Mechanistic Multilayer Quantitative Model for Nonlinear Pharmacokinetics, Target Occupancy and Pharmacodynamics (PK/TO/PD) Relationship of D-Amino Acid Oxidase Inhibitor, TAK-831 in Mice.

PURPOSE: TAK-831 is a highly selective and potent inhibitor of D-amino acid oxidase (DAAO) currently under clinical development for schizophrenia. In this study, a mechanistic multilayer quantitative model that parsimoniously connects pharmacokinetics (PK), target occupancy (TO) and D-serine concentrations as a pharmacodynamic (PD) readout was established in mice. METHODS: PK, TO and PD time-profiles were obtained in mice and analyzed by mechanistic binding kinetics model connected with an indirect response model in a step wise fashion. Brain distribution was investigated to elucidate a possible mechanism driving the hysteresis between PK and TO. RESULTS: The observed nonlinear PK/TO/PD relationship was well captured by mechanistic modeling framework within a wide dose range of TAK-831 in mice. Remarkably different brain distribution was observed between target and reference regions, suggesting that the target-mediated slow binding kinetics rather than slow penetration through the blood brain barrier caused the observed distinct kinetics between PK and TO. CONCLUSION: A quantitative mechanistic model for concentration- and time-dependent nonlinear PK/TO/PD relationship was established for TAK-831 in mice with accounting for possible rate-determining process. The established mechanistic modeling framework will provide a quantitative means for multilayer biomarker-assisted clinical development in multiple central nervous system indications.

Pharmacokinetic and pharmacodynamic modeling of the metastin/kisspeptin analog, TAK-448, for its anti-tumor efficacy in a rat xenograft model.

TAK-448 is the investigational metastin/kisspeptin analog, which is known to have an anti-tumor effect through suppression of androgen hormones (luteinizing hormone and testosterone) levels. This study developed pharmacokinetic-pharmacodynamic (PK/PD) models of TAK-448 and leuprorelin acetate (TAP-144) in a rat vertebral-cancer of the prostate (VCaP) androgen-sensitive prostate cancer xenograft model to quantitatively assess and compare the anti-tumor effects of both drugs. A potential contribution of the hormone-independent direct effects of TAK-448 to the tumor growth inhibition was also investigated in the in vivo rat xenograft model, because our in vitro experiments revealed that TAK-448 may also directly suppress VCaP cellular proliferation. The PK/PD model successfully described the time course of tumor growth inhibition after drug treatment as well as the development of resistance to the inhibition of androgen hormones, following drug treatment or castration. The EC50 of the hormone-dependent inhibitory effect of TAK-448 was much lower than that of TAP-144, and TAK-448 also has a faster onset of anti-tumor effect than TAP-144, demonstrating that TAK-448 has a stronger overall anti-tumor effect than TAP-144. In addition, model inference, by incorporating a hormone-independent inhibition pathway of TAK-448 into the PK-PD model, suggested that such a direct inhibition pathway for TAK-448 cannot be excluded, as also indicated by in vitro studies, but its EC50 would be approximately three orders of magnitude higher than that of the hormone-dependent pathway. This study helps to understand the potential and mechanism of TAK-448 as a prostate cancer treatment.

The Enhancement of Subcutaneous First-Pass Metabolism Causes Nonlinear Pharmacokinetics of TAK-448 after a Single Subcutaneous Administration to Rats.

2-(N-acetyl-D-tyrosyl-trans-4-hydroxy-L-prolyl-L-asparaginyl-L-threonyl-L-phenylalanyl) hydrazinocarbonyl-L-leucyl-Nω-methyl-L-arginyl-L-tryptophanamide monoacetate (TAK-448, RVT-602), a kisspeptin analog, has been developed as a therapeutic agent for prostate cancer. The purpose of the present study is to clarify the mechanism of the less than dose-proportional nonlinear pharmacokinetics of TAK-448 after subcutaneous administration to rats. The plasma pharmacokinetics of TAK-448 and radiolabeled TAK-448 ([14C]TAK-448) were examined after subcutaneous and intravenous administrations to rats. [14C]TAK-448 was also subcutaneously injected together with protease inhibitors. The effects of the protease inhibitors on the in vitro metabolism of [14C]TAK-448 were investigated using rat skin homogenates. In a dose-ascending study, less than dose-proportional nonlinear pharmacokinetics were observed after subcutaneous administration with limited absorption of TAK-448 at the highest dose level contrary to the linear pharmacokinetics following intravenous dosing, indicating enhancement of subcutaneous metabolism with dose escalation. The systemic absorption of unchanged TAK-448 recovered when protease inhibitors were subcutaneously coadministered, suggested the involvement of subcutaneous proteases in the first-pass metabolism. An in vitro metabolism study suggests that serine protease could be responsible for the subcutaneous metabolism of TAK-448. Dose-dependent enhancement of first-pass metabolism appears to contribute to the less than dose-proportional nonlinear pharmacokinetics of TAK-448 after subcutaneous administrations to rats.

