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Significance Glucagon-like peptide-1 receptor agonists have become established as a leading class of diabetes medications. However, these peptide-based drugs are administered by subcutaneous injection or, in one case, by a complex oral dosing regimen. We now report the discovery of LY3502970, a potent and selective small-molecule GLP-1R agonist. LY3502970 exhibits preclinical pharmacology equivalent to a marketed injectable GLP-1R agonist and possesses pharmacokinetic properties compatible with oral dosing in humans. Cryoelectron microscopy (cryo-EM) studies reveal an ECD-driven receptor binding mode for LY3502970 that provides a favorable pharmacological profile., Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over β-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein–coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.

Activation of glucagon-like peptide-1 receptors (GLP-1R) is a promising approach for the treatment of type 2 diabetes mellitus (T2D), and several peptide analogues of GLP-1 have been successfully developed. Here we identified a new orally active non-peptide GLP-1R agonist, OWL833, and examined its efficacy in cynomolgus monkeys. The agonistic activity of OWL833 was evaluated by measuring cAMP accumulation in cells expressing mammal GLP-1Rs and other glucagon family receptors. In the cells expressing human or cynomolgus monkey GLP-1R, OWL833 promoted cAMP accumulation to the same level achieved by GLP-1, and its EC50 values for human and cynomolgus monkey GLP-1Rs were the same at 1.1 nmol/L. In contrast, OWL833 showed no stimulatory activity on human glucagon, GIP, and GLP-2 receptors. The predicted bioavailability and T1/2 of OWL833 in human based on rodent and non-rodent experimental data are 30% and ∼18 hour, respectively. After OWL833 followed by glucose was intravenously injected in cynomolgus monkeys, OWL833 decreased the blood glucose level via insulin secretion to the same extent as effective concentrations of exenatide, a GLP-1 peptide mimetic, in clinic. Doses of 0.and 0.1 mg/kg OWL833 given orally to cynomolgus monkeys reduced their food intake by 25% and 45%, respectively, as compared with vehicle group, and the effect on food intake was equal to that of 0.3 and 0.6 µg/kg subcutaneous injections of exenatide. No critical finding was observed in the subchronic toxicity studies in rats and cynomolgus monkeys. These findings demonstrate that OWL833 has full agonistic activity on human and cynomolgus GLP-1R and improves glucose tolerance by stimulating insulin secretion, and that it exhibits an anti-feeding activity similar to exenatide. In conclusion, OWL833 has potential as a once-daily, orally active, non-peptide GLP-1 receptor agonist. Disclosure T. Kawai: Employee; Self; Chugai Pharmaceutical Co., Ltd. F. Tanino: Employee; Self; Chugai Pharmaceutical Co., Ltd. M. Fukazawa: Employee; Self; Chugai Pharmaceutical Co., Ltd. K. Ogawa: Employee; Self; Chugai Pharmaceutical Co., Ltd. S. Nagao: Employee; Self; Chugai Pharmaceutical Co., Ltd. H. Yoshino: Employee; Self; Chugai Pharmaceutical Co., Ltd. S. Komatsu: Employee; Self; Chugai Pharmaceutical Co., Ltd. Y. Suzuki: Employee; Self; Chugai Pharmaceutical Co., Ltd. Y. Kawabe: Employee; Self; Chugai Pharmaceutical Co., Ltd..

