Changes in glucose-induced plasma active glucagon-like peptide-1 levels by co-administration of sodium-glucose cotransporter inhibitors with dipeptidyl peptidase-4 inhibitors in rodents.

We investigated whether structurally different sodium-glucose cotransporter (SGLT) 2 inhibitors, when co-administered with dipeptidyl peptidase-4 (DPP4) inhibitors, could enhance glucagon-like peptide-1 (GLP-1) secretion during oral glucose tolerance tests (OGTTs) in rodents. Three different SGLT inhibitors-1-(ß-d-Glucopyranosyl)-4-chloro-3-[5-(6-fluoro-2-pyridyl)-2-thienylmethyl]benzene (GTB), TA-1887, and canagliflozin-were examined to assess the effect of chemical structure. Oral treatment with GTB plus a DPP4 inhibitor enhanced glucose-induced plasma active GLP-1 (aGLP-1) elevation and suppressed glucose excursions in both normal and diabetic rodents. In DPP4-deficient rats, GTB enhanced glucose-induced aGLP-1 elevation without affecting the basal level, whereas metformin, previously reported to enhance GLP-1 secretion, increased both the basal level and glucose-induced elevation. Oral treatment with canagliflozin and TA-1887 also enhanced glucose-induced aGLP-1 elevation when co-administered with either teneligliptin or sitagliptin. These data suggest that structurally different SGLT2 inhibitors enhance plasma aGLP-1 elevation and suppress glucose excursions during OGTT when co-administered with DPP4 inhibitors, regardless of the difference in chemical structure. Combination treatment with DPP4 inhibitors and SGLT2 inhibitors having moderate SGLT1 inhibitory activity may be a promising therapeutic option for improving glycemic control in patients with type 2 diabetes mellitus.

Intestinal Sodium Glucose Cotransporter 1 Inhibition Enhances Glucagon-Like Peptide-1 Secretion in Normal and Diabetic Rodents.

The sodium glucose cotransporter (SGLT) 1 plays a major role in glucose absorption and incretin hormone release in the gastrointestinal tract; however, the impact of SGLT1 inhibition on plasma glucagon-like peptide-1 (GLP-1) levels in vivo is controversial. We analyzed the effects of SGLT1 inhibitors on GLP-1 secretion in normoglycemic and hyperglycemic rodents using phloridzin, CGMI [3-(4-cyclopropylphenylmethyl)-1-(ß-d-glucopyranosyl)-4-methylindole], and canagliflozin. These compounds are SGLT2 inhibitors with moderate SGLT1 inhibitory activity, and their IC50 values against rat SGLT1 and mouse SGLT1 were 609 and 760 nM for phloridzin, 39.4 and 41.5 nM for CGMI, and 555 and 613 nM for canagliflozin, respectively. Oral administration of these inhibitors markedly enhanced and prolonged the glucose-induced plasma active GLP-1 (aGLP-1) increase in combination treatment with sitagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, in normoglycemic mice and rats. CGMI, the most potent SGLT1 inhibitor among them, enhanced glucose-induced, but not fat-induced, plasma aGLP-1 increase at a lower dose compared with canagliflozin. Both CGMI and canagliflozin delayed intestinal glucose absorption after oral administration in normoglycemic rats. The combined treatment of canagliflozin and a DPP4 inhibitor increased plasma aGLP-1 levels and improved glucose tolerance compared with single treatment in both 8- and 13-week-old Zucker diabetic fatty rats. These results suggest that transient inhibition of intestinal SGLT1 promotes GLP-1 secretion by delaying glucose absorption and that concomitant inhibition of intestinal SGLT1 and DPP4 is a novel therapeutic option for glycemic control in type 2 diabetes mellitus.

Novel Indole-N-glucoside, TA-1887 As a Sodium Glucose Cotransporter 2 Inhibitor for Treatment of Type 2 Diabetes.

Inhibition of the renal sodium glucose cotransporter (SGLT) increases urinary glucose excretion (UGE) and thus reduces blood glucose levels during hyperglycemia. To explore the potential of new antihyperglycemic agents, we synthesized and determined the human SGLT2 (hSGLT2) inhibitory potential of novel substituted 3-benzylindole-N-glucosides 6. Optimization of 6 resulted in the discovery of 3-(4-cyclopropylbenzyl)-4-fluoroindole-N-glucoside 6a-4 (TA-1887), a highly potent and selective hSGLT2 inhibitor, with pronounced antihyperglycemic effects in high-fat diet-fed KK (HF-KK) mice. Our results suggest the potential of indole-N-glucosides as novel antihyperglycemic agents through inhibition of renal SGLT2.