Investigation of disposition for TAK-448, a synthetic peptide of kisspeptin analog, in rats and dogs using the radiolabeled TAK-448 suitable for pharmacokinetic study.

Disposition of 2-(N-acetyl-d-tyrosyl-trans-4-hydroxy-l-prolyl-l-asparaginyl-l-threonyl-l-phenylalanyl) hydrazinocarbonyl-L-leucyl-Nω-methyl-l-arginyl-l-tryptophanamide monoacetate (TAK-448, RVT-602), a synthetic kisspeptin analog, was investigated after parenteral dosing of radiolabeled TAK-448 ([d-Tyr-14C]TAK-448) to rats and dogs, and it was confirmed if the radiolabeling position at d-Tyr was eligible for assessment of in vivo disposition. Dosed radioactivity was rapidly and well absorbed after subcutaneous administration and an appreciable amount of unchanged TAK-448 (TAK-448F) and a hydrolyzed metabolite, M-I, were detected in the plasma of rats and dogs. After intravenous administration of [d-Tyr-14C]TAK-448 to rats, the radioactivity widely distributed to tissues with relatively higher concentrations in kidney and urinary bladder. The radioactivity was decreased rapidly from the tissues. After subcutaneous administration of [d-Tyr-14C]TAK-448 to rats and dogs, the dosed radioactivity was almost completely recovered by 48 and 72 h in rats and dogs, respectively, and most of the radioactivity was excreted in urine after extensive metabolism in the two species. These results suggest that TAK-448 has an acceptable pharmacokinetic profile for clinical evaluation and development, and demonstrate that the synthesized [D-Tyr-14C]TAK-448 used in this study represents a favorable labeling position to evaluate disposition properties of this compound.

Mechanism for Covalent Binding of MLN3126, an Oral Chemokine C-C Motif Receptor 9 Antagonist, to Serum Albumins.

N-{4-Chloro-2-[(1-oxidopyridin-4-yl)carbonyl]phenyl}-4-(propan-2-yloxy)benzenesulfonamide (MLN3126) is an orally available chemokine C-C motif receptor 9 selective antagonist. In nonclinical pharmacokinetic studies of MLN3126, nonextractable radioactivity was observed in plasma after oral administration of 14C-labeled MLN3126 ([14C]MLN3126) to Sprague-Dawley (SD) rats. In this study, the nonextractable radioactive component was digested with trypsin or a nonspecific protease, pronase, after chemical reduction to obtain drug-peptide adducts or drug-amino acid adducts. The chemical structure of these adducts was characterized by liquid chromatography/mass spectrometry. The results demonstrated that the major part of the nonextractable radioactivity was accounted for by covalent binding via the Schiff base formed specifically between the ε-amino group of lysine residue 199 in rat serum albumin and the carbonyl group of MLN3126. The half-life (t1/2) of the total radioactivity in plasma during and after 21 daily multiple oral administrations of [14C]MLN3126 to SD rats was approximately 5-fold shorter than the reported t1/2 of albumin in rats. The data indicated that the covalent binding was reversible under physiologic conditions. The formation of the covalent binding was also confirmed in in vitro incubations with serum albumins from rats, humans, and dogs in the same manner, indicating that there are no qualitative interspecies differences in the formation of the Schiff base.

Long-Term Stability of Cryopreserved Human Hepatocytes: Evaluation of Phase I and II Drug-Metabolizing Enzyme Activities and CYP3A4/5 Induction for More than a Decade.