Background and purpose Although inhibition of renal sodium-glucose co-transporter 2 (SGLT2) has a stable glucose-lowering effect in patients with type 2 diabetes, the effect of SGLT2 inhibition on renal dysfunction in type 2 diabetes remains to be determined. To evaluate the renoprotective effect of SGLT2 inhibition more precisely, we compared the effects of tofogliflozin (a specific SGLT2 inhibitor) with those of losartan (an angiotensin II receptor antagonist) on renal function and beta-cell function in db/db mice. Experimental approach The effects of 8-week tofogliflozin or losartan treatment on renal and beta-cell function were investigated in db/db mice by quantitative image analysis of glomerular size, mesangial matrix expansion and islet beta-cell mass. Blood glucose, glycated Hb and insulin levels, along with urinary albumin and creatinine were measured Key results Tofogliflozin suppressed plasma glucose and glycated Hb and preserved pancreatic beta-cell mass and plasma insulin levels. No improvement of glycaemic conditions or insulin level was observed with losartan treatment. Although the urinary albumin/creatinine ratio of untreated db/db mice gradually increased from baseline, tofogliflozin or losartan treatment prevented this increase (by 50-70%). Tofogliflozin, but not losartan, attenuated glomerular hypertrophy. Neither tofogliflozin nor losartan altered matrix expansion. Conclusions and implications Long-term inhibition of renal SGLT2 by tofogliflozin not only preserved pancreatic beta-cell function, but also prevented kidney dysfunction in a mouse model of type 2 diabetes. These findings suggest that long-term use of tofogliflozin in patients with type 2 diabetes may prevent progression of diabetic nephropathy.

To understand the risk of hypoglycemia associated with urinary glucose excretion (UGE) induced by sodium-glucose cotransporter (SGLT) inhibitors, it is necessary to know the relationship between the ratio of contribution of SGLT2 vs. SGLT1 to renal glucose reabsorption (RGR) and the glycemic levels in vivo. To examine the contributions of SGLT2 and SGLT1 in normal rats, we compared the RGR inhibition by tofogliflozin, a highly specific SGLT2 inhibitor, and phlorizin, an SGLT1 and SGLT2 (SGLT1/2) inhibitor, at plasma concentrations sufficient to completely inhibit rat SGLT2 (rSGLT2) while inhibiting rSGLT1 to different degrees. Under hyperglycemic conditions by glucose titration, tofogliflozin and phlorizin achieved ≥50% inhibition of RGR. Under hypoglycemic conditions by hyperinsulinemic clamp, RGR was reduced by 20–50% with phlorizin and by 1–5% with tofogliflozin, suggesting the smaller contribution of rSGLT2 to RGR under hypoglycemic conditions than under hyperglycemic conditions. Next, to evaluate the hypoglycemic potentials of SGLT1/2 inhibition, we measured the plasma glucose (PG) and endogenous glucose production (EGP) simultaneously after UGE induction by SGLT inhibitors. Tofogliflozin (400 ng/ml) induced UGE of about 2 mg·kg−1·min−1 and increased EGP by 1–2 mg·kg−1·min−1, resulting in PG in the normal range. Phlorizin (1,333 ng/ml) induced UGE of about 6 mg·kg−1·min−1 and increased EGP by about 4 mg·kg−1·min−1; this was more than with tofogliflozin, but the minimum PG was lower. These results suggest that the contribution of SGLT1 to RGR is greater under lower glycemic conditions than under hyperglycemic conditions and that SGLT2-selective inhibitors pose a lower risk of hypoglycemia than SGLT1/2 inhibitors.

Although mitogen-activated protein kinase kinase (MEK) is a key signaling molecule and a negative regulator of insulin action, it is still uncertain whether MEK can be a therapeutic target for amelioration of insulin resistance (IR) in type 2 diabetes (T2D)in vivo. To clarify whether MEK inhibition improves T2D, we examined the effect of continuous MEK inhibition with two structurally different MEK inhibitors, RO5126766 and RO4987655, in mouse models of T2D. RO5126766 and RO4987655 were administered via dietary admixture. Both compounds decreased blood glucose and improved glucose tolerance in doses sufficient to sustain inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation downstream of MEK in insulin-responsive tissues indb/dbmice. A hyperinsulinemic-euglycemic clamp test showed increased glucose infusion rate (GIR) indb/dbmice treated with these compounds, and about 60% of the increase was attributed to the inhibition of endogenous glucose production, suggesting that the liver is responsible for the improvement of IR. By means of adenovirus-mediatedMek1shRNA expression, we confirmed that blood glucose levels are reduced by suppression of MEK1 expression in the liver ofdb/dbmice. Taken together, these results suggested that the MEK signaling pathway could be a novel therapeutic target for novel antidiabetic agents.

