Minoru Uchiyama

Inhibition of in vitro metabolism of simvastatin by itraconazole in humans and prediction of in vivo drug-drug interactions

AbstractPurpose. To evaluate an interaction between simvastatin and itraconazole in in vitro studies and to attempt a quantitative prediction of in vivo interaction in humans. Methods. The inhibitory effect of itraconazole on simvastatin metabolism was evaluated using human liver microsomes and the Ki values were calculated for the unbound drug in the reaction mixture. A physiologically-based pharmacokinetic model was used to predict the maximum in vivo drug-drug interaction. Results. Itraconazole competitively inhibited the metabolism of simvastatin to M-1 and M-2 with Ki values in the nM range. The area under the curve (AUC) of simvastatin after concomitant dosing with itraconazole was predicted to increase ca. 84-101-fold compared with that without administration of itraconazole. Taking into consideration the fact that this method predicts the maximum interaction, this agrees well with the clinical observation of a 19-fold increase. A similar prediction, based on the Ki value without taking into account the drug adsorption to microsomes, led to an underevaluation of the interaction. Conclusions. It was demonstrated that the competitive inhibition of CYP3A4-mediated simvastatin metabolism by itraconazole is the main cause of the drug interaction and that a Ki value corrected for drug adsorption to microsomes is the key factor in quantitatively predicting the maximum in vivo drug interactions.

Characterization of phenotypes in Gstm1-null mice by cytosolic and in vivo metabolic studies using 1,2-dichloro-4-nitrobenzene.

Glutathione S-transferase Mu 1 (GSTM1) has been regarded as one of the key enzymes involved in phase II reactions in the liver, because of its high expression level. In this study, we generated mice with disrupted glutathione S-transferase Mu 1 gene (Gstm1-null mice) by gene targeting, and characterized the phenotypes by cytosolic and in vivo studies. The resulting Gstm1-null mice appeared to be normal and were fertile. Expression analyses for the Gstm1-null mice revealed a deletion of Gstm1 mRNA and a small decrease in glutathione S-transferase alpha 3 mRNA. In the enzymatic study, GST activities toward 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) in the liver and kidney cytosols were markedly lower in Gstm1-null mice than in the wild-type control. Gstm1-null mice had GST activities of only 6.1 to 21.0% of the wild-type control to DCNB and 26.0 to 78.6% of the wild-type control to CDNB. After a single oral administration of DCNB to Gstm1-null mice, the plasma concentration of DCNB showed larger AUC0–24 (5.1–5.3 times, versus the wild-type control) and higher Cmax (2.1–2.2 times, versus the wild-type control), with a correspondingly lower level of glutathione-related metabolite (AUC0–24, 9.4–17.9%; and Cmax, 9.7–15.6% of the wild-type control). In conclusion, Gstm1-null mice showed markedly low ability for glutathione conjugation to DCNB in the cytosol and in vivo and would be useful as a deficient model of GSTM1 for absorption, distribution, metabolism, and excretion/toxicology studies.

Identification of novel metabolic pathways of pioglitazone in hepatocytes: N-glucuronidation of thiazolidinedione ring and sequential ring opening pathway

The metabolism of [14C]pioglitazone was studied in vitro in incubations with freshly isolated human, rat, and monkey hepatocytes. Radioactivity detection high-performance liquid chromatography analysis of incubation extracts showed the detection of 13 metabolites (M1–M13) formed in incubations with human hepatocytes. An identical set of metabolites (M1–M13) was also detected in monkey hepatocytes. However, in rat hepatocytes, M1 through M3, M5 through M7, M9 through M11, and M13 were also detected, but M4, M8, and M12 were not detected. The structures of the metabolites were elucidated by liquid chromatography/tandem mass spectrometry using electrospray ionization. Novel metabolites of pioglitazone detected using these methods included thiazolidinedione ring-opened methyl sulfoxide amide (M1), thiazolidinedione ring-opened N-glucuronide (M2), thiazolidinedione ring-opened methyl sulfone amide (M3), thiazolidinedione ring N-glucuronide (M7), thiazolidinedione ring-opened methylmercapto amide (M8), and thiazolidinedione ring-opened methylmercapto carboxylic acid (M11). In summary, based on the results from these studies, two novel metabolic pathways for pioglitazone in hepatocytes are proposed to be as follows: 1) N-glucuronidation of the thiazolidinedione ring of pioglitazone to form M7 followed by hydrolysis to M2, and methylation of the mercapto group of the thiazolidinedione ring-opened mercapto carboxylic acid to form M11; and 2) methylation of the mercapto group of the thiazolidinedione ring-opened mercapto amide to form M8, oxidation of M8 to form M1, and oxidation of M1 to form M3.