N-Glucosides as human sodium-dependent glucose cotransporter 2 (hSGLT2) inhibitors.

Inhibition of renal sodium-dependent glucose cotransporter 2 (SGLT2) increases urinary glucose excretion (UGE), and thus reduces blood glucose levels in hyperglycemia. A series of N-glucosides was synthesized for biological evaluation as human SGLT2 (hSGLT2) inhibitors. Among these compounds, N-glucoside 9d possessing an indole core structure showed good in vitro activity (IC50=7.1 nM against hSGLT2). Furthermore, 9d exhibited favorable in vivo potency with regard to UGE in rats based on good pharmacokinetic profiles.

C-Glucosides with heteroaryl thiophene as novel sodium-dependent glucose cotransporter 2 inhibitors.

Canagliflozin (1), a novel inhibitor for sodium-dependent glucose cotransporter 2 (SGLT2), has been developed for the treatment of type 2 diabetes. To investigate the effect of replacement of the phenyl ring in 1 with heteroaromatics, C-glucosides 2 were designed, synthesized, and evaluated for their inhibitory activities against SGLT2. Of these, 3-pyridyl, 2-pyrimidyl or 5-membered heteroaryl substituted derivatives showed highly potent inhibitory activity against SGLT2, while 5-pyrimidyl substitution was associated with slightly reduced activity. In particular, 2g (TA-3404) had remarkable anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice.

Impact of intestinal glucuronidation on the pharmacokinetics of raloxifene.

Raloxifene is extensively glucuronidated in humans, effectively reducing its oral bioavailability (2%). It was also reported to be glucuronidated in preclinical animals, but its effects on the oral bioavailability have not been fully elucidated. In the present study, raloxifene and its glucuronides in the portal and systemic blood were monitored in Gunn rats deficient in UDP-glucuronosyltransferase (UGT) 1A, Eisai hyperbilirubinemic rats (EHBRs), which hereditarily lack multidrug resistance-associated protein (MRP) 2, and wild-type rats after oral administration. The in vitro-in vivo correlation (IVIVC) of four UGT substrates (raloxifene, biochanin A, gemfibrozil, and mycophenolic acid) in rats was also evaluated. In Gunn rats, the product of fraction absorbed and intestinal availability and hepatic availability of raloxifene were 0.63 and 0.43, respectively; these values were twice those observed in wild-type Wistar rats, indicating that raloxifene was glucuronidated in both the liver and intestine. The ratio of glucuronides to unchanged drug in systemic blood was substantially higher in EHBRs (129-fold) than in the wild-type Sprague-Dawley rats (10-fold), suggesting the excretion of raloxifene glucuronides caused by MRP2. The IVIVC of the other UGT substrates in rats displayed a good relationship, but the oral clearance values of raloxifene and biochanin A, which were extensively glucuronidated by rat intestinal microsomes, were higher than the predicted clearances using rat liver microsomes, suggesting that intestinal metabolism may be a great contributor to the first-pass effect. Therefore, evaluation of intestinal and hepatic glucuronidation for new chemical entities is important to improve their pharmacokinetic profiles.

Discovery of canagliflozin, a novel C-glucoside with thiophene ring, as sodium-dependent glucose cotransporter 2 inhibitor for the treatment of type 2 diabetes mellitus.

We discovered that C-glucosides 4 bearing a heteroaromatic ring formed metabolically more stable inhibitors for sodium-dependent glucose cotransporter 2 (SGLT2) than the O-glucoside, 2 (T-1095). A novel thiophene derivative 4b-3 (canagliflozin) was a highly potent and selective SGLT2 inhibitor and showed pronounced anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice.

Effect of plasma protein binding on in vitro-in vivo correlation of biliary excretion of drugs evaluated by sandwich-cultured rat hepatocytes.