Sodium glucose cotransporter 2 inhibitors have attracted attention as they exert antidiabetic and antiobesity effects. In this study, we investigated the effects of tofogliflozin on glucose homeostasis and its metabolic consequences and clarified the underlying molecular mechanisms. C57BL/6 mice were fed normal chow containing tofogliflozin (0.005%) for 20 weeks or a high-fat diet containing tofogliflozin (0.005%) for 8 weeks ad libitum. In addition, the animals were pair-fed in relation to controls to exclude the influence of increased food intake. Tofogliflozin reduced the body weight gain, mainly because of fat mass reduction associated with a diminished adipocyte size. Glucose tolerance and insulin sensitivity were ameliorated. The serum levels of nonesterified fatty acid and ketone bodies were increased and the respiratory quotient was decreased in the tofogliflozin-treated mice, suggesting the acceleration of lipolysis in the white adipose tissue and hepatic β-oxidation. In fact, the phosphorylation of hormone-sensitive lipase and the adipose triglyceride lipase protein levels in the white adipose tissue as well as the gene expressions related to β-oxidation, such as Cpt1α in the liver, were significantly increased. The hepatic triglyceride contents and the expression levels of lipogenic genes were decreased. Pair-fed mice exhibited almost the same results as mice fed an high-fat diet ad libitum. Moreover, a hyperinsulinemic-euglycemic clamp revealed that tofogliflozin improved insulin resistance by increasing glucose uptake, especially in the skeletal muscle, in pair-fed mice. Taken together, these results suggest tofogliflozin ameliorates insulin resistance and obesity by increasing glucose uptake in skeletal muscle and lipolysis in adipose tissue.

Granulocyte-colony stimulating factor (G-CSF) has been reported to mobilize bone marrow multi-potent stem cells, which differentiate into cardiac myocytes after myocardial infarction (MI). However, there have not been any reports regarding the effect of G-CSF on stem cell infiltration in the MI site. Hearts of mice that had undergone coronary occlusion were isolated and digested with collagenase. Infiltrating cells in the heart were collected using Percoll density gradients. The infiltrating cells were sorted for side population (SP) cells using Hoechst 33342 dye. Hundreds of infiltrating SP cells were found in the heart from 1 to 14 d after MI. There were only a few SP cells in hearts without infarction. Infiltrating SP cells were increased in the 4-d G-CSF treated group compared with the vehicle group (1106 +/- 106 vs. 323 +/- 26/heart, P < 0.05). The infiltration of inflammatory cells was not influenced by the G-CSF treatment. In a separate series of experiments, we confirmed that the infiltrating SP cells were derived from bone marrow. That is, SP cells in the infarcted hearts of mice, which had been transplanted with bone marrow from ROSA 26 (beta-galactosidase transgenic) mice, were positive for beta-galactosidase. In the immunohistochemical examination, Sca-1(+)/CD45(-) cells were existed in the infarcted site after MI. Therefore, SP cells may infiltrate into infarcted heart. G-CSF augmented this kind of stem cell infiltration without increasing inflammatory cells. These results suggest that G-CSF may enhance myocardial regeneration without aggravated inflammation in the infarcted heart.

The anti-anginal effect of CP-060S, a new cardioprotective agent that prevents myocardial Na+-, Ca2+-overload and has Ca2+-channel blocking activity, was evaluated in a rat model of arginine8-vasopressin (AVP)-induced cardiac ischaemia. Infusion of AVP (0.2 IU kg−1) depressed the electrocardiogram (ECG) ST segment, an index of myocardial ischaemia. Vehicle, CP-060S and diltiazem were given orally 1, 2, 4, 8, 12 and 24 h before the administration of AVP. CP-060S, at 3 mg kg−1 and 10 mg kg−1, suppressed AVP-induced ST-segment depression for 2 h and 12 h, respectively. In contrast, diltiazem, at 10 and 30 mg kg−1, suppressed AVP-induced ST-segment depression for only 1 h. The persistent suppression of the AVP-induced ST-segment depression by CP-060S correlated with the time course of changes in its plasma concentration. The minimum effective concentration of CP-060S was estimated to be 30 ng mL−1 (≅ 50 nwi), consistent with its vasorelaxant potency in rat isolated aortic strips (concentration producing 50% relaxation of KCl contraction, IC50 = 32.6 ± 8.3 nM). Intravenously administered CP-060S, at 300 μg kg−1 and diltiazem at 500 μg kg−1 showed similar haemodynamic changes, whereas CP-060S, at 300 μg kg−1, significantly suppressed AVP-induced ST-segment depression and diltiazem, at 500 μg kg−1, had no effect on AVP-induced ST-segment depression. In summary, orally administered CP-060S exerted a long-lasting anti-anginal effect proportionate to the time course of changes in its plasma concentration in a rat model of AVP-induced ischaemia.

