Oral SSTR5 Antagonist SCO-240 for Growth Hormone Stimulation: A Phase I Single-Dose Study in Healthy Individuals.

Somatostatin inhibits endocrine and exocrine secretion in various tissues by acting on five somatostatin receptor subtypes (SSTR1-5). The clinical effects of SSTR5 antagonism remain unknown. Herein, we evaluated the effects of SCO-240, an oral SSTR5 antagonist, in healthy individuals. This randomized, single-center, double-blind, placebo-controlled, phase I study included healthy Japanese and White individuals. The effects of ascending single oral doses of SCO-240 were evaluated in healthy individuals. The main outcome measures were safety, tolerability, pharmacokinetics, and pharmacodynamics (gallbladder contractions and levels of serum insulin and plasma glucagon-like peptide-1 (GLP-1)). The levels of pituitary hormones were evaluated in our exploratory analysis. The results indicated that SCO-240 was safe and well-tolerated at all tested doses. Oral SCO-240 was readily absorbed, with its systemic exposure increasing in a dose-dependent manner. The median time to maximum concentration and mean terminal half-life of SCO-240 were 3-4 and 10.2-12.6 hours, respectively, in the ascending dose section. No clinically meaningful changes in SCO-240 pharmacokinetic profiles were observed between fed and fasted or between Japanese and White individuals. No increase in gallbladder contractions or levels of insulin and GLP-1 were detected. SCO-240 induced robust growth hormone (GH) secretion without altering the levels of other pituitary hormones. In conclusion, the study is the first to demonstrate that SSTR5 antagonism stimulates GH secretion in humans. SCO-240 was safe and well-tolerated and exhibited once-daily oral dosing potential. The robust effects of SCO-240 on GH secretion suggest that it may be a treatment option for GH-related disorders.

An Exploratory Randomized Trial of SCO-792, an Enteropeptidase Inhibitor, in Patients With Type 2 Diabetes and Albuminuria.

Introduction: Elevated plasma amino acid levels overload kidney function by increasing glomerular filtration rate (GFR). Inhibiting gut amino acid intake may have therapeutic benefits for patients with kidney dysfunction. For a prospective phase 2a trial, we carried out an exploratory evaluation of the safety and efficacy of SCO-792, an enteropeptidase inhibitor that blocks gut amino acid intake, in patients with type 2 diabetes mellitus (T2DM) and albuminuria. Methods: Seventy-two patients with T2DM, a urine albumin-to-creatinine ratio (UACR) of 200-5000 mg/g, and an estimated GFR >30 ml/min per 1.73 m2 were included. Patients were randomly assigned (1:2:2) to the following groups and received treatment for 12 weeks: placebo (n = 15), SCO-792 500 mg once daily (SCO-792 QD; n = 29), or SCO-792 500 mg 3 times daily (SCO-792 3 times a day (TID); n = 28) by following a double-blind approach. We evaluated UACR changes from the baseline along with safety as the primary end points and other parameters as secondary or exploratory end points. Results: SCO-792 was safe and well tolerated up to 1500 mg/day for 12 weeks. UACR changes from baseline were -14% (P = 0.4407), -27% (P = 0.0271), and -28% (P = 0.0211) in placebo, SCO-792 QD, and SCO-792 TID, respectively, whereas UACR changes in SCO-792 groups were not statistically significant compared with placebo. The hemoglobin A1c (HbA1c) levels from baseline, an exploratory end point, decreased in the SCO-792 TID group. Conclusion: SCO-792 was safe and well tolerated for 12 weeks and may be associated with decreased UACR in patients with T2DM and albuminuria. Further clinical studies are essential to confirm our findings.

Selective somatostatin receptor 5 inhibition improves hepatic insulin sensitivity.