We evaluated the long-term stability of hepatocytes stored in the vapor phase of liquid nitrogen for their viability, cytochrome P450 (CYP) 1A2 activity, CYP3A4/5 activity, uridine diphosphate-glucuronosyl transferase (UGT) activity, sulfotransferase (SULT) activity, and CYP3A4/5 induction during 14 years of preservation. No substantial degradation of viability, CYP1A2 activity, UGT activity, or CYP3A4/5 induction was observed. CYP3A4/5 activity showed a slight decrease after 7 years of storage, and SULT activity gradually decreased during storage, although substantial activities remained even after 14 years. These results indicate that cryopreserved human hepatocytes can be stored stably for more than a decade with little or no change in viability, activity of drug-metabolizing enzymes, or CYP3A4/5 induction, and can be widely applicable to qualitative research in drug metabolism.

In vitro metabolism of TAK-438, vonoprazan fumarate, a novel potassium-competitive acid blocker.

1. TAK-438, vonoprazan fumarate, is a novel orally active potassium-competitive acid blocker, developed as an antisecretory drug. In this study, we investigated the in vitro metabolism of 14C-labeled TAK-438. In human hepatocytes, M-I, M-II, M-III and M-IV-Sul were mainly formed, and these were also detected in clinical studies. N-demethylated TAK-438 was also formed as an in vitro specific metabolite. Furthermore, CYP3A4 mainly contributed to the metabolism of TAK-438 to M-I, M-III, and N-demethylated TAK-438, and CYP2B6, CYP2C19 and CYP2D6 partly catalyzed the metabolism of TAK-438. The sulfate conjugation by SULT2A1 also contributed to the metabolism of TAK-438 to form TAK-438 N-sulfate, and CYP2C9 mediated the formation of M-IV-Sul from TAK-438 N-sulfate. The metabolite M-IV, which could be another possible intermediate in the formation of M-IV-Sul, was not observed as a primary metabolite of TAK-438 in any of the in vitro studies. 2. In conclusion, TAK-438 was primarily metabolized by multiple metabolizing enzymes including CYP3A4, CYP2B6, CYP2C19, CYP2D6, and a non-CYP enzyme SULT2A1, and the influence of the CYP2C19 genotype status on gastric acid suppression post TAK-438 dosing could be small. The multiple metabolic pathways could also minimize the effects of co-administrated CYP inhibitors or inducers on the pharmacokinetics of TAK-438.

Disposition and metabolism of TAK-438 (vonoprazan fumarate), a novel potassium-competitive acid blocker, in rats and dogs.

1. Following oral administration of [14C]TAK-438, the radioactivity was rapidly absorbed in rats and dogs. The apparent absorption of the radioactivity was high in both species. 2. After oral administration of [14C]TAK-438 to rats, the radioactivity in most tissues reached the maximum at 1-hour post-dose. By 168-hour post-dose, the concentrations of the radioactivity were at very low levels in nearly all the tissues. In addition, TAK-438F was the major component in the stomach, whereas TAK-438F was the minor component in the plasma and other tissues. High accumulation of TAK-438F in the stomach was observed after oral and intravenous administration. 3. TAK-438F was a minor component in the plasma and excreta in both species. Its oxidative metabolite (M-I) and the glucuronide of a secondary metabolite formed by non-oxidative metabolism of M-I (M-II-G) were the major components in the rat and dog plasma, respectively. The glucuronide of M-I (M-I-G) and M-II-G were the major components in the rat bile and dog urine, respectively, and most components in feces were other unidentified metabolites. 4. The administered radioactive dose was almost completely recovered. The major route of excretion of the drug-derived radioactivity was via the feces in rats and urine in dogs.

Impact of acute fat mobilisation on the pharmacokinetics of the highly fat distributed compound TAK-357, investigated by physiologically based pharmacokinetic (PBPK) modeling and simulation.

