactivation, whereas weak inactivation was observed following coincubation of ABT with diclofenac [(S)-warfarin probe substrate]. The kinetic parameters of time-dependent inhibition of ABT for CYP2C9 in the absence and presence of (S)-warfarin (20 µM) were 0.0826 and 0.273 min(-1) for kinact and 3.49 and 0.157 mM for KI, respectively. In addition, a 73.4-fold shift was observed in the in vitro potency (kinact/KI ratio), with an increase from 23.7 ml/min/mmol (ABT alone) to 1740 ml/min/mmol [ABT with (S)-warfarin (20 µM)]. These findings were supported by in silico structural modeling, which showed ABT preferentially binding to the warfarin-binding pocket and the displacement of ABT to the active site in the presence of (S)-warfarin.

Discovery of the 1,7-diazacarbazole class of inhibitors of checkpoint kinase 1.

Checkpoint kinase 1 (ChK1) is activated in response to DNA damage, acting to temporarily block cell cycle progression and allow for DNA repair. It is envisaged that inhibition of ChK1 will sensitize tumor cells to treatment with DNA-damaging therapies, and may enhance the therapeutic window. High throughput screening identified carboxylate-containing diarylpyrazines as a prominent hit series, but with limited biochemical potency and no cellular activity. Through a series of SAR investigations and X-ray crystallographic analysis the critical role of polar contacts with conserved waters in the kinase back pocket was established. Structure-based design, guided by in silico modeling, transformed the series to better satisfy these contacts and the novel 1,7-diazacarbazole class of inhibitors was discovered. Here we present the genesis of this novel series and the identification of GNE-783, a potent, selective and orally bioavailable inhibitor of ChK1.

Learning and confirming with preclinical studies: modeling and simulation in the discovery of GDC-0917, an inhibitor of apoptosis proteins antagonist.

The application of modeling and simulation techniques is increasingly common in the preclinical stages of the drug development process. GDC-0917 [(S)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-N-(2-(oxazol-2-yl)-4-phenylthiazol-5-yl)pyrrolidine-2-carboxamide] is a potent second-generation antagonist of inhibitor of apoptosis (IAP) proteins that is being developed for the treatment of various cancers. GDC-0917 has low to moderate clearance in the mouse (12.0 ml/min/kg), rat (27.0 ml/min/kg), and dog (15.3 ml/min/kg), and high clearance in the monkey (67.6 ml/min/kg). Accordingly, oral bioavailability was lowest in monkeys compared with other species. Based on our experience with a prototype molecule with similar structure, in vitro-in vivo extrapolation was used to predict a moderate clearance (11.5 ml/min/kg) in humans. The predicted human volume of distribution was estimated using simple allometry at 6.69 l/kg. Translational pharmacokinetic-pharmacodynamic (PK-PD) analysis using results from MDA-MB-231-X1.1 breast cancer xenograft studies and predicted human pharmacokinetics suggests that ED50 and ED90 targets can be achieved in humans using acceptable doses (72 mg and 660 mg, respectively) and under an acceptable time frame. The relationship between GDC-0917 concentrations and pharmacodynamic response (cIAP1 degradation) was characterized using an in vitro peripheral blood mononuclear cell immunoassay. Simulations of human GDC-0917 plasma concentration-time profile and cIAP1 degradation at the 5-mg starting dose in the phase 1 clinical trial agreed well with observations. This work shows the importance of leveraging information from prototype molecules and illustrates how modeling and simulation can be used to add value to preclinical studies in the early stages of the drug development process.

On the maintenance of hepatocyte intracellular pH 7.0 in the in-vitro metabolic stability assay.