Diabetes is a metabolic disorder with an increasing global prevalence. Somatostatin (SST), a peptide hormone, regulates hormone secretion via five SST receptor (SSTR) subtypes (SSTR1-5) in a tissue-specific manner. As SSTR5 is expressed in pancreatic ß-cells and intestinal L-cells, studies have suggested that SSTR5 regulates glucose tolerance through insulin and incretin secretion, thereby having a prominent role in diabetes. Moreover, SSTR5 knockout (KO) mice display enhanced insulin sensitivity; however, the underlying mechanism has not been clarified. Therefore, in this study, we investigate the effect of SSTR5 blockade on insulin resistance and the target organ using SSTR5 KO mice and a selective SSTR5 antagonist (compound-1). High-fat diet (HFD)-fed SSTR5 KO mice exhibited significantly lower homeostasis model assessment of insulin resistance (HOMA-IR) than HFD-fed wild-type mice. Two-week oral administration of compound-1 dose-dependently and significantly reduced changes in the levels of glycosylated hemoglobin (GHb), plasma glucose, plasma insulin, and HOMA-IR in male KK-Ay /Ta Jcl mice (KK-Ay mice), a model of obese type 2 diabetes with severe insulin resistance. Additionally, compound-1 significantly increased the glucose infusion rate while decreasing hepatic glucose production in male KK-Ay mice, as evidenced by hyperinsulinemic-euglycemic clamp analyses. In addition, compound-1 ameliorated the insulin-induced Akt phosphorylation suppression by octreotide in the liver of male C57BL/6J mice. Collectively, our results demonstrate that selective SSTR5 inhibition can improve insulin sensitivity by enhancing liver insulin action; thus, selective SSTR5 antagonists represent potentially novel therapeutic agents for type 2 diabetes.

Selective Acetyl-CoA Carboxylase 1 Inhibitor Improves Hepatic Steatosis and Hepatic Fibrosis in a Preclinical Nonalcoholic Steatohepatitis Model.

Acetyl-CoA carboxylase (ACC) 1 and ACC2 are essential rate-limiting enzymes that synthesize malonyl-CoA (M-CoA) from acetyl-CoA. ACC1 is predominantly expressed in lipogenic tissues and regulates the de novo lipogenesis flux. It is upregulated in the liver of patients with nonalcoholic fatty liver disease (NAFLD), which ultimately leads to the formation of fatty liver. Therefore, selective ACC1 inhibitors may prevent the pathophysiology of NAFLD and nonalcoholic steatohepatitis (NASH) by reducing hepatic fat, inflammation, and fibrosis. Many studies have suggested ACC1/2 dual inhibitors for treating NAFLD/NASH; however, reports on selective ACC1 inhibitors are lacking. In this study, we investigated the effects of compound-1, a selective ACC1 inhibitor for treating NAFLD/NASH, using preclinical in vitro and in vivo models. Compound-1 reduced M-CoA content and inhibited the incorporation of [14C] acetate into fatty acids in HepG2 cells. Additionally, it reduced hepatic M-CoA content and inhibited de novo lipogenesis in C57BL/6J mice after a single dose. Furthermore, compound-1 treatment of 8 weeks in Western diet-fed melanocortin 4 receptor knockout mice-NAFLD/NASH mouse model-improved liver hypertrophy and reduced hepatic triglyceride content. The reduction of hepatic M-CoA by the selective ACC1 inhibitor was highly correlated with the reduction in hepatic steatosis and fibrosis. These findings support further investigations of the use of this ACC1 inhibitor as a new treatment of NFLD/NASH. SIGNIFICANCE STATEMENT: This is the first study to demonstrate that a novel selective inhibitor of acetyl-CoA carboxylase (ACC) 1 has anti-nonalcoholic fatty liver disease (NAFLD) and anti-nonalcoholic steatohepatitis (NASH) effects in preclinical models. Treatment with this compound significantly improved hepatic steatosis and fibrosis in a mouse model. These findings support the use of this ACC1 inhibitor as a new treatment for NAFLD/NASH.

Enteropeptidase inhibitor SCO-792 effectively prevents kidney function decline and fibrosis in a rat model of chronic kidney disease.