Pharmacokinetics, metabolism, and disposition of rivoglitazone, a novel peroxisome proliferator-activated receptor γ agonist, in rats and monkeys

The pharmacokinetics, metabolism, and excretion of rivoglitazone [(RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride], a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, were evaluated in male F344/DuCrlCrlj rats and cynomolgus monkeys. The total body clearance and volume of distribution of rivoglitazone were low in both animals (0.329–0.333 ml per min/kg and 0.125–0.131 l/kg for rats and 0.310–0.371 ml per min/kg and 0.138–0.166 l/kg for monkeys), and the plasma half-life was 4.55 to 4.84 h for rats and 6.21 to 6.79 h for monkeys. The oral bioavailability was high (>95% in rats and >76.1% in monkeys), and the exposure increased dose proportionally. After administration of [14C]rivoglitazone, radioactivity was mainly excreted in feces in rats, whereas radioactivity was excreted in urine and feces with the same ratio in monkeys. Because excreted rivoglitazone in urine and bile was low, metabolism was predicted to be the main contributor to total body clearance. The structures of 20 metabolites (M1–M20) were identified, and 5 initial metabolic pathways were proposed: O-demethylation, TZD ring opening, N-glucuronidation, N-demethylation, and TZD ring hydroxylation. O-Demethylation was the main metabolic pathway in both animals, but N-demethylation and TZD ring hydroxylation were observed only in monkeys. N-Glucuronide (M13) was nonenzymatically hydrolyzed to TZD ring-opened N-glucuronide (M9), and the amount of these metabolites in monkeys was larger than that in rats. In plasma, rivolitazone was observed as the main component in both animals, and O-demethyl-O-sulfate (M11) was observed as the major metabolite in rats but as many minor metabolites in monkeys.

In Vitro Metabolism of Rivoglitazone, a Novel Peroxisome Proliferator-Activated Receptor γ Agonist, in Rat, Monkey, and Human Liver Microsomes and Freshly Isolated Hepatocytes

The in vitro metabolism of rivoglitazone, (RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride, a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, was studied in liver microsomes and freshly isolated hepatocytes of rat, monkey, and human as well as cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes. Fourteen metabolites were detected, and these structures were elucidated by liquid chromatography-tandem mass spectrometry. Five initial metabolic pathways of rivoglitazone consisting of four oxidation pathways and one N-glucuronidation pathway were predicted in correspondence with those proposed for in vivo studies using rats and monkeys. In metabolization using liver microsomes, the TZD ring-opened mercapto amide (M22) and TZD ring-opened mercapto carboxylic acid (M23) were identified as the primary metabolite of the TZD ring-opening pathway and its sequential metabolite, which have not been detected previously from in vivo studies. Combination with S-adenosyl-l-methionine was useful to obtain the sequential S-methylated metabolites from the oxidative metabolites. N-Glucuronide and sequential TZD ring-opened metabolites were also found in liver microsomes in the presence of UDP-glucuronic acid. The O-demethyl-O-sulfate (M11), which is the major in vivo metabolite in rats and monkeys, was detected in all species of hepatocytes. In addition, a TZD ring-opened S-cysteine conjugate (M15) was detected in human hepatocytes. From these results, the in vivo metabolic pathways in humans were predicted to be the four oxidation and one N-glucuronidation pathways. The four oxidative metabolites were formed by multiple human P450 enzymes, and N-glucuronide was formed by UGT1A3 and UGT2B7.

Pharmacokinetics of DS-5565, a novel α2δ ligand, in rats and monkeys and assessment of DDI risk