We examined the growth regulating activities of enantiomers of inabenfide [4'-chloro-2'-(α-hydroxybenzyl)isonicotinanilide, 82211-24-3 (CAS RN Number)], an inhibitor of gibberellin biosynthesis, on rice plants (Oryza sativa L. cv. Koshihikari) with pot and field experiments. The (S)-form of inabenfide was more effective in reducing culm length than the racemate, whereas the (R)-form showed little effect. In treatment during the tillering stage, the (S)-form was about two times as potent as inabenfide (racemate) in its dwarfing effects on culms but also reduced the spikelet density to a greater extent than did the racemate. In addition, in treatments near the heading stage, the (S)-form (0.06 g/m 2 ) reduced culm length, especially at the upper internodes, by 15% of the control and decreased the degree of lodging at maturity, whereas the racemate (0.24 g/m 2 ) showed a lesser dwarfing effect. We conclude that: (I) the (S)-form is the active ingredient of inabenfide in reducing the culm length and lodging of rice plants.; (2) the (S)-form may be absorbed and translocated to elongating upper internodes more rapidly than inabenfide when applied near the heading stage.; and (3) treatment with the (S)-form near the heading stage will reduce the amount of ingredient required.

We compared the antianginal effect of CP-060S, a novel cardioprotective drug with Na+ and Ca2+ overload-preventing activity as well as Ca2+ channel antagonistic activity, with that of diltiazem, in an experimental model of vasospastic angina induced by methacholine in anaesthetized rats. Intra-aortic injection of methacholine at the coronary ostium provoked the ST-segment elevation of the electrocardiogram (ECG), indicating myocardial ischemia. CP-060S (3, 5 and 10 mg/kg, i.d.) significantly and dose-dependently suppressed the methacholine-induced ST-elevation, with the duration of action being at least 3 h at the highest dose. In addition, CP-060S at 3 mg/kg could inhibit the ST-elevation without producing significant changes in blood pressure, heart rate or rate-pressure product (RPP). In contrast, diltiazem (10 and 30 mg/kg, i.d.) significantly decreased the RPP, a significant suppression of the ST-elevation could only be achieved at the highest dose and its duration of action was about 2 h. Similar results were obtained with i.v. administration of the drugs, i.e. CP-060S given i.v. could inhibit the ST-elevation with less haemodynamic changes than diltiazem. In conclusion, CP-060S exerted a more potent and sustained protection against myocardial ischemia evoked by methacholine than diltiazem. The characteristics of the effects of CP-060S observed here suggest that this drug may be a desirable drug for the treatment of vasospastic angina.

In the field experiments from 1987 to 1992, the treatment with inabenfide at 0.20-0.24 g a.i. m-2 reduced the culm length by 4-14% of the control, and prevented breaking-type lodging. The dwarfing effect of inabenfide was evident especially at the lower internodes and at the upper three leaf blades. The yield, percentage of ripened grains and 1000 grain weight of rice plants treated with inabenfide were greater than those of control. Furthermore, we observed an increasein the percentage of ripened grains and the 1000 grain weight by the treatment with inabenfide under lodging-inhibiting conditions and in 1990 and 1992, when lodging was not so much influenced by inabenfide treatment. At 17-18 days after heading, the relative light intensity in the canopy of the inabenfide-treated plants was higher than in the canopy of the control plants, and light extinction coefficient (K) was lower in the former than in the latter. Although the number of grains per panicle was decreased, the number of panicles per hill and the leaf area per grain at early ripening stage were increased by the treatment with inabenfide. The starch content in leaf-sheaths and culms in the inabenfide-treated plants was significantly higher than that in the control from heading to maturity. It is suggested that: (1) Inabenfide promotes the ripening of rice.; (2) The effect is related not only to the direct effect on lodging but also to the “plant type”, improved by the treatment with inabenfide.