BACKGROUND: Inhibiting enteropeptidase, a gut serine protease regulating protein digestion, suppresses food intake and ameliorates obesity and diabetes in mice. However, the effects of enteropeptidase inhibition on kidney parameters are largely unknown. Here, we evaluated the chronic effects of an enteropeptidase inhibitor, SCO-792, on kidney function, albuminuria and kidney pathology in spontaneously hypercholesterolaemic (SHC) rats, a rat chronic kidney disease (CKD) model. METHODS: SCO-792, an orally available enteropeptidase inhibitor, was administered [0.03% and 0.06% (w/w) in the diet] to 20-week-old SHC rats showing albuminuria and progressive decline in glomerular filtration rate (GFR) for five weeks. The effects of SCO-792 and the contribution of amino acids to these effects were evaluated. RESULTS: SCO-792 increased the faecal protein content, indicating that SCO-792 inhibited enteropeptidase in SHC rats. Chronic treatment with SCO-792 prevented GFR decline and suppressed albuminuria. Moreover, SCO-792 improved glomerulosclerosis and kidney fibrosis. Pair feeding with SCO-792 (0.06%) was less effective in preventing GFR decline, albuminuria and renal histological damage than SCO-792 treatment, indicating the enteropeptidase-inhibition-dependent therapeutic effects of SCO-792. SCO-792 did not affect the renal plasma flow, suggesting that its effect on GFR was mediated by an improvement in filtration fraction. Moreover, SCO-792 increased hydrogen sulphide production capacity, which has a role in tissue protection. Finally, methionine and cysteine supplementation to the diet abrogated SCO-792-induced therapeutic effects on albuminuria. CONCLUSIONS: SCO-792-mediated inhibition of enteropeptidase potently prevented GFR decline, albuminuria and kidney fibrosis; hence, it may have therapeutic potential against CKD.

Enteropeptidase inhibition improves kidney function in a rat model of diabetic kidney disease.

AIM: To examine the effects of an enteropeptidase inhibitor, SCO-792, on kidney function in rats. MATERIALS AND METHODS: The pharmacological effects of SCO-792 were evaluated in Wistar fatty (WF) rats, a rat model of diabetic kidney disease (DKD). RESULTS: Oral administration of SCO-792 increased faecal protein content and improved glycaemic control in WF rats. SCO-792 elicited a rapid decrease in urine albumin-to-creatinine ratio (UACR). SCO-792 also normalized glomerular hyperfiltration and decreased fibrosis, inflammation and tubular injury markers in the kidneys. However, pioglitazone-induced glycaemic improvement had no effect on kidney variables. Dietary supplementation of amino acids (AAs), which bypass the action of enteropeptidase inhibition, mitigated the effect of SCO-792 on UACR reduction, suggesting a pivotal role for enteropeptidase. Furthermore, autophagy activity in the glomerulus, which is impaired in DKD, was elevated in SCO-792-treated rats. Finally, a therapeutically additive effect on UACR reduction was observed with a combination of SCO-792 with irbesartan, an angiotensin II receptor blocker. CONCLUSIONS: This study is the first to demonstrate that enteropeptidase inhibition is effective in improving disease conditions in DKD. SCO-792-induced therapeutic efficacy is likely to be independent of glycaemic control and mediated by the regulation of AAs and autophagy. Taken together with a combination effect of irbesartan, SCO-792 may be a novel therapeutic option for patients with DKD.

Enteropeptidase inhibition improves obesity by modulating gut microbiota composition and enterobacterial metabolites in diet-induced obese mice.

Enteropeptidase is a transmembrane serine protease localized in the lumen of the duodenum that acts as a key enzyme for protein digestion. SCO-792 is an orally available enteropeptidase inhibitor that has been reported to have therapeutic effects on obesity and diabetes in mice. However, the mechanism underlying the therapeutic effect of SCO-792 has not yet been fully elucidated. In this study, we evaluated the role of gut microbiota on SCO-792-induced body weight (BW) reduction in high-fat diet-induced obese (DIO) mice. Chronic administration of SCO-792 substantially decreased BW and food intake in DIO mice. While the pair-fed study uncovered food intake-independent mechanisms of BW reduction by SCO-792. Interestingly, antibiotics-induced microbiota elimination in the gut canceled SCO-792-induced BW reduction by nearly half without affecting the anorectic effect, indicating the involvement of gut microbiota in the anti-obesity mechanism that is independent of food intake reduction. Microbiome analysis revealed that SCO-792 altered the gut microbiota composition in DIO mice. Notably, it was found that the abundance of Firmicutes decreased while that of Verrucomicrobia increased at the phylum level. Increased abundance of Akkermansia muciniphila, a bacterium known to be useful for host metabolism, was observed in SCO-792-treated mice. Fecal metabolome analysis revealed increased amino acid levels, indicating gut enteropeptidase inhibition. In addition, SCO-792 was found to increase the level of short-chain fatty acids, including propionate, and bile acids in the feces, which all help maintain gut health and improve metabolism. Furthermore, it was found that SCO-792 induced the elevation of colonic immunoglobulin A (IgA) concentration, which may maintain the microbiota condition, in DIO mice. In conclusion, this study demonstrates the contribution of microbiota to SCO-792-induced BW reduction. Enteropeptidase-mediated regulation of microbiota, enterobacterial metabolites, and IgA in the gut may coordinately drive the therapeutic effects of SCO-792 in obesity.