OBJECTIVE: We elucidated the pharmacokinetic profiles of DS-5565 in animals and drug interaction risk as perpetrator. BACKGROUND: DS-5565 is an oral analgesic drug that binds to the α 2 δ subunit of voltage-dependent Ca 2+ channels. DESIGN/METHODS: DS-5565 is the salt and the free form of DS-5565 is active moiety. Plasma concentration of the active moiety was determined by validated LC-MS/MS method. The profiles were investigated in F344 rats, streptozotocin-induced diabetic BN rats as neuropathic pain model, and cynomolgus monkeys. Distribution was assessed by whole body autoradioluminography following an oral administration of 14 C-labeled active moiety in F344 rats. Induction potential for CYP1A2 and CYP3A4 was assessed using fresh human hepatocytes. Inhibition potential for various CYP isoforms and drug transporters was tested using human liver microsomes and transporter overexpressing cells, respectively. RESULTS: The plasma exposure increased proportionally with the investigated dose in both strain and species. The bioavailability was higher than 85% in both species. The radioactivity was detected in almost tissues at 30 min but was primarily detectable in limited organs at 24 h post-dose. The plasma protein binding in rats, monkeys and humans in vitro was low. A few metabolites, which have not pharmacological activity, were detected qualitatively at low levels in plasma in both after oral administration. The primary excretion route of the radioactivity was urine; 蠅 87% of the dose was recovered within 7 days after an oral administration of the 14 C-labeled compound in rats and monkeys. Renal clearance of DS-5565 was higher than GFR in both animals, suggesting active secretion in the kidney. DS-5565 did not induce CYP1A2/3A4 in human hepatocytes, and not inhibit various CYP isoforms in human liver microsomes and drug transporters in overexpressing cells. CONCLUSIONS: Pharmacokinetics of DS-5565 in animals was favorable. DS-5565 has low potential to be perpetrator in drug-drug interaction. Disclosure: Dr. Yamamura has received personal compensation for activities with Daiichi Sankyo. Dr. Yamada has received personal compensation for activities with Daiichi Sankyo. Dr. Takahashi has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Uchiyama has received personal compensation for activities with Daiichi Phamaceutical Corp. Dr. Koda has received personal compensation for activities with Daiichi Pharmaceutical Corp. Dr. Mikkaichi has received personal compensation for activities with Daiichi Pharmaceutical Corp. Dr. Nishiya has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Honda has received personal compensation for activities with Daiichi Pharmaceutical Corp. Dr. Arakawa has received personal compensation for activities with Daiichi Pharmaceutical Corporation as an employee. Dr. Domon has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Fischer has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Mueller has received personal compensation for activities with Daiichi Pharmaceutical Corp.

DS11960558, A Water Soluble Prodrug of DS-2969b, for Intravenous Treatment of Clostridium difficile Infection

Abstract Background Clostridium difficile infection (CDI) is the most common cause of diarrhea in hospitals. The only available intravenous (IV) therapy for CDI is metronidazole, which showed only a 52.4% cure rate in a prospective observational study. DS11960558 is a water-soluble prodrug of DS-2969b, which is a novel GyrB inhibitor in clinical development for oral treatment of CDI and has been found to be safe and tolerable in its phase 1 study. The in vivo efficacy and physicochemical, pharmacokinetic, and toxicological profiles of the prodrug were evaluated in this study. Methods Efficacy was evaluated in a hamster CDI model. The animals were primed with a single subcutaneous (SC) clindamycin injection. Then, the infection was induced by oral gavage of C. difficile 2009155 (NAP1/027) spores. Treatment was initiated 6 hours post infection and repeated for 5 days. The animals were observed for survival and death once daily for 35 days. The physicochemical, pharmacokinetic, and toxicological profiles were evaluated by standard methods. Results SC administration of the prodrug showed comparable and superior efficacy to oral (PO) administration of DS-2969b and the combination of metronidazole (SC) and vancomycin (PO), respectively, in the hamster CDI model (Figure 1). The solubility of DS-2969b was 0.4 mg/mL while that of the prodrug was much higher, >100 mg/mL. The prodrug was converted to DS-2969a (free form of DS-2969b) rapidly after IV administration, and the conversion ratio was >80% (Figure 2). The main metabolites in rat urine and feces were oxidized forms of DS-2969a, and there were no prodrug-specific metabolites. Fecal excretion of DS-2969a was similar between IV administration of the prodrug and PO administration of DS-2969b. Most of the radioactivity was recovered after IV administration of the 14C-labeled prodrug in rats. The radioactivity was distributed widely in most tissues including the intestinal lumen, similar to the distribution of DS-2969b in rats. In safety pharmacology, genotoxicity, and rat 14-day repeated dose toxicity studies, the prodrug as well as DS-2969b did not show any significant findings. Conclusion These results support development of DS11960558 as an alternative IV treatment option for CDI patients who cannot take medicine orally. Disclosures M. Yamada, Daiichi Sankyo Co., Ltd.: Employee, Salary; M. Uchiyama, Daiichi Sankyo Co., Ltd.: Employee, Salary; S. I. Inoue, Daiichi Sankyo Co., Ltd.: Employee, Salary; T. Deguchi, Daiichi Sankyo Co., Ltd.: Employee, Salary; Y. Furuta, Daiichi Sankyo Co., Ltd.: Employee, Salary; K. Yabe, Daiichi Sankyo Co., Ltd.: Employee, Salary; N. Masuda, Daiichi Sankyo Co., Ltd.: Employee, Salary

