The Effects of Drug Metabolizing Enzyme Inhibitors on Hepatic Efflux and Uptake Transporters.

BACKGROUND: Non-selective chemical inhibitors of phase I and phase II enzymes are commonly used in in vitro metabolic studies to elucidate the biotransformation pathways of drugs. However, the inhibition of the inhibitors on efflux and uptake transporters is not well investigated, potentially leading to unexpected and ambiguous results in these studies. OBJECTIVE: The commonly used metabolizing enzyme inhibitors, 1-aminobenzotriazole (ABT), SKF- 525A, pargyline, allopurinol, menadione, methimazole, piperine and raloxifene, were examined for their potential inhibition of the major hepatic ABC (ATP binding cassette) and SLC (solute carrier) transporters. METHODS: Different concentrations of the metabolizing enzyme inhibitors were used to study their effects on ABC and SLC transporters expressed in MDR1-MDCKI, Bcrp1-MDCKII, OATP1B1-HEK, OATP1B3-HEK, OCT1-HEK, OCT3-HEK cells and MRP2 vesicles. RESULTS: ABT, allopurinol and methimazole had no inhibitory effects on MDR1, Bcrp1, MRP2 or on OATP1B1, OATP1B3, OCT1 or OCT3. Pargyline did not inhibit OATP1B1 or OATP1B3, but weakly inhibited OCT1 and OCT3. In contrast, SKF-525A showed inhibition of not only MDR1, Bcrp1 and MRP2 but also OATP1B1, OATP1B3 and OCT1. Menadione and raloxifene weakly inhibited Bcrp1, but the inhibition of raloxifene on MDR1 was as potent as on the xanthine oxidase pterin oxidation. Piperine showed inhibition of MDR1, Bcrp1, OATP1B1, OCT1 and OCT3. CONCLUSION: ABT, pargyline, allopurinol and methimazole have no inhibitory effects on the studied ABC and SLC transporters, suggesting the inhibitors are unlikely to cause confounding inhibition of transporters when used in metabolism studies. However, SKF525A, menadione, raloxifene and piperine can inhibit the activities of ABC and/or SLC transporters.

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.

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.

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.

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.

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.

Inhibitory properties of trapping agents: glutathione, potassium cyanide, and methoxylamine, against major human cytochrome p450 isoforms.

In the early stages of drug discovery, the formation of reactive metabolites is often assessed by co-incubating the drug in liver microsomes with a trapping agent in the presence of NADPH. Our group assessed the capability of commonly used trapping agents to reversibly inhibit major cytochrome P450 (CYP) isoforms. Glutathione and cyanide did not inhibit the enzymes at concentrations up to 10 mM; however methoxylamine did show inhibition, with IC(50) values of 0.53 mM for CYP1A2, 4.12 mM for CYP2C9, 2.04 mM for CYP2C19, 9.72 mM for CYP2D6, and 1.26 and >10 mM for CYP3A4/5 (for testosterone and midazolam, respectively, as substrates).

Comparative pharmacokinetic and disposition studies of [1-14C]1-eicosanylcyclohexane, a surrogate mineral hydrocarbon, in female Fischer-344 and Sprague-Dawley rats.

White oils or waxes [mineral hydrocarbons (MHCs)] with substantial levels of saturated hydrocarbons in the range of C18 to C32 have produced hepatic microgranulomas and lymph node microgranulomas (also referred to as histiocytosis) after repeated administration to female Fischer-344 (F-344) rats. Female Sprague-Dawley (S-D) rats are less sensitive to these MHC-induced hepatic and lymph node effects. Studies reported herein characterized the pharmacokinetics and disposition of a representative C-26 MHC, [1-(14)C]1-eicosanylcyclohexane ([(14)C]EICO), in these two rat strains. Female F-344 and S-D rats were administered by oral gavage either a high (1.80 g/kg) or a low (0.18 g/kg) dose of MHC in olive oil (1:4, v/v) containing [(14)C]EICO as a tracer. Blood, urine, feces, liver, and mesenteric lymph nodes (MLNs) were analyzed for [(14)C]EICO and (14)C-metabolites. After the high dose, F-344 rats had a higher blood C(max) of [(14)C]EICO, a longer time to C(max), and a greater area under the systemic blood concentration-time curve from zero to time infinity compared with S-D rats. After the low dose, F-344 rats displayed a unique triphasic blood concentration-time profile, meaning two distinct C(max) values were observed. Fecal excretion was the major route of [(14)C]EICO elimination for both rat strains (70-92% of the dose). S-D rats eliminated the majority of [(14)C]EICO metabolites recovered in the urine by 16 h (8-17% of the dose), whereas F-344 rats did not excrete the same amount until 72 to 96 h. Beyond 24 h, a greater level of [(14)C]EICO was recovered in livers of F-344 rats; at 96 h, 3 and 0.1% of the dose was retained in livers of F-344 and S-D rats, respectively. The major urinary metabolites of EICO in both rat strains were identified as 12-cyclohexyldodecanoic acid and 10-cyclohexyldecanoic acid. Based on the pharmacokinetic parameters and disposition profiles, the data indicate inherent strain differences in the total systemic exposure, rate of metabolism, and hepatic and lymph node retention of [(14)C]EICO, which may be associated with the different strain sensitivities to the formation of liver granulomas and MLN histiocytosis.