The account of pH difference between hepatocytes (intracellular pH 7.0) and extracellular water (pH 7.4) leads to the novel equation for hepatic clearance (Berezhkovskiy, J Pharma Sci 100:1167-1683, 2011). The metabolic stability assay using hepatocytes is commonly performed in the incubation buffer of pH 7.4. If hepatocytes retain their physiological pH 7.0 in these conditions, then the assay would mimic the in vivo condition, that is pH 7.4 for plasma and extracellular water, and pH 7.0 in hepatocytes. In this case the rate of drug elimination, taken as proportional to unbound drug concentration in buffer, would correspond to the in vivo rate of drug elimination as proportional to the unbound drug concentration in the extracellular water. Consequently the commonly used PBPK equation for the rate of hepatic elimination, and the equation for hepatic clearance would be valid. However, the experiment designed to determine hepatocyte internal pH indicated that it was not maintained in the in vitro stability assay, so that hepatocytes acquire the same pH as the incubation buffer. Thus, the novel equations for hepatic clearance (that include an ionization factor) should be applied regardless if the intrinsic clearance was obtained either from microsomal or hepatocyte stability assay.

GTP1 metabolic stability assessment: A study of the tau PET tracer [18F]GTP1.

Tau PET imaging using the tau specific PET tracer [18F]GTP1 has been and is part of therapeutic trials in Alzheimer's disease to monitor the accumulation of tau aggregates in the brain. Herein, we examined the metabolic processes of GTP1 and assessed the influence of smoking on its metabolism through in vitro assays. The tracer metabolic profile was assessed by incubating GTP1 with human liver microsomes (HLM) and human hepatocytes. Since smoking strongly stimulates the CYP1A2 enzyme activity, we incubated GTP1 with recombinant CYP1A2 to evaluate the role of the enzyme in tracer metabolism. It was found that GTP1 could form up to eleven oxidative metabolites with higher polarity than the parent. Only a small amount (2.6 % at 60 min) of a defluorinated metabolite was detected in HLM and human hepatocytes incubations highlighting the stability of GTP1 with respect to enzymatic defluorination. Moreover, the major GTP1 metabolites were not the product of CYP1A2 activity suggesting that smoking may not impact in vivo tracer metabolism and subsequently GTP1 brain kinetics.

Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates.

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease.

Significant species difference in amide hydrolysis of GDC-0834, a novel potent and selective Bruton's tyrosine kinase inhibitor.

(R)-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide (GDC-0834) is a potent and selective inhibitor of Bruton's tyrosine kinase (BTK), investigated as a potential treatment for rheumatoid arthritis. In vitro metabolite identification studies in hepatocytes revealed predominant formation of an inactive metabolite (M1) via amide hydrolysis in human. The formation of M1 appeared to be NADPH-independent in human liver microsomes. M1 was found in only minor to moderate quantities in plasma from preclinical species dosed with GDC-0834. Human clearance predictions using various methodologies resulted in estimates ranging from low to high. In addition, GDC-0834 exhibited low clearance in PXB chimeric mice with humanized liver. Uncertainty in human pharmacokinetic prediction and high interest in a BTK inhibitor for clinical evaluation prompted an investigational new drug strategy, in which GDC-0834 was rapidly advanced to a single-dose human clinical trial. GDC-0834 plasma concentrations in humans were below the limit of quantitation (<1 ng/ml) in most samples from the cohorts dosed orally at 35 and 105 mg. In contrast, substantial plasma concentrations of M1 were observed. In human plasma and urine, only M1 and its sequential metabolites were identified. The formation kinetics of M1 was evaluated in rat, dog, monkey, and human liver microsomes in the absence of NADPH. The maximum rate of M1 formation (V(max)) was substantially higher in human compared with that in other species. In contrast, the Michaelis-Menten constant (K(m)) was comparable among species. Intrinsic clearance (V(max)/K(m)) of GDC-0834 from M1 formation in human was 23- to 169-fold higher than observed in rat, dog, and monkey.

Novel mechanism for dehalogenation and glutathione conjugation of dihalogenated anilines in human liver microsomes: evidence for ipso glutathione addition.

