Epxoide hydrolase -polymorphism and role in toxicology

Abstract Microsomal epoxide hydrolase is a critical biotransformation enzyme that catalyzes the conversion of a broad array of xenobiotic epoxide substrates to more polar diol metabolites. The gene has been shown previously to exhibit polymorphism, including variation in the coding region leading to amino acid substitutions at positions 113 (Y/H) and 139 (H/R). To better evaluate the phenotype associated with the structural region genetic polymorphisms associated with mEH, we performed enzymatic analyses using purified mEH proteins that were expressed using a baculovirus system, or with microsomal preparations obtained from liver tissues that were derived from individuals with homozygous mEH allelic status. Benzo[a]pyrene-4,5-oxide and cis-stilbene oxide were employed as substrates for the enzymatic determinations. Results obtained with the purified enzymes suggested that the reaction velocity catalyzed by the wild type (Y113/H139) protein was approximately two-fold greater than the corresponding velocities for the variant forms of the enzyme. However, when reaction rates were analyzed using human liver microsomal preparations, the maximal velocities generated among the variant mEH proteins were not statistically different. Collectively, these results indicate that the structural differences coded by the mEH genetic variants may have only modest impact on the enzyme's specific activity in vivo

Evaluation of pharmaceutical excipients as cosolvents in 4-methyl umbelliferone glucuronidation in human liver microsomes: An application for compounds with low solubility

Introduction: In order to minimize the potential inhibitory effects of organic solvents on metabolic activity, standard incubation procedures for carrying out microsomal assays involve the use of less than 1% w/v organic solvents. Often, solvents needed to dissolve the substrate add-up nearly to this concentration. This presents a practical limitation for poorly soluble xenobiotics, which cannot be incubated at concentrations high enough to obtain a Vmax, and therefore subsequent values for Km and Clint cannot be calculated. Our goal was to study the application of a variety of pharmaceutical excipients to aid the solubilization of compounds in vitro in glucuronidation incubations, without affecting the reaction kinetics. Methods: In vitro glucuronidation incubations were carried out in human liver microsomes with 4-methylumbelliferone (4-MU) and the kinetics of 4-MU glucuronidation in the presence of excipients were compared to that in control incubations without any excipients. In addition, IC75 values were calculated for each excipient. Results and Summary: We observed that HPBCD may be employed in in vitro glucuronidation incubations up to 0.5% w/v without affecting the Clint of 4-MU. Although NMP and DMA showed low IC75 values approximately 0.1% w/v each, neither excipients altered the Clint of 4-MUG formation. Our studies point toward a possible application of pharmaceutical excipients to carry out in vitro glucuronidation of substrates with poor aqueous solubility, in order to estimate Clint and subsequently scaled organ clearance values

Identification of a novel N-carbamoyl glucuronide: In vitro, In vivo and mechanistic studies

1-[4-Aminomethyl-4-(3-chloro-phenyl )-cyclohexyl]-tetrahydro-pyrimidin-2-one, 1, was developed as an inhibitor of dipeptidyl peptidase-4 enzyme (DPP-4). Biotransformation studies with 1 revealed the presence of an N-carbamoyl glucuronide metabolite (M1) in rat bile and urine. N-carbamoyl glucuronides are rarely observed, and little is understood regarding the mechanism of N-carbamoyl glucuronidation. The objectives of the current investigation were to elucidate the structure of the novel N-carbamoyl glucuronide, to investigate the mechanism of N-carbamoyl glucuronide formation in vitro using stable labeled CO2, UGT reaction phenotyping, and to assess whether M1 was formed to the same extent in vitro across species – mouse, rat, hamster, dog, monkey and human. Structure elucidation was carried out on a Thermo LTQ-Orbitrap® with accurate mass measurement and MSn capabilities. 13C-labeled carbon dioxide (13CO2) was used for identification of the mechanism of N-carbamoyl glucuronidation. Mechanistic studies with 13C-labeled CO2 in rat liver microsomes revealed that CO2 from the bicarbonate buffer (in equilibrium with exogenous CO2) may be responsible for the formation of M1. M1 was formed in vitro in liver microsomes from multiple species – mainly rat and hamster, followed by similar formation in dog, monkey, mouse, human. M1 could be detected in UGT1A1, UGT1A3 and UGT2B7 Supersomes® in a CO2 rich environment. In conclusion our study demonstrates that formation of M1 was observed in microsomal incubations across various species and strongly suggests the incorporation of CO2 from the bicarbonate buffer, in equilibrium with exogenous CO2 into the carbamoyl moiety of the formed N-carbamoyl glucuronide