In a dog toxicokinetic study, an unusual plasma concentration increase of the highly lipophilic compound TAK-357 was observed 2 weeks after termination of a 2-week repeated dosing in one dog with acute body weight loss. The present study investigates the cause of this increase. A physiologically based pharmacokinetic (PBPK) model was constructed using the rat and dog pharmacokinetic data. Using the constructed model, the TAK-357 concentration profile in the case of body weight change was simulated. The PBPK model-derived simulation suggested that redistribution from adipose tissues to plasma due to a loss of body fat caused the observed concentration increase of TAK-357 in dog plasma. The analysis demonstrates that the disposition of a highly lipophilic and fat-distributed compound can be affected by acute changes in adipose tissue mass. PBPK modeling and simulation proved to be efficient tools for the quantitative hypothesis testing of apparently atypical PK phenomena resulting from acute physiological changes.

Influence of body composition on disposition of the highly fat distributed compound as analysed by physiologically based pharmacokinetic (PBPK) modeling and simulation.

A recent study suggested that the pharmacokinetics (PK) of highly fat distributed compounds can be affected by acute changes in the volume of adipose tissue. The present study investigates possible influences of body composition on the disposition of the highly lipophilic compound TAK-357 in two rat strains. Physiologically based PK (PBPK) modeling and simulation was applied on single and multiple dose PK data of TAK-357 in obese Wistar fatty rats and Wistar lean rats having approximately 45% and 13% body fat, respectively. The observed effects of an elevated fat mass in Wistar fatty rats on the plasma concentrations appeared to be partly compensated for by other differences between the two rat strains. A decrease in the tissue to blood partition coefficients under high body fat conditions was identified as another factor contributing to the difference in PK. A higher lipid content in the plasma in high body fat animals may result in relatively lower tissue to blood partition coefficients. PBPK-based simulations indicate that the plasma concentrations of lipophilic compounds in high body fat conditions can differ by up to two-times at steady-state. This confirms that there is only a small impact of body composition change on the plasma concentration of highly lipophilic drugs and that the need for therapeutic dose adjustments may be limited.

The effects of the concentration-dependent erythrocyte distribution of TAK-802, a potent acetylcholinesterase inhibitor, on rat pharmacokinetics.

The purpose of this study was to investigate the effect of the concentration-dependent erythrocyte distribution of TAK-802, a potent acetylcholinesterase inhibitor, on rat pharmacokinetics. In an ascending oral dose study, the maximum plasma concentration (Cmax ) of TAK-802 increased in a dose-dependent manner. The time to reach Cmax decreased as the dose increased, whereas the total clearance was independent of the tested dose range. In this intravenous (i.v.) ascending dose study in rats, the apparent distribution volumes at steady state decreased, and the apparent terminal elimination rate constants increased with TAK-802 dose escalation. A marked concentration dependency was observed in an associated in vitro erythrocyte distribution study. The in vitro erythrocyte distribution study results and a relationship analysis between the plasma and blood concentrations of TAK-802 after i.v. dosing revealed that the characteristics of the erythrocyte distribution could be expressed by Langmuir's adsorption formula. The concentration-time profiles of TAK-802 in plasma and whole blood calculated using a nonlinear pharmacokinetic model incorporating the concentration-dependent erythrocyte distribution with optimized parameters fit well to the observed plasma and blood concentration profiles obtained from the i.v. ascending dose study. These results indicate that the concentration-dependent erythrocyte distribution plays a major role in the nonlinear pharmacokinetics of TAK-802 in rats. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacokinetic and pharmacodynamic modeling of hedgehog inhibitor TAK-441 for the inhibition of Gli1 messenger RNA expression and antitumor efficacy in xenografted tumor model mice.

6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-3-(2,2,2-trifluoroethoxy)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (TAK-441) is a potent, selective hedgehog signaling pathway inhibitor that binds to Smo and is being developed for the treatment of cancer. The objectives of these studies were to explore the possibility of establishing of a link between the pharmacokinetics of TAK-441 and the responses of Gli1 mRNA in tumor-associated stromal or skin cells and the antitumor effect of hedgehog inhibition. To this end, we built pharmacokinetic and pharmacodynamic models that describe the relationship of the concentrations of TAK-441 plasma to the responses of Gli1 mRNA in the tumor (target) and skin (surrogate) and to tumor growth inhibition in mice bearing xenografts of human pancreatic tumors (PAN-04). The responses of Gli1 mRNA and tumor growth were described by an indirect response model and an exponential tumor growth model, respectively. The IC50 values for Gli1 mRNA inhibition in the tumor and skin by TAK-441 were estimated to be 0.0457 and 0.113 µg/ml, respectively. The IC90 value for tumor growth inhibition was estimated to be 0.68 µg/ml. These results suggest that a >83% inhibition of Gli1 mRNA expression in the skin or a >94% inhibition of Gli1 mRNA expression in the tumor would be required to sufficiently inhibit (>90%) hedgehog-related tumor growth in the xenografted model mice. We conclude that Gli1 mRNA expression in the tumor and skin could be a useful biomarker for predicting the antitumor effect of hedgehog inhibitors.