CP-060 S, (-)-( S)-2-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3 ,4-methylenedioxyphenoxy)ethyl]-amino]propyl]-1,3-thiazolidin++ +-4-one hydrogen fumarate, is a novel cardioprotective drug which prevents Na+-, Ca2+-overload and has Ca2+ channel blocking activity. We compared the anti-ischemic effects of CP-060S with those of diltiazem, a Ca2+ channel blocker, and R56865, N-[1-[4-(4-fluorophenoxy)butyl]-4-piperidinyl]-N-methyl-2-benzothiazo lamine, a Na+-, Ca2+-overload inhibitor, in a canine pacing-induced ischemia model. CP-060S 100 microg kg(-1) significantly suppressed the pacing-induced ischemic epicardial ST-segment elevation by maximally 75%, while diltiazem 100 microg kg(-1) suppressed it by maximally 35%. R56865 100 microg kg(-1) significantly suppressed the ST-segment elevation by maximally 30%. In addition, diltiazem 100 microg kg(-1) caused synergistic suppression of ST-segment elevation by 70% when administered simultaneously with R56865 100 microg kg(-1). These results suggest that a Na+-, Ca2+-overload preventive action and a Ca2+ channel blocking action independently contribute to the suppression of the ST-segment elevation. Therefore, CP-060S may suppress pacing-induced ST-segment elevation by a dual action by preventing Na+-, Ca2+-overload and the Ca2+ channel blockade.

CP-060S is a novel sodium and calcium overload inhibitor, and is also characterized as a calcium channel blocker. As these activities have each been shown independently to ameliorate ischaemia damage in the myocardium, the combination may synergistically exert cardioprotection. In this study, therefore, the protective effect of CP-060S against ischaemia- and reperfusion-induced arrhythmia was evaluated in anesthetized rats. Rats were anaesthetized with pentobarbitone, and the left anterior descending coronary artery was occluded for either 5 min with subsequent reperfusion (a reperfusion-induced arrhythmia model) or 30 min without (an ischaemia-induced arrhythmia model). All drugs were intravenously administered 1 min before the onset of occlusion. In the reperfusion-induced arrhythmia model, the animals in the vehicle-treated group exhibited ventricular tachycardia (VT) in 100%, ventricular fibrillation (VF) in 89%, and death caused by sustained VF in 56%. CP-060S (30–300 μg kg−1) dose-dependently suppressed the incidences of arrhythmias. Significant decreases occurred at 100 μg kg−1 in VF (incidence: 42%) and mortality (8%), and at 300 μg kg−1 in VT (50%), VF (33%) and mortality (8%). This protective effect of CP-060S was 10 times more potent than that of a pure calcium channel blocker, diltiazem (30–1000 μg kg−1) we tested, in terms of effective dose ranges. As both drugs decreased myocardial oxygen consumption estimated by rate-pressure product to a similar extent, the calcium channel blocking activity of CP-060S would not seem to be sufficient to explain its potency. In the same model, co-administration of ineffective doses of diltiazem (300 μg kg−1) and a sodium and calcium overload inhibitor, R56865 (100 μg kg−1), produced significant suppression of VT (incidence: 62%), VF (46%) and mortality (8%). By contrast, co-administration of R56865 at the same dose with CP-060S (300 μg kg−1) did not add to the effect of a single treatment of CP-060S. In the ischaemia-induced arrhythmia model, CP-060S (300 μg kg−1) significantly decreased the incidence of VF from 75% to 29%, whereas diltiazem (1 mg kg−1) was ineffective. These results suggest that CP-060S inhibits both ischaemia- and reperfusion-induced arrhythmia. The combination of the calcium channel blocking effect and the calcium overload inhibition was hypothesized to contribute to these potently protective effects. British Journal of Pharmacology (1998) 123, 1409–1417; doi:10.1038/sj.bjp.0701742

