Satoru Asahi

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.

Establishment of the transformants expressing human cytochrome P450 subtypes in HepG2, and their applications on drug metabolism and toxicology

Transformants with stable expression of a series of human cytochrome P450 (CYP) subtypes in the human hepatic cell line, HepG2, were established. These transformants are designated Hepc/1A1.4, Hepc/1A2.9, Hepc/2A6L.14, Hepc/2B6.68, Hepc/2C8.46, Hepc/2C9.1, Hepc/2C19.12, Hepc/2D6.39, Hepc/2E1.3-8 and Hepc/3A4.2-30, which stably expressed human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively. The expression of the CYP subtypes in the transformants was confirmed by both determination of enzyme activities and the reverse transcriptase polymerase chain reaction (RT-PCR) procedure. The apparent K(m) values of the expressed CYP subtypes for their specific substrates were close to those of human liver microsomes. In addition to their CYP activities, these transformants retained glucuronide- and sulfate-conjugating activities. Furthermore, the activities of CYP2C9, CYP2D6 and CYP3A4 were inhibited by their specific inhibitors. The cytotoxicity of acetaminophen (APAP), cyclophosphamide (CPA) and benz[a]anthracene (BA) were analyzed by CYP-expressing transformants. The cytotoxicity depended on the expression of CYP subtypes and increased in a dose-dependent manner. These results show the metabolic activation of APAP, CPA and BA by the specific CYP subtypes expressed in the transformants and demonstrate the usefulness of these transformants for in vitro metabolic and toxicological studies in human liver.

Establishment and characterization of the transformants stably-expressing MDR1 derived from various animal species in LLC-PK1

PurposeStable transformants expressing human multidrug resistance 1 (MDR1), monkey MDR1, canine MDR1, rat MDR1a, rat MDR1b, mouse mdr1a, and mouse mdr1b in LLC-PK1 were established to investigate species differences in P-glycoprotein (P-gp, ABCB1) mediated efflux activity.MethodsThe seven cDNAs of MDR1 from five animals were cloned, and their transformants stably expressing the series of MDR1 in LLC-PK1 were established. Transport studies of clarithromycin, daunorubicin, digoxin, erythromycin, etoposide, paclitaxel, propranolol, quinidine, ritonavir, saquinavir, verapamil, and vinblastine were performed by using these cells, and efflux activity was compared among the species.ResultsExcept for propranolol, all compounds showed efflux activity in all transformants, and were judged to be substrates of P-gp. There were slight interspecies and interisoforms differences in the substrate recognition. However, the efflux ratio among the series of the MDR1 stably expressing cells showed good correlation as represented between human and monkey MDR1, and poor correlation as represented between human MDR1 and mouse mdr1a, and human and canine MDR1.ConclusionsResults in the present study indicate that all MDR1 stably expressing cells have efflux activity for various P-gp substrates, and that interspecies differences and similarities of the P-gp substrate efflux activity may exist.

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT

Abstract Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently, 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.

Hypothermia enhances heat-shock protein 70 production in ischemic brains.

Although moderate hypothermia is one of the most robust and effective techniques available for reducing ischemic injury, its key mechanism still remains unclear. Our proteomic analysis of the brains of rats treated with a 2-h middle cerebral artery occlusion showed that postischemic hypothermia markedly potentiated a sustained increase in heat-shock protein 70 (Hsp70). The elevated Hsp70 level was confirmed by enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemical staining. Expression of other Hsp proteins was unaffected by hypothermia. Interestingly, hypothermia did not increased, even decreased, the upregulation of hsp70 mRNA expression by ischemia, suggesting that Hsp70 abundance is controlled by an unknown posttranscriptional regulation. As Hsp70 exerts a protective role against ischemic damage, the specific increase in Hsp70 production may contribute to the neuroprotective effect of hypothermia.

Advantages of Human Hepatocyte-Derived Transformants Expressing a Series of Human Cytochrome P450 Isoforms for Genotoxicity Examination

Metabolites of chemicals can often be ultimate genotoxic species; thus, in vitro routine testing requires the use of rat liver S9. However, there is a question as to whether this represents an appropriate surrogate for human metabolism. We have previously demonstrated the usefulness of HepG2 transformants expressing major human cytochrome P450 (CYP) isoforms to assess the genotoxicity of metabolites. We further assessed the advantages of these transformants from the following three aspects. First, the sensitivity of these transformants was confirmed with micronucleus (MN) induction by 7,12-dimethylbenz[a]anthracene or ifosfamide in transformants expressing the corresponding CYP1A1 or CYP2B6 and CYP2C9, respectively. Second, by using these transformants, beta-endosulfan, a chemical for which the CYP isoforms contributing to its genotoxicity are unknown, was found to induce MN through the CYP3A4-mediated pathway. This result was confirmed by the facts that the decreased CYP3A4 activity using a inhibitor or short interfering RNA (siRNA) repressed MN induction by beta-endosulfan and that endosulfan sulfate, one of the metabolites produced by CYP3A4, induced MN in the transformants harboring an empty vector. Third, the interaction between phase I and II drug-metabolizing enzymes was demonstrated by MN induction with inhibitors of uridine diphosphate (UDP)-glucuronosyltransferases in tamoxifen-treated transformants harboring the corresponding CYP3A4 or with inhibitors of glutathione S-transferase in safrole-treated transformants harboring the corresponding CYP2D6, whereas neither tamoxifen nor safrole alone induced MN in any transformant. These advantages provide the benefits of newly established transformants for in vitro genotoxicity testing that reflects comprehensive metabolic pathways including not only human CYP isoforms but also the phase II enzymes.

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

Abstract 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.

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‐14 C]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). Copyright

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

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

Abstract 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.