The objective of the present study was to investigate the influence of halogen position on the formation of reactive metabolites from dihalogenated anilines. Herein we report on a proposed mechanism for dehalogenation and glutathione (GSH) conjugation of a series of ortho-, meta-, and para-dihalogenated anilines observed in human liver microsomes. Of particular interest were conjugates formed in which one of the halogens on the aniline was replaced by GSH. We present evidence that a (4-iminocyclohexa-2,5-dienylidene)halogenium reactive intermediate (QX) was formed after oxidation, followed by ipso addition of GSH at the imine moiety. The ipso GSH thiol attacks at the ortho-carbon and eventually leads to a loss of a halogen and GSH replacement. The initial step of GSH addition at the ipso position is also supported by density functional theory, which suggests that the ipso carbon of the chloro, bromo, and iodo (but not fluoro) containing 2-fluoro-4-haloanilines is the most positive carbon and that these molecules have the favorable highest occupied molecular orbital of the aniline and the lowest unoccupied orbital from GSH. The para-substituted halogen (chloro, bromo, or iodo but not fluoro) played a pivotal role in the formation of the QX, which required a delocalization of the positive charge on the para-halogen after oxidation. This mechanism was supported by structure-metabolism relationship analysis of a series of dihalogenated and monohalogenated aniline analogues.

Preclinical pharmacokinetics of the novel PI3K inhibitor GDC-0941 and prediction of its pharmacokinetics and efficacy in human.

The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation is associated with the development of many cancers. GDC-0941, a potent and selective inhibitor of PI3K, was characterised preclinically in in vitro and in vivo studies. Plasma protein binding was extensive, with free fraction less than 7%, and blood-to-plasma ratio ranged from 0.6 to 1.2 among the species tested. GDC-0941 human hepatic clearance was predicted to be moderate by liver microsomal incubations. GDC-0941 had high permeability in Madin-Darby canine kidney cells. The clearance of GDC-0941 was high in mouse (63.7 mL/min/kg), rat (49.3 mL/min/kg) and cynomolgus monkey (58.6 mL/min/kg), and moderate in dog (11.9 mL/min/kg). The volume of distribution ranged from 2.52 L/kg in rat to 2.94 L/kg in monkey. Oral bioavailability ranged from 18.6% in monkey to 77.9% in mouse. Predicted human clearance and volume of distribution using allometry were 6 mL/min/kg and 2.9 L/kg, respectively. The human efficacious doses were predicted based on results from preclinical pharmacokinetic studies and xenograft models. GDC-0941 preclinical characterisation and predictions of its properties in human supported its progression towards clinical development. GDC-0941 is currently in phase II clinical trials.

Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity.

The majority of marketed small-molecule drugs undergo metabolism by hepatic Cytochrome P450 (CYP) enzymes (Rendic 2002). Since these enzymes metabolize a structurally diverse number of drugs, metabolism-based drug-drug interactions (DDIs) can potentially occur when multiple drugs are coadministered to patients. Thus, a careful in vitro assessment of the contribution of various CYP isoforms to the total metabolism is important for predicting whether such DDIs might take place. One method of CYP phenotyping involves the use of potent and selective chemical inhibitors in human liver microsomal incubations in the presence of a test compound. The selectivity of such inhibitors plays a critical role in deciphering the involvement of specific CYP isoforms. Here, we review published data on the potency and selectivity of chemical inhibitors of the major human hepatic CYP isoforms. The most selective inhibitors available are furafylline (in co-incubation and pre-incubation conditions) for CYP1A2, 2-phenyl-2-(1-piperidinyl)propane (PPP) for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, (-)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine for CYP2D6. As for CYP2A6, tranylcypromine is the most widely used inhibitor, but on the basis of initial studies, either 3-(pyridin-3-yl)-1H-pyrazol-5-yl)methanamine (PPM) and 3-(2-methyl-1H-imidazol-1-yl)pyridine (MIP) can replace tranylcypromine as the most selective CYP2A6 inhibitor. For CYP3A4, ketoconazole is widely used in phenotyping studies, although azamulin is a far more selective CYP3A inhibitor. Most of the phenotyping studies do not include CYP2E1, mostly because of the limited number of new drug candidates that are metabolized by this enzyme. Among the inhibitors for this enzyme, 4-methylpyrazole appears to be selective.

Case Study 9: Probe-Dependent Binding Explains Lack of CYP2C9 Inactivation by 1-Aminobenzotriazole (ABT).