Evaluation of low background liquid scintillation counter for non-clinical ADME studies.

1. The performance of low background (BG) liquid scintillation counter (LSC) was evaluated for practical purposes of non-clinical drug absorption, distribution, metabolism and excretion (ADME) studies. The measurement conditions for the radioactivity for low BG LSC were investigated and metabolite profiling in rat plasma after single oral administration of (14)C-labeled compounds was performed. Metabolite profiling was also conducted using conventional LSC and accelerator mass spectrometer (AMS), and the performances of these measuring instruments were compared. 2. The established measurement conditions showed good linearity of the calibration curve over the concentration range from 3 to 300 dpm/vial. Metabolite profiling using low BG LSC for the plasma samples diluted to 5-55 times was comparable to that using conventional LSC for the undiluted plasma samples. Meanwhile, metabolite profiling using AMS for the plasma samples diluted to 250-2000 times was comparable to that using conventional LSC. 3. These results suggest that the application of low BG LSC is one of the useful tools for non-clinical ADME studies and that it is possible to select conventional LSC, low BG LSC or AMS for radioactivity measurement by considering the specific radioactivity of the compounds and the predicted concentrations of radioactivity in biological samples in ADME studies.

Absorption of TAK-491, a new angiotensin II receptor antagonist, in animals.

The absorption process in animals of TAK-491, designed as ester-based prodrug with medoxomil moiety, was evaluated. In the plasma of rats and dogs, TAK-536, the pharmacologically active metabolite, was present as the main component with hardly detectable concentrations of TAK-491 after oral administration of TAK-491. In the rat portal plasma, TAK-536 was also present as the main component with hardly detectable concentrations of TAK-491 after jejunal loop injection of TAK-491, suggesting TAK-491 was absorbed from small intestine and hydrolyzed almost completely during absorption. Caco-2 study indicated the permeability of TAK-491 was improved by prodrug modification and the compound could be mainly transferred as TAK-491. This is well consistent with the facts that the AUC and T(max) of TAK-536 after oral administration of TAK-491 were higher and shorter than those after oral administration of TAK-536 in dogs Hydrolysis of TAK-491 is observed not only by the intestinal and hepatic S9 fraction, but also by plasma and human serum albumin. However, medoxomil alcohol wasn't detected during the hydrolysis of TAK-491. These metabolic features of TAK-491 were similar to olmesartan medoxomil, suggesting the hydrolytic pathway and enzymes for TAK-491 when catalyzing to TAK-536 would be the same as olmesartan medoxomil.

Species differences of organic anion transporters involved in the renal uptake of 4-amino-3-chlorophenyl hydrogen sulfate, a metabolite of resatorvid, between rats and dogs.

Previous studies on the metabolic fate of resatorvid (TAK-242) have shown that species differences in the pharmacokinetics of 4-amino-3-chlorophenyl hydrogen sulfate (M-III), a metabolite of TAK-242, between rats and dogs are mainly attributable to the urinary excretion process. In the present study, the renal uptake mechanism of M-III was investigated using kidney slices and Xenopus laevis oocytes expressing rat organic anion transporter 1 (rOat1; Slc22a6) and rOat3 (Slc22a8). The uptake of p-aminohippuric acid (PAH), a substrate for Oats, by kidney slices from rats and dogs increased at 37 °C and M-III inhibited the uptake. The initial uptake clearance of M-III by rat kidney slices was 0.295 and 0.0114 ml/min/g at 37 °C and 4 °C, respectively. The Eadie-Hofstee plot of M-III uptake at 37 °C revealed two-component transport processes with K(m) values being 6.48 and 724 µmol/l. The uptake was inhibited by probenecid (PBC), PAH and benzylpenicillin (PCG). In contrast, in dog kidney slices, the initial uptake clearance of M-III was 8.70 × 10(-3) and 9.00 × 10(-3) ml/min/g at 37 °C and 4 °C, respectively, and the uptake was not inhibited by PBC. Furthermore, rOat1- and rOat3-expressing oocytes mediated M-III uptake and the uptake was inhibited by PAH and PCG, respectively. These results suggest that rOat1 and rOat3 are responsible for the renal uptake of M-III in rats. Moreover, it is speculated that Oat(s) is unable to transport M-III in dogs and that the difference in the substrate recognition of Oat(s) contributes to the species difference in the pharmacokinetics of M-III between rats and dogs.