In the present study, we examined in vitro biliary clearance of several compounds in sandwich-cultured rat hepatocytes (SCRH) and compared it with that observed in vivo in rats; the effect of plasma protein binding on in vitro-in vivo correlation of biliary excretion was also assessed. The in vitro biliary excretion was determined by differential cumulative uptake of compounds in SCRH preincubated in the presence and absence of Ca(2+)/Mg(2+). The cumulative uptake study of radiolabeled substrates revealed that the function of canalicular efflux transporters such as bile salt export pump, multidrug resistance-associated protein 2, breast cancer resistance protein, and multidrug resistance 1 was adequately maintained in SCRH. Unlabeled test compounds, pravastatin, rosuvastatin, valsartan, cefmetazole, and cefoperazone exhibited varying degrees of in vitro biliary excretion in the cumulative uptake study using SCRH. In vivo biliary excretions of these compounds were measured in common bile duct-cannulated rats. Whereas their biliary excretion ratios were all more than 60% of the dose, the in vivo intrinsic biliary clearances varied from 10.5 to 1787.2 ml/min/kg. The in vitro intrinsic biliary clearances of test compounds were well correlated with their corresponding in vivo intrinsic clearances calculated on the basis of the plasma unbound concentration (r(2) = 0.984), whereas less correlation was observed when they were calculated on the basis of plasma total concentration (r(2) = 0.217). These results indicate that SCRH is a useful in vitro model for predicting in vivo intrinsic biliary clearance in rats. In addition, for an accurate prediction, it is necessary to evaluate the in vivo intrinsic biliary clearance based on plasma unbound concentration but not total concentration.

Characterization of hepatobiliary transport systems of a novel alpha4beta1/alpha4beta7 dual antagonist, TR-14035.

PURPOSE: Our previous pharmacokinetic studies have demonstrated that TR-14035, a novel dual antagonist for alpha4beta1/alpha4beta7 integrin, selectively and strongly accumulated in the liver and was mainly excreted in bile as an unchanged drug. In the present study, we investigated the hepatobiliary transport system in detail. MATERIALS AND METHODS: Uptake by hepatocytes and organic anion transporting polypeptide (OATP)-expressing Xenopus laevis oocytes or Flp-In-293 cells was performed in vitro. Biliary excretion was investigated in mdr1a/b-knockout mice, Bcrp-knockout mice and Mrp2-defective Eisai hyperbilirubinemic rats (EHBRs). RESULTS: TR-14035 was taken up by rat and human hepatocytes by an apparently single saturable mechanism with K(m) of 6.7 and 2.1 microM, respectively, and taurocholate and digoxin reduced this uptake. OATP1B1/OATP-C and OATP1B3/OATP8 expressed in oocytes mediated the TR-14035 uptake with K(m) of 7.5 and 5.3 microM, respectively. OATP1B1*15, a genetic variant of OATP1B1, exhibited a decreased transport of TR-14035 compared with OATP1B1*1a. Biliary excretion and total body clearance of unchanged TR-14035 in EHBRs were significantly lower than those in normal rats, while there was no difference in the clearances between wild and mdr1a/b- or Bcrp-knockout mice. CONCLUSION: These results indicate that OATP1B1 and OATP1B3 are at least partly responsible for the accumulation of TR-14035 into hepatocytes, and Mrp2 principally mediates the biliary excretion of TR-14035. Furthermore, genetic polymorphisms of OATP1B1 may cause an interindividual variability in the pharmacokinetics of TR-14035.

Role of human liver cytochrome P450 2C9 in the metabolism of a novel alpha4beta1/alpha4beta7 dual antagonist, TR-14035.

The metabolism of a novel dual antagonist for alpha4beta1/alpha4beta7 integrin, TR-14035, and the role of polymorphic enzyme responsible for this metabolism were investigated. Human liver microsomes catalyzed the NADPH-dependent metabolism of TR-14035 to a primary metabolite, O-desmethyl TR-14035. This formation was completely blocked by both sulfaphenazole, a selective CYP2C9 inhibitor, and CYP2C9 antibody, whereas potent inhibitors selective for other CYPs exhibited little effects. Of 12 recombinant CYPs examined, O-desmethyl metabolite was principally formed by CYP2C9. CYP1A1, an extrahepatic enzyme, also had this activity (about one-fourth of CYP2C9). Utilizing recombinant CYP2C9*1, K(m) and V(max)/K(m) values of 23.3 microM and 0.284 microL/min/pmol CYP2C9, respectively, were obtained for the O-desmethyl formation, which were quite similar to those in CYP2C9*2 enzyme. In contrast, V(max)/K(m) value in recombinant CYP2C9*3 was approximately one-sixth of CYP2C9*1 and *2. In agreement, kinetics studies using human liver microsomes with CYP2C9*1/*1, *2/*2 and *3/*3 genotypes revealed that the V(max)/K(m) value in *2/*2 microsomes was comparable to that in wild type microsomes, in contrast, that in *3/*3 microsomes was reduced. These results demonstrate CYP2C9 is a primary enzyme mediating the O-desmethylation of TR-14035 in human liver. In homozygotes of CYP2C9*3, the metabolic clearance of TR-14035 should be decreased compared with homozygotes of CYP2C9*1 or 2.