A Phase I, Single‐Ascending‐Dose Study in Healthy Subjects to Assess the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of DS‐2969b, a Novel GyrB Inhibitor

DS‐2969b is a novel GyrB inhibitor in development for the treatment of Clostridium difficile infection. The aim of this study was to assess the safety, tolerability, pharmacokinetics, and effects on normal gastrointestinal microbiota groups of single daily oral ascending doses of DS‐2969b in healthy subjects. The study enrolled 6 sequential ascending dose cohorts (6 mg, 20 mg, 60 mg, 200 mg, 400 mg, and 600 mg). In each cohort, 6 subjects were administered DS‐2969b and 2 subjects were administered matching placebo. DS‐2969b was safe and well tolerated at all dose levels examined. All adverse events related to DS‐2969b were mild to moderate in severity and predominantly related to the gastrointestinal tract. DS‐2969a (free form of DS‐2969b) plasma concentrations increased with increasing doses; however, both the maximum serum concentration and area under the plasma concentration–time curve generally increased less than dose proportionally. DS‐2969a was predominantly eliminated in the urine, with feces as a minor route of elimination. While the overall proportion of DS‐2969a eliminated in the feces was low, target fecal levels sufficient for C. difficile eradication were achieved within 24 hours of administration with doses of 60 mg or higher. In exploratory analyses, DS‐2969b appeared to reduce bacterial counts in 8 of the 25 groups of normal intestinal microbiota examined, suggesting that DS‐2969b has only a mild effect on intestinal microbiota. Data from this study support and encourage further development of DS‐2969b as a novel treatment for C. difficile infection.

Abstract 1311: Disposition and biotransformation of [14C]-radiolabeled CS-7017 in healthy male subjects

Background: CS-7017 is a novel, oral, highly potent and selective peroxisome proliferator-activated receptor gamma agonist with demonstrated anticancer activity in preclinical models. Currently, CS-7017 is in phase 2 trials for the treatment of non-small cell lung and colorectal cancer. CS-7017 disposition and biotransformation were investigated in healthy male subjects following oral administration of [14C]-radiolabeled CS-7017. Methods: Blood, plasma, urine, and fecal samples were collected and analyzed for radioactive levels to determine CS-7017 disposition following a single 0.5 mg oral dose containing approximately 50 µCi of [14C]-radiolabeled CS-7017 in 6 healthy male subjects. Metabolite separation, identification, and quantification were performed using radio/HPLC followed by LC-ESI/MS and LC-MS/MS. Plasma samples were also analyzed using LC-MS/MS for CS-7017 and by accelerator mass spectrometry for metabolite profiling. Results: CS-7017 was rapidly absorbed (plasma Tmax: 1.52 h) and steadily eliminated following oral administration. The majority of radioactivity was eliminated in feces (∼70% vs ∼20% in urine). In blood, the radioactivity was mainly distributed in plasma with little or no association with blood cells, as indicated by a blood to plasma ratio of 0.5. The parent molecule is the main circulating analyte in plasma contributing 60-70% of the total exposure. R-239457 (dearyl form) and its sulphate were the two highest circulating metabolites contributing 4.5% and 6.3% of the total radioactivity at 12 h post-dose, respectively. Several other radioactive peaks (∼12) were detected in plasma at a low level ( Conclusion: CS-7017 was rapidly absorbed and steadily eliminated with the parent molecule contributing the majority of the exposure; this indicates that CS-7017 itself contributes toward in vivo pharmacological activity. Traces of the parent molecule in urine and feces indicate that the majority of the drug is eliminated by metabolism. Therefore, CS-7017 exposure may be impacted by hepatic impairment, but may not be significantly changed in subjects with impaired renal function as renal excretion contributes to a relatively low proportion of clearance (∼20%). Based on the metabolite profiles, the metabolic routes in human and animals (rats and monkeys) are similar. The two highest metabolites detected in human plasma were also present in the plasma of at least one of the preclinical toxicology species, therefore, safety concerns raised by the metabolites would be minimal in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1311. doi:10.1158/1538-7445.AM2011-1311