Differences in the pharmacokinetics of 4-amino-3-chlorophenyl hydrogen sulfate, a metabolite of resatorvid, in rats and dogs.

The pharmacokinetics of 4-amino-3-chlorophenyl hydrogen sulfate, M-III of resatorvid, in rats and dogs were investigated using radiolabeled M-III ([(14)C]M-III). The elimination half-life of (14)C in the plasma of rats was approximately 1/30 of that of dogs after intravenous dosing of [(14)C]M-III at 0.5 mg/kg to rats and dogs. The in vitro and in vivo plasma protein binding ratios of M-III were relatively high and were the same in both species. The intrinsic clearance (CL(int)) of M-III in rats was much higher than the glomerular filtration rate in rats. Furthermore, the concentration of [(14)C]M-III in the kidney of rats was much higher than that in the plasma. On the contrary, in dogs, the concentration of [(14)C]M-III in the kidney was very much lower than that in the plasma. These results indicated that M-III was effectively taken up into the kidney and was excreted into the urine in rats; however, in dogs, ineffective renal uptake of M-III was presumed. When [(14)C]M-III and probenecid were simultaneously and continually infused intravenously to rats, the CL(int) of M-III decreased with increasing plasma concentrations of probenecid, indicating that kidney uptake of M-III in rats was inhibited by probenecid. It was also thought that uptake by the organic anion transport system(s) in the basolateral membrane is involved in the renal uptake of M-III in rats. The pharmacokinetic differences of M-III between rats and dogs are considered to be mainly caused by the difference in the urinary excretion via the renal distribution processes.

An unusual metabolic pathway of sipoglitazar, a novel antidiabetic agent: cytochrome P450-catalyzed oxidation of sipoglitazar acyl glucuronide.

Animal pharmacokinetic studies of sipoglitazar, a novel antidiabetic agent, showed that the deethylated metabolite (M-I) and the glucuronide conjugate of sipoglitazar (sipoglitazar-G) appeared to be the key metabolites in the elimination process. M-I was also measured as the main metabolite in the plasma of humans administered sipoglitazar. In vitro metabolic studies were performed to investigate the metabolic pathways from sipoglitazar to M-I in humans. The metabolic profile with human hepatocytes and hepatic microsomes indicated that M-I was not formed directly from sipoglitazar and that sipoglitazar-G was involved in the metabolism from sipoglitazar to M-I. Further studies of the metabolism of sipoglitazar-G revealed that the properties of the glucuronide conjugate and its metabolism are as follows: high-performance liquid chromatography, liquid chromatography-tandem mass spectrometry, and NMR analyses showed that sipoglitazar-G was composed of two glucuronides, sipoglitazar-G1, a ß-1-O-acyl glucuronide, and sipoglitazar-G2, an α-2-O-acyl glucuronide. The stability study of these glucuronides suggested that sipoglitazar-G1 could be converted to sipoglitazar-G2 and sipoglitazar, but sipoglitazar-G2 could not be converted to sipoglitazar-G1. The oxidative metabolic study of sipoglitazar-G1 and -G2 with human hepatic microsomes and cytochrome P450-expressing microsomes revealed that M-I was formed only from sipoglitazar-G1, not from sipoglitazar-G2, and that CYP2C8 was mainly involved in this process. From these results, it is shown that the metabolic pathway from sipoglitazar to M-I is an unusual one, in which sipoglitazar is initially metabolized to sipoglitazar-G1 by UDP-glucuronosyltransferase and then sipoglitazar-G1 is metabolized to M-I by O-dealkylation by CYP2C8 and deconjugation. Sipoglitazar-G2 is sequentially formed by the migration of the ß-site of sipoglitazar-G1.