Preclinical investigations and a first-in-human phase I trial of M4112, the first dual inhibitor of indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase 2, in patients with advanced solid tumors

Background M4112 is an oral, potent, and selective indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) dual inhibitor. Here, we report preclinical data and first-in-human phase I data, including safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary efficacy, of M4112 monotherapy in patients with advanced solid tumors.Methods In preclinical studies, M4112 was administered to mice with IDO1-expressing tumors to determine tumor IDO1 and liver TDO2 inhibition. In the phase I trial, patients received doses of M4112 two times per day in 28-day cycles until progression, toxicity, or withdrawal of consent. The primary objective was to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D). The primary endpoint was the incidence of dose-limiting toxicities (DLTs), treatment-emergent adverse events (TEAEs), and treatment-emergent changes in safety parameters. Other endpoints included pharmacokinetics, pharmacodynamics, and antitumor effects.Results In mice, M4112 significantly decreased the kynurenine:tryptophan ratio in the liver and tumor. Fifteen patients received M4112 at five distinct dose levels (three patients per cohort: 100, 200, 400, 600, and 800 mg two times per day orally). Initially, all doses inhibited IDO1 ex vivo, but plasma kynurenine levels returned to or exceeded baseline levels after day 15. Despite initial changes in kynurenine, there was no significant reduction of plasma kynurenine at steady state. There was one DLT (grade 3 allergic dermatitis; 800 mg two times per day) and one grade 2 QT prolongation (800 mg two times per day), resulting in dose reduction (not a DLT). M4112 was well tolerated, and neither the MTD nor the RP2D was established. TEAEs included fatigue, nausea, and vomiting. The best overall response was stable disease (n=9, 60%).Conclusions There were no serious safety concerns at any dose. Although M4112 inhibited IDO1 activity ex vivo, plasma kynurenine levels were not reduced despite achieving target exposure.Trial registration number NCT03306420

Evaluation of the metabolism, bioactivation and pharmacokinetics of triaminopyrimidine analogs toward selection of a potential candidate for antimalarial therapy

<p>1. During the course of metabolic profiling of lead Compound <b>1</b>, glutathione (GSH) conjugates were detected in rat bile, suggesting the formation of reactive intermediate precursor(s). This was confirmed by the identification of GSH and <i>N</i>-acetylcysteine (NAC) conjugates in microsomal incubations.</p> <p>2. It was proposed that bioactivation of Compound <b>1</b> occurs via the formation of a di-iminoquinone reactive intermediate through the involvement of the C-2 and C-5 nitrogens of the pyrimidine core.</p> <p>3. To further investigate this hypothesis, structural analogs with modifications at the C-5 nitrogen were studied for metabolic activation in human liver microsomes supplemented with GSH/NAC.</p> <p>4. Compounds <b>1</b> and <b>2</b>, which bear secondary nitrogens at the C-5 of the pyrimidine core, were observed to form significant amounts of GSH/NAC-conjugates <i>in vitro</i>, whereas compounds with tertiary nitrogens at C-5 (Compound <b>3</b> and <b>4</b>) formed no such conjugates.</p> <p>5. These observations provide evidence that electron/hydrogen abstraction is required for the bioactivation of the triaminopyrimidines, potentially via a di-iminoquinone intermediate. The lack of a hydrogen and/or steric hindrance rendered Compound <b>3</b> and <b>4</b> incapable of forming thiol conjugates.</p> <p>6. This finding enabled advancement of compound <b>4</b>, with a desirable potency, safety and PK profile, as a lead candidate for further development in the treatment of malaria.</p

Preliminary Studies of 3,5-Diarylazoles as Novel and Selective Inhibitors of Protein Kinase D

Preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D (PKD) are reported. Notably, compounds bearing an α-aminonitrile moiety have been found to be active in cellular assays of HDAC5 nuclear localization, orally biovailable, and highly selective versus a panel of additional putative histone deacetylase (HDAC) kinases, providing potential tools for the further study of PKD / HDAC5 signalin