In vitro evaluation of potential drug interactions mediated by cytochrome P450 and transporters for luseogliflozin, an SGLT2 inhibitor.

1. We evaluated potential in vitro drug interactions of luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, mediated by CYP inhibition, CYP induction and drug transporters using human liver microsomes, primary hepatocytes and recombinant cells-expressing efflux or uptake transporters, respectively. 2. Human CYP inhibition studies indicated that luseogliflozin was a weak inhibitor for CYP2C19 with an IC50 value of 58.3 µM, whereas it was not an inhibitor of the other eight major isoforms that were tested. The exposure of primary hepatocytes to luseogliflozin for 72 hrs weakly induced CYP3A4 at a concentration of 10 µM, whereas it did not induce CYP1A2 or CYP2B6 at concentrations of 0.1-10 µM. 3. An in vitro transport study suggested that luseogliflozin is a substrate for human P-glycoprotein (P-gp), but not for breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1 and OATP1B3, organic anion transporter (OAT) 1 and OAT3, or organic cation transporter (OCT) 2. Luseogliflozin weakly inhibited OATP1B3 with an IC50 value of 93.1 µM, but those for other transporters are greater than 100 µM. 4. Based on the therapeutic plasma concentration of the drug, clinically relevant drug interactions are unlikely to occur between luseogliflozin and coadministered drugs mediated by CYPs and/or transporters.

Preclinical metabolism and disposition of luseogliflozin, a novel antihyperglycemic agent.

1. We investigated the metabolism and disposition of luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, in rats and dogs, as well as in vitro metabolism in rats, dogs and humans. In addition, we studied its localization in the rat kidney. 2. [14C]Luseogliflozin was rapidly and well absorbed (>86% of the dose) after oral administration to rats and dogs. The drug-derived radioactivity was mainly excreted via the feces in both species. 3. The predominant radioactivity component in the excreta was associated with the metabolites, with only a minor fraction of unchanged luseogliflozin. The major metabolites were two glucuronides (M8 and M16) in the rats, and the O-deethylated form (M2) and other oxidative metabolites (M3 and M17) in the dogs. 4. The in vitro metabolism in dog and human hepatocytes was significantly slower than that in the rat hepatocytes. The biotransformation in animal hepatocytes was similar to that observed in vivo. Incubation with human hepatocytes resulted in the formation of metabolites, including M2, M3, M8 and M17, via multiple metabolic pathways. 5. [14C]Luseogliflozin was well-distributed to its target organ, the kidney, and was found to be localized in the renal cortex, which shows SGLT2 expression. This characteristic distribution was inhibited by preinjection of phlorizin, an SGLT inhibitor, suggesting that the renal radioactivity was associated with SGLT2.

Lymphopenia induced by a novel selective S1P(1) antagonist structurally unrelated to S1P.

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P(1)) and participates in many pathological conditions. We developed a novel type S1P(1)-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P(1) resulting in reduced signaling downstream of S1P(1), including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P(1)-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P(1) antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P(1) agonists on lymphocyte sequestration results from their functional antagonism.

(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.

In vitro and in vivo evaluation of the metabolism and bioavailability of ester prodrugs of mgs0039 (3-(3,4-dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic Acid), a potent metabotropic glutamate receptor antagonist.

MGS0039 (3-(3,4-dichlorobenzyloxy)-2-amino-6-fluorobicyclo-[3.1.0]hexane-2,6-dicarboxylic acid) has been identified as a potent and selective antagonist for metabotropic glutamate receptors. However, the oral bioavailability of MGS0039 is 10.9% in rats, due to low absorption. Several prodrugs, synthesized to improve absorption, exhibited 40 to 70% bioavailability in rats. This study investigated in vitro metabolism using liver S9 fractions from both cynomolgus monkeys and humans and oral bioavailability in cynomolgus monkeys to select the prodrug most likely to exhibit optimal pharmacokinetic profiles in humans. In monkeys, transformation to active substance was observed (5.9-72.8%) in liver S9 fractions, and n-butyl, n-pentyl, 3-methylbutyl, and 4-methylpentyl ester prodrugs exhibited high transformation ratios (>64%). Cmax levels and F values after oral dosing increased to 4.1- to 6.3-fold and 2.4- to 6.3-fold, respectively, and a close relationship between transformation ratios and Cmax and F values was observed, indicating that the hydrolysis rate in liver S9 fractions is the key factor in determining oral bioavailability in monkeys. In humans, n-hexyl, n-heptyl, n-octyl, 5-methylbutyl, and 6-methylpentyl ester prodrugs exhibited high transformation ratios (>65%) in liver S9 fractions. With these prodrugs, n-hexyl, n-heptyl, and 5-methylpentyl ester, almost complete recovery (96-99%) was obtained. Given the transformation ratio, we anticipated that the n-heptyl alkyl ester prodrug would exhibit the highest oral bioavailability of active substances in humans, if the hydrolysis rate in liver S9 fractions is indeed the key factor in determining oral bioavailability in humans. On this basis, MGS0210 (3-(3,4-dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid n-heptyl ester) seems to be a promising candidate among MGS0039 prodrugs.

mRNA expression and amino acid transport characteristics of cultured human brain microvascular endothelial cells (hBME).

An in vitro cell culture system for estimating the human blood-brain barrier (BBB) permeability of drugs is required for the development of drugs with effects on the central nervous system. In this study, cultured human brain microvascular endothelial cells (hBME) were characterized. hBME cells exhibited concentration-dependent uptake of L-Leu, L-Glu and L-Lys with K(m) values of 51.1+/-23.1 microM, 163.3+/-79.8 microM and 72.4+/-56.6 microM, respectively. The cellular accumulation of rhodamine123 in hBME cells was unaffected by P-glycoprotein (P-gp) substrates (cyclosporin A, quinidine and verapamil), while the accumulation in human P-gp-overexpressing cells was significantly increased in the presence of these P-gp substrates. RT-PCR revealed that hBME cells expressed large neutral amino acid transporter 1 (LAT1) and its associated molecule (4F2hc), excitatory amino acid transporter 3 (EAAT3), cationic amino acid transporter 1 (CAT1), glucose transporter 1 (GLUT1), monocarboxylic acid transporter 1 (MCT1) and multidrug resistance-associated protein 1 (MRP1). However, no expression of multidrug resistance protein 1 (MDR1) was detected. The results suggest that these amino acid transporters are functionally expressed at the human BBB, and that hBME cells retain the in vivo BBB transport functions and expression characteristics. Consequently, hBME cells should be a useful tool for studies of the human BBB.