Involvement of organic cation transporters in the clearance and milk secretion of thiamine in mice.

PURPOSE: To investigate the role of organic cation transporters (Octs) and multidrug and toxin extrusion protein 1 (Mate1) in the disposition of thiamine. METHODS: The uptake of [(3)H]thiamine was determined in Oct1-, Oct2-, and Oct3-expressing HEK293 cells and freshly isolated hepatocytes. A pharmacokinetic study of thiamine-d3 following intravenous infusion (1 and 100 nmol/min/kg) was conducted in male Oct1/2(+/+) and Oct1/2(-/-) mice. A MATE inhibitor, pyrimethamine, (5 mg/kg) was administered intravenously. The plasma and breast milk concentrations of thiamine were determined in female mice. RESULTS: Thiamine is a substrate of Oct1 and Oct2, but not Oct3. Oct1/2 defect caused a significant reduction in the uptake of [(3)H]thiamine by hepatocytes in vitro, and elevated the plasma thiamine concentration by 5.8-fold in vivo. The plasma clearance of thiamine-d3 was significantly decreased in Oct1/2(-/-) mice. At the higher infusion rate of 100 nmol/min/kg thiamine-d3, Oct1/2 defect or pyrimethamine-treatment caused a significant reduction in the renal clearance of thiamine-d3. The total thiamine and thiamine-d3 concentrations were moderately reduced in the intestine of Oct1/2(-/-) mice but were unchanged in the kidney, liver, or brain. The milk-to-plasma concentration ratio of thiamine was decreased by 28-fold in the Oct1/2(-/-) mice. CONCLUSIONS: Oct1 is possibly responsible for the plasma clearance of thiamine via tissue uptake and for milk secretion. Oct1/2 and Mate1 are involved in the renal tubular secretion of thiamine.

Metabolism of N,N-dipropyl-2-[4-methoxy-3-(2-phenyl-ethoxy)-phenyl]-ethyl-amine-monohydrochloride (NE-100), a novel sigma ligand: contribution of cytochrome P450 forms involved in the formation of individual metabolites in human liver and small intestine.

In the present study, human cytochrome P450 (CYP) forms involved in producing the primary metabolites of NE-100 were identified. Major metabolites of NE-100 in human liver microsomes (HLM) were N-depropylation of NE-100 (NE-098), p-hydroxylation of phenyl group of NE-100 (NE-152), m-hydroxylation of phenyl group of NE-100 (NE-163) and O-demethylation of NE-100 (NE-125). Judging from the correlation and inhibition studies, NE-125 and NE-152+163mix formations were predominantly mediated by CYP2D6 and NE-098 formation was mediated by multiple CYP forms at a low NE-100 concentration (0.1 microM) in the HLM. According to relative activity factor (RAF) approaches, all these reactions were predominantly catalyzed by CYP2D6 at a substrate concentration assuming a plasma level of NE-100 (K(m)>>S) in case of the human liver. Depending on the increase in NE-100 concentrations, the rate of contribution for NE-098 and NE-152+163mix formations increased in CYP3A4, although the predominant contribution of CYP2D6 for NE-125 formation did not change. In human intestinal microsomes (HIM), NE-100 was mainly metabolized to NE-098 and NE-152+163mix by CYP3A4. The intrinsic clearance for their formations in HIM was 3.2 and 14.9 times less than those in HLM, respectively, and no formation of NE-125 was observed in HIM. These results strongly suggest that CYP2D6 is the predominant form for NE-100 metabolism in the human liver in in vivo conditions (K(m)>>S) and the liver plays a more important role than does the small intestine in the first pass metabolism.

(1S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio-D-glucitol (TS-071) is a potent, selective sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor for type 2 diabetes treatment.

Derivatives of a novel scaffold, C-phenyl 1-thio-D-glucitol, were prepared and evaluated for sodium-dependent glucose cotransporter (SGLT) 2 and SGLT1 inhibition activities. Optimization of substituents on the aromatic rings afforded five compounds with potent and selective SGLT2 inhibition activities. The compounds were evaluated for in vitro human metabolic stability, human serum protein binding (SPB), and Caco-2 permeability. Of them, (1S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio-D-glucitol (3p) exhibited potent SGLT2 inhibition activity (IC(50) = 2.26 nM), with 1650-fold selectivity over SGLT1. Compound 3p showed good metabolic stability toward cryo-preserved human hepatic clearance, lower SPB, and moderate Caco-2 permeability. Since 3p should have acceptable human pharmacokinetics (PK) properties, it could be a clinical candidate for treating type 2 diabetes. We observed that compound 3p exhibits a blood glucose lowering effect, excellent urinary glucose excretion properties, and promising PK profiles in animals. Phase II clinical trials of 3p (TS-071) are currently ongoing.

Deuterium-resistant algal cell line for D labeling of heterotrophs expresses enhanced level of Hsp60 in D2O medium.

Fully deuterated components from autotrophic cell lysate are useful materials for labeling of heterotrophs with deuterium. To facilitate the faster production of deuterated algal lysate, we selected a mutant Chlorella strain that grows faster in heavy water than the wild type. The mutant DR-17 was found to have a higher level of Hsp60 and an elevated level of protein synthesis. We previously isolated a deuterium-resistant yeast cell line that was also found to express elevated level of Hsp70 (K. Unno, T. Kishido, M. Morioka, S. Okada, and N. Oku, Biol. Pharm. Bull. 26:799-802, 2003). This suggests that the overexpression of heat shock proteins is required to compensate for the deuterium isotope effect.

Differences in cytochrome P450 forms involved in the metabolism of N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a novel sigma ligand, in human liver and intestine.

N,N-Dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100) has been developed to treat subjects with schizophrenia. This drug is mainly excreted in the form of oxidative metabolites. In the present study, identification of p450 forms involved in the metabolism was carried out using human livers and intestinal microsomes (HLM and HIM). Eadie-Hofstee plots for NE-100 disappearance in HLM were biphasic, thus indicating the involvement of at least two p450 forms. The metabolism of NE-100 was mediated with recombinant CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. A significant correlation was observed between activities of NE-100 metabolism and dextromethorphan O-demethylation (a specific activity for CYP2D6) or testosterone 6beta-hydroxylation (a specific activity for CYP3A4) in HLM. The activity of NE-100 metabolism was inhibited by approximately 80% by an anti-CYP2D6 antibody and only by quinidine among the p450-selective inhibitors at a low substrate concentration (0.1 microM). In contrast, with a high substrate concentration (10 microM), the activity was inhibited by an anti-CYP3A4 antibody and by ketoconazole. On the other hand, in HIM, the Eadie-Hofstee plots for NE-100 disappearance were monophasic, and the metabolism was strongly inhibited by an anti-CYP3A4 antibody and by ketoconazole but not by other inhibitors used. These results strongly suggest that NE-100 has different profiles regarding metabolism between liver and intestine. During absorption, NE-100 is mainly metabolized by CYP3A4 in the intestine and thereafter by CYP2D6 in the liver in the presence of therapeutic doses.