The myocardial infarct size (IS)-limiting effect of CP-060S, a novel cardioprotective drug that prevents Na + -, Ca 2+ -overload and has Ca 2+ channel-blocking activity, was compared with that of diltiazem, a pure Ca 2+ antagonist, to determine whether the prevention of Na + -, Ca 2+ -overload contributes to this IS-limiting effect. Dogs were subjected to 90 min of left circumflex coronary artery (LCx) occlusion followed by 5 h of reperfusion. Either CP-060S (300 μg/kg) or diltiazem (600 μg/kg) was administered intravenously 20 min before the occlusion. CP-060S significantly limited IS compared with that of vehicle (percentage of the area at risk: vehicle, 50.64 ± 6.08%; CP-060S, 21.13 ± 3.75%; p < 0.01 vs. vehicle). Although diltiazem exerted a significant decrease in rate-pressure product (RPP; an index of myocardial oxygen consumption) during occlusion equal to that of CP-060S, diltiazem did not significantly reduce IS (33.90 ± 4.30%). Regional myocardial blood flow (RBF) was not significantly different between any of the groups. Therefore the IS-limiting effect of CP-060S cannot be explained in terms of changes in RPP or RBF. Thus the IS limitation induced by CP-060S is probably the consequence of a direct cardioprotective effect on myocytes. The prevention of Na + -, Ca 2+ -overload may be the primary reason for this IS-limiting effect.

CP-060S, (−)-(S)-2-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3,4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thiazolidin-4-one hydrogen fumarate is a novel cardioprotective drug, which is able to prevent Na+-, Ca2+-overload and also has Ca2+ channel blocking activity. The latter action of CP-060S was characterized by radioligand binding experiments with rat cardiac membranes in terms of the interaction with the three principal binding sites on the L-type Ca2+ channel, which bind such drugs as the 1,4-dihydropyridines, phenylalkylamines and benzothiazepines. CP-060S exhibited complete and concentration-dependent inhibition of [ 3 H ](+)-PN200-110, [ 3 H ](−)-desmethoxyverapamil and [ 3 H ]cis-(+)-diltiazem binding to their specific binding sites. Saturation studies showed that CP-060S increased the Kd of [ 3 H ](+)-PN200-110 and [ 3 H ](−)-desmethoxyverapamil without causing a significant change in the maximum binding density. The dissociation kinetics of the three radioligands were accelerated by CP-060S. These results suggest that CP-060S interacts with a novel binding site on the L-type Ca2+ channel and has a negative allosteric interaction with the three principal binding sites for the 1,4-dihydropyridines, phenylalkylamines and benzothiazepines.

Aims Urinary tract infection (UTI) is a common clinical problem in diabetic patients; however, the relationship between UTI and glucosuria remains uncertain. To investigate the relationship, we examined the effect of glucosuria induced by sodium glucose cotransporter 2 (SGLT2) inhibitors on the progression of UTI in mice. Methods From 1 day before transurethral inoculation with Candida albicans, female mice were treated orally once a day with an SGLT2 inhibitor in different treatment regimens: (i) dapagliflozin at 10 mg/kg for 2, 3 or 7 days, (ii) dapagliflozin at 0.1, 1 or 10 mg/kg for 3 days and (iii) dapagliflozin, canagliflozin or tofogliflozin at 10 mg/kg for 3 days. To evaluate the ascending UTI, the kidneys were removed 6 days after the inoculation, and the number of viable C. albicans cells in kidney was measured as colony-forming units (CFU). Results In mice treated with dapagliflozin, the number of C. albicans CFU in kidney increased in accordance with both treatment duration and dose. The number of CFU significantly increased when mice were treated with 10 mg/kg dapagliflozin or canagliflozin but not tofogliflozin. With dapagliflozin and canagliflozin, urine glucose concentration (UGC) significantly increased up to 24 h after drug administration; with tofogliflozin, UGC significantly increased only up to 12 h after drug administration. Conclusions Our data indicate that increased susceptibility to UTI is associated with a persistent increase in UGC.