The potential for new chemical entities to inhibit the major cytochrome P450 (CYP) isoforms is routinely evaluated to minimize the risk of developing drugs with drug-drug interaction liabilities. CYP inhibition assays are routinely performed in a high-throughput format to efficiently screen large numbers of compounds. In evaluating a time-saving assay using diclofenac as the CYP2C9 probe substrate, a discrepancy was observed in which minimal inhibition was detected using diclofenac whereas using (S)-warfarin resulted in potent inhibition, supporting the presence of dual-binding sites in the relatively large CYP2C9 active site cavity.These observations provided further insights into explaining the reported ineffective inactivation of CYP2C9 for the pan-CYP inactivator 1-aminobenzotriazole (ABT). Mechanistic reversible and time-dependent inhibition experiments revealed that the ineffective CYP2C9 inactivation by ABT was also probe-dependent, with utilization of (S)-warfarin as the probe substrate resulting in more potent CYP2C9 inhibition by ABT compared to diclofenac. Addition of (S)-warfarin to the reversible and time-dependent inhibition experiments between ABT and diclofenac resulted in an attenuation of the inhibitory effects of ABT on CYP2C9-mediated diclofenac metabolism. Molecular docking studies further confirmed that (S)-warfarin and diclofenac preferentially bind in different regions of the CYP2C9 active site, with (S)-warfarin occupying a distal "warfarin-binding pocket" and diclofenac occupying a binding site close to the active heme moiety. ABT preferentially binds in the distal warfarin-binding pocket, supporting that diclofenac is minimally deterred from access to the CYP2C9 active site in the presence of ABT, thus resulting in minimal inactivation. Simultaneously docking of (S)-warfarin and ABT revealed that (S)-warfarin outcompetes ABT for the distal binding site and results in the binding of ABT to the CYP2C9 active site, supporting the observations of potent inactivation of CYP2C9 when (S)-warfarin is the probe substrate.These results highlight that probe selection is crucial when evaluating CYP inhibition potential, and it is recommended that multiple probes be utilized for CYP2C9, similar to the approach routinely employed for CYP3A4. Further, utilization of ABT as a pan-inhibitor of CYP activity for investigational compounds, both in vitro and in vivo, should be accompanied with the understanding that residual CYP-mediated oxidative metabolism could potentially be observed for CYP2C9 substrates and should not necessarily be attributed to non-P450-mediated metabolism.

Formation of a quinoneimine intermediate of 4-fluoro-N-methylaniline by FMO1: carbon oxidation plus defluorination.

Here, we report on the mechanism by which flavin-containing monooxygenase 1 (FMO1) mediates the formation of a reactive intermediate of 4-fluoro-N-methylaniline. FMO1 catalyzed a carbon oxidation reaction coupled with defluorination that led to the formation of 4-N-methylaminophenol, which was a reaction first reported by Boersma et al. (Boersma et al. (1993) Drug Metab. Dispos. 21 , 218 - 230). We propose that a labile 1-fluoro-4-(methylimino)cyclohexa-2,5-dienol intermediate was formed leading to an electrophilic quinoneimine intermediate. The identification of N-acetylcysteine adducts by LC-MS/MS and NMR further supports the formation of a quinoneimine intermediate. Incubations containing stable labeled oxygen (H(2)(18)O or (18)O(2)) and ab initio calculations were performed to support the proposed reaction mechanism.

Second generation 2-pyridyl biphenyl amide inhibitors of the hedgehog pathway.

Potent and efficacious inhibitors of the hedgehog pathway for the treatment of cancer have been prepared using the 2-pyridyl biphenyl amide scaffold common to the clinical lead GDC-0449. Analogs with polar groups in the para-position of the aryl amide ring optimized potency, had minimal CYP inhibition, and possessed good exposure in rats. Compounds 9d and 14f potently inhibited hedgehog signaling as measured by Gli1 mRNA and were found to be equivalent or more potent than GDC-0449, respectively, when studied in a Ptch(+/-) medulloblastoma allograft model, that is, highly dependent on hedgehog signaling.