Discovery of orteronel (TAK-700), a naphthylmethylimidazole derivative, as a highly selective 17,20-lyase inhibitor with potential utility in the treatment of prostate cancer.

A novel naphthylmethylimidazole derivative 1 and its related compounds were identified as 17,20-lyase inhibitors. Based on the structure-activity relationship around the naphthalene scaffold and the results of a docking study of 1a in the homology model of 17,20-lyase, the 6,7-dihydro-5H-pyrrolo[1,2-c]imidazole derivative (+)-3c was synthesized and identified as a potent and highly selective 17,20-lyase inhibitor. Biological evaluation of (+)-3c at a dose of 1mg/kg in a male monkey model revealed marked reductions in both serum testosterone and dehydroepiandrosterone concentrations. Therefore, (+)-3c (termed orteronel [TAK-700]) was selected as a candidate for clinical evaluation and is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer.

Chemical reactivity of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) in vitro.

Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) was metabolized to cyclohexene and phenyl ring moieties in non-clinical pharmacokinetic studies and it was suggested that the cyclohexene ring moiety of TAK-242 is tightly bound to endogenous macromolecules. After incubation of TAK-242 and glutathione (GSH) in phosphate buffer (pH 7.4) at 37 °C, TAK-242 reacted with GSH to produce a glutathione conjugate of the cyclohexene ring moiety of TAK-242, which had been observed as a metabolite (M-SG) in non-clinical pharmacokinetic studies. Formation of M-SG was time dependent with a first order reaction and M-I, a metabolite from the phenyl ring moiety of TAK-242, was also produced in parallel. The formation of M-SG was accelerated with increasing pH, therefore it was indicated that TAK-242 reacted with GSH by a nucleophilic substitution reaction. Because glutathione transferase (GST) enhanced M-SG formation in vitro, it is expected that the conjugation of TAK-242 with GSH is also facilitated by GST in vivo in addition to a spontaneous chemical reaction. When radio-labeled TAK-242 ([cyclohexene ring-U-¹4C]TAK-242) was incubated with rat serum albumin (RSA) or human serum albumin (HSA) in vitro, the radioactive material was covalently bound to RSA and HSA, and M-I was generated simultaneously in the reaction mixture. The chemical structure of the TAK-242 adduct covalently bound to HSA was characterized by the accurate mass spectra that cyclohexene ring moiety of TAK-242 was covalently bound to the lysine residue in HSA. The adduct was also detected in the plasma of rats and humans after single i.v. dosing of TAK-242 (in vivo).

In vitro micronucleus test in HepG2 transformants expressing a series of human cytochrome P450 isoforms with chemicals requiring metabolic activation.

It is known that many genotoxic chemicals require oxidative metabolism to elicit genotoxicity. Induced rat liver S9 fraction has been employed as a 'metabolite factory' in in vitro genotoxicity testing. However, the relevance of the induced rat liver S9 fraction has been called into question due to the differences in the rat and human cytochrome P450 (CYP) activities. In the present study, we used a series of ten transformants expressing major human CYP isoforms such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 in HepG2 cells. To elucidate the usefulness and feasibility of these transformants, genotoxicity was tested without using rat S9. Among these transformants, benzo(a)pyrene-induced or cyclophosphamide-produced micronucleus (MN) frequency was markedly increased in transformants expressing CYP1A2 or CYP2C9, respectively. To explore the possibility that these transformants can be used for screening the possible genotoxicity of newly developed drugs, a chemical which is known to enhance genotoxicity in the presence of external metabolic activation system, okadaic acid (OA), was investigated. OA-induced MN frequency was significantly induced in transformants expressing CYP1A2 compared with the other CYP isoforms. The induced MN frequency was suppressed by treatment with a CYP1A2 specific inhibitor and CYP1A2 to siRNA. In control HepG2 cells harboring an empty vector, OA was treated with microsomes expressing CYP1A2 to induce MN. These results demonstrated that this screening system worked well and OA was found to be metabolically activated by CYP1A2 to induce MN. Based on the results obtained in the present study, this system of transformants is useful to elucidate the genotoxicity involving human CYP metabolism in the process of drug discovery.