Although excessive fructose intake is epidemiologically linked with dyslipidemia, obesity, and diabetes, the mechanisms regulating plasma fructose are not well known. Cells transfected with sodium/glucose cotransporter 5 (SGLT5), which is expressed exclusively in the kidney, transport fructose in vitro; however, the physiological role of this transporter in fructose metabolism remains unclear. To determine whether SGLT5 functions as a fructose transporter in vivo, we established a line of mice lacking the gene encoding SGLT5. Sodium-dependent fructose uptake disappeared in renal brush border membrane vesicles from SGLT5-deficient mice, and the increased urinary fructose in SGLT5-deficient mice indicated that SGLT5 was the major fructose reabsorption transporter in the kidney. From this, we hypothesized that urinary fructose excretion induced by SGLT5 deficiency would ameliorate fructose-induced hepatic steatosis. To test this hypothesis we compared SGLT5-deficient mice with wild-type mice under conditions of long-term fructose consumption. Paradoxically, however, fructose-induced hepatic steatosis was exacerbated in the SGLT5-deficient mice, and the massive urinary fructose excretion was accompanied by reduced levels of plasma triglycerides and epididymal fat but fasting hyperinsulinemia compared with fructose-fed wild-type mice. There was no difference in food consumption, water intake, or plasma fructose between the two types of mice. No compensatory effect by other transporters reportedly involved in fructose uptake in the liver and kidney were indicated at the mRNA level. These surprising findings indicated a previously unrecognized link through SGLT5 between renal fructose reabsorption and hepatic lipid metabolism.

Sodium/glucose cotransporter 2 (SGLT2) is the predominant mediator of renal glucose reabsorption and is an emerging molecular target for the treatment of diabetes. We identified a novel potent and selective SGLT2 inhibitor, tofogliflozin (CSG452), and examined its efficacy and pharmacological properties as an antidiabetic drug. Tofogliflozin competitively inhibited SGLT2 in cells overexpressing SGLT2, and K(i) values for human, rat, and mouse SGLT2 inhibition were 2.9, 14.9, and 6.4 nM, respectively. The selectivity of tofogliflozin toward human SGLT2 versus human SGLT1, SGLT6, and sodium/myo-inositol transporter 1 was the highest among the tested SGLT2 inhibitors under clinical development. Furthermore, no interaction with tofogliflozin was observed in any of a battery of tests examining glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase reactions. A single oral gavage of tofogliflozin increased renal glucose clearance and lowered the blood glucose level in Zucker diabetic fatty rats. Tofogliflozin also improved postprandial glucose excursion in a meal tolerance test with GK rats. In db/db mice, 4-week tofogliflozin treatment reduced glycated hemoglobin and improved glucose tolerance in the oral glucose tolerance test 4 days after the final administration. No blood glucose reduction was observed in normoglycemic SD rats treated with tofogliflozin. These findings demonstrate that tofogliflozin inhibits SGLT2 in a specific manner, lowers blood glucose levels by increasing renal glucose clearance, and improves pathological conditions of type 2 diabetes with a low hypoglycemic potential.

