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.
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.
This article is available online at http://dmd.aspetjournals.org ABSTRACT: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 Odemethylation (a specific activity for CYP2D6) or testosterone 6-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 M). In contrast, with a high substrate concentration (10 M), 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.
1. It has previously been reported that N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) was predominantly metabolized by cytochrome P450 (CYP) 2D6 in human liver microsomes (HLM). In the present study, the contribution of CYP forms involved in the formation of the major metabolites of NE-100 in human liver lacking CYP2D6 activity (PM-HLM) has been predicted by use of in vitro kinetic data on recombinant CYPs microsomes (rCYPs). 2. In PM-HLM, NE-100 is predicted to be metabolized to N-despropyl-NE-100 (NE-098), p-hydroxy-NE-100 (NE-152) and m-hydroxyl-NE-100 (NE-163), but not to O-demethy-NE-100 (NE-125), which is a major metabolite in pooled human liver microsomes (EM-HLM). The relative activity factor approach assumed that NE-098 formation is predominantly catalysed by CYP3A4 and CYP2C9 and the NE-152+163mix (a mixture of two hydroxylated metabolites, NE-152 and NE-163) formation is only catalysed by CYP3A4. 3. The predicted contribution rates of CYP3A4 and CYP2C9 for NE-098 formation were 58.1 and 34.6%, respectively, in PM-HLM. These predicted results were strongly supported by kinetic and inhibition studies using PM-HLM. The intrinsic clearance of NE-100 predicted from rCYPs (the predicted CLint-HLM-total) corresponded to those observed from EM- and PM-HLM (the observed CLint-HLM). 4. The in vivo oral clearance (CLoral) of NE-100 in extensive metabolizers and poor metabolizers of CYP2D6 was predicted to be 50times higher in extensive metabolizers than poor metabolizers using in vitro-in vivo scaling method based on the dispersion model. These data suggest that polymorphism of CYP2D6 might greatly affect NE-100 metabolism in vivo.
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.Stable isotope labeling is an essential tool for studying the functional structure and assembly of proteins by nuclear magnetic resonance (NMR). While high deuteration levels of nonexchangeable sites in a protein are beneficial for main chain assignment, the complete exchange of D in exchangeable sites to H is required for proton detection. If amide protons involved in strong hydrogen bonds or buried inside the protein are not accessible to H in the solvent, the amides will remain in the deuterated form. Although this problem may be resolved by unfolding the protein using chemical denaturants followed by refolding in the presence of H 2 O, complete exchange and refolding is not always possible.The expression of proteins in fully deuterated algal lysate medium in 100% H 2 O has recently been described (6). Using this technique, deuterated samples were uniformly protonated at their amide sites, irrespective of the solvent exchange characteristics of the folded protein. We isolated a mutant clone to facilitate the production of deuterated growth media from algal Chlorella. 14 C-␣ aminoisobutyric acid (␣-AIB), which is an unavailable amino acid for protein synthesis, in 60% D 2 O medium at 25°C. The relative radioactivity of each precursor was 18.5 kBq/0.2 M/ml. The incubated cells were washed with icecold water. The incorporation of amino acid into cells was determined from the radioactivity of cells using a liquid scintillation counter (LSC). That of nucleic acids was determined from the radioactivity in the ethanol-insoluble fraction of cells. MATERIALS AND METHODS PreparationThe effect of D 2 O on protein synthesis was investigated using 35 S-L-methionine ( 35 S-Met; 185 kBq/0.2 M/ml) in 60 or 100% D 2 O medium. Labeled cells (2 ϫ 10 8 cells) were homogenized with glass beads. The homogenate was centrifuged at 200 ϫ g for 10 min and then at 9,000 ϫ g for 30 min, and the supernatant was applied to a Sephadex G-25 column (PD-10; Pharmacia Biotech). The radioactivity and protein concentration of these fractions were measured using LSC and a protein assay kit (Bio-Rad), respectively.Carbon fixation and amount of photosynthetic pigments. Fixation of 14 C was measured as described previously (13). Briefly, Chlorella cells (2 ϫ 10 7 cells/190 l) were preilluminated with 2.5 klx for 3 min at 25°C, and then 10 l of 10 mM Na...
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