The anti-anginal effect of CP-060S, a new cardioprotective agent that prevents myocardial Na+-, Ca2+-overload and has Ca2+-channel blocking activity, was evaluated in a rat model of arginine8-vasopressin (AVP)-induced cardiac ischaemia. Infusion of AVP (0.2 IU kg(-1)) depressed the electrocardiogram (ECG) ST segment, an index of myocardial ischaemia. Vehicle, CP-060S and diltiazem were given orally 1, 2, 4, 8, 12 and 24 h before the administration of AVP. CP-060S, at 3 mg kg(-1) and 10 mg kg(-1), suppressed AVP-induced ST-segment depression for 2 h and 12 h, respectively. In contrast, diltiazem, at 10 and 30 mg kg(-1), suppressed AVP-induced ST-segment depression for only 1 h. The persistent suppression of the AVP-induced ST-segment depression by CP-060S correlated with the time course of changes in its plasma concentration. The minimum effective concentration of CP-060S was estimated to be 30 ng mL(-1) (approximately 50 nM), consistent with its vasorelaxant potency in rat isolated aortic strips (concentration producing 50% relaxation of KCl contraction, IC50 = 32.6+/-8.3 nM). Intravenously administered CP-060S, at 300 microg kg(-1) and diltiazem at 500 microg kg(-1) showed similar haemodynamic changes, whereas CP-060S, at 300 microg kg(-1), significantly suppressed AVP-induced ST-segment depression and diltiazem, at 500 microg kg(-1), had no effect on AVP-induced ST-segment depression. In summary, orally administered CP-060S exerted a long-lasting anti-anginal effect proportionate to the time course of changes in its plasma concentration in a rat model of AVP-induced ischaemia.

Objective: Tofogliflozin, a highly selective inhibitor of sodium/glucose cotransporter 2 (SGLT2), induces urinary glucose excretion (UGE), improves hyperglycemia and reduces body weight in patients with Type 2 diabetes (T2D). The mechanisms of tofogliflozin on body weight reduction were investigated in detail with obese and diabetic animal models. Methods: Diet-induced obese (DIO) rats and KKAy mice (a mouse model of diabetes with obesity) were fed diets containing tofogliflozin. Body weight, body composition, biochemical parameters and metabolic parameters were evaluated. Results: In DIO rats tofogliflozin was administered for 9 weeks, UGE was induced and body weight gain was attenuated. Body fat mass decreased without significant change in bone mass or lean body mass. Food consumption (FC) increased without change in energy expenditure, and deduced total calorie balance (deduced total calorie balance=FC−UGE−energy expenditure) decreased. Respiratory quotient (RQ) and plasma triglyceride (TG) level decreased, and plasma total ketone body (TKB) level increased. Moreover, plasma leptin level, adipocyte cell size and proportion of CD68-positive cells in mesenteric adipose tissue decreased. In KKAy mice, tofogliflozin was administered for 3 or 5 weeks, plasma glucose level and body weight gain decreased together with a reduction in liver weight and TG content without a reduction in body water content. Combination therapy with tofogliflozin and pioglitazone suppressed pioglitazone-induced body weight gain and reduced glycated hemoglobin level more effectively than monotherapy with either pioglitazone or tofogliflozin alone. Conclusion: Body weight reduction with tofogliflozin is mainly due to calorie loss with increased UGE. In addition, tofogliflozin also induces a metabolic shift from carbohydrate oxidation to fatty acid oxidation, which may lead to prevention of fat accumulation and inflammation in adipose tissue and liver. Tofogliflozin may have the potential to prevent obesity, hepatic steatosis and improve insulin resistance as well as hyperglycemia.

Inhibition of the biosynthesis of gibberellins by 4'-chloro-2'-(a-hydroxybenzyl)-isonicotinanilide (inabenfide) was investigated with cell-free systems prepared from immature seeds of Cucurbita maxima, Phaseolus vulgaris and Pisum sativum. Inabenfide specifically inhibited the oxidation of kaurene, kaurenol and kaurenal but it did not inhibit the oxidation steps that lead to kaurenoic acid. The (S)- and (R)-forms of inabenfide and 2'-benzoyl-4'-chloro-isonicotinanilide (compound [1]) also inhibited the same oxidation steps. The (S)-form was the most active in inhibition of the oxidation of kaurene; the racemic form (inabenfide) was one half as effective; and the (R)-form and compound [1] had little effect on the biosynthesis of gibberellins. Growth-retardant activities of these compounds were also examined using seedlings of C. maxima. Since the growth-retardant activity of these compounds was correlated with their ability to inhibit the oxidation of kaurene in cell-free systems, we suggest that the growth-retardant activity of inabenfide is due to inhibition of the three oxidation steps from kaurene to kaurenoic acid in the biosynthesis of gibberellins.