SCO-792, an enteropeptidase inhibitor, improves disease status of diabetes and obesity in mice.

AIMS: Enteropeptidase is a serine protease localized on the duodenal brush border that catalyzes the conversion of inactive trypsinogen into active trypsin, thereby regulating protein breakdown in the gut. We evaluated the effects of SCO-792, a novel enteropeptidase inhibitor, in mice. MATERIALS AND METHODS: In vivo inhibition of enteropeptidase was evaluated via an oral protein challenge. Pharmacological effects were evaluated in normal mice, in diet-induced obese (DIO) mice and in obese and diabetic ob/ob mice. RESULTS: A single oral administration of SCO-792 inhibited plasma branched-chain amino acids (BCAAs) in an oral protein challenge test in mice, indicating in vivo inhibition of enteropeptidase. Repeated treatment with SCO-792 induced reduction in food intake and decrease in body weight in DIO and ob/ob mice. Plasma FGF21 levels were increased in SCO-792-treated DIO mice, an observation that was probably independent of reduction in food intake. Hyperglycaemia was markedly improved in SCO-792-treated ob/ob mice. A hyperinsulinaemic-euglycaemic clamp study revealed improved muscle insulin sensitivity in SCO-792-treated ob/ob mice. SCO-792 also improved plasma and liver lipid profiles and decreased plasma alanine transaminase, suggesting a potential treatment for liver diseases. Dietary supplementation with essential amino acids attenuated the effect of SCO-792 on reduction in food intake and decrease in body weight in normal mice, suggesting a pivotal role for enteropeptidase in these biological phenomena. CONCLUSIONS: SCO-792 inhibited enteropeptidase in vivo, reduced food intake, decreased body weight, increased insulin sensitivity, improved glucose and lipid control, and ameliorated liver parameters in mouse models with obesity and/or diabetes. SCO-792 may exhibit similar effects in patients.

Discovery of novel 5-oxa-2,6-diazaspiro[3.4]oct-6-ene derivatives as potent, selective, and orally available somatostatin receptor subtype 5 (SSTR5) antagonists for treatment of type 2 diabetes mellitus.

Somatostatin receptor subtype 5 (SSTR5) has emerged as a novel attractive drug target for type 2 diabetes mellitus. Starting from N-benzyl azetidine derivatives 1 and 2 as in-house hit compounds, we explored the introduction of a carboxyl group into the terminal benzene of 1 to enhance SSTR5 antagonistic activity by the combination of the substituents at the 3-position of the isoxazoline. Incorporation of a carboxyl group at the 4-position of the benzene ring resulted in a significant enhancement in potency, however, the 4-benzoic acid derivative 10c exhibited moderate human ether-a-go-go related gene (hERG) inhibitory activity. A subsequent optimization study revealed that replacement of the 4-benzoic acid with an isonipecotic acid dramatically reduced hERG inhibition (5.6% inhibition at 30µM) by eliminating π-related interaction with hERG K+ channel, which resulted in the identification of 1-(2-((2,6-diethoxy-4'-fluorobiphenyl-4-yl)methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)piperidin-4-carboxylic acid 25a (hSSTR5/mSSTR5 IC50=9.6/57nM). Oral administration of 25a in high-fat diet fed C57BL/6J mice augmented insulin secretion in a glucose-dependent manner and lowered blood glucose concentration.

Discovery of novel somatostatin receptor subtype 5 (SSTR5) antagonists: Pharmacological studies and design to improve pharmacokinetic profiles and human Ether-a-go-go-related gene (hERG) inhibition.

Somatostatin (SST) is a peptide hormone comprising 14 or 28 amino acids that inhibits endocrine and exocrine secretion via five distinct G-protein-coupled receptors (SSTR1-5). SSTR5 has an important role in inhibiting the secretion of pancreatic and gastrointestinal hormones (e.g., insulin, GLP-1, PYY) through the binding of SSTs; hence, SSTR5 antagonists are expected to be novel anti-diabetic drugs. In the course of our lead generation program of SSTR5 antagonists, we have discovered a novel spiroazetidine derivative 3a. However, pharmacological evaluation of 3a revealed that it had to be administered at a high dose (100mg/kg) to show a persistent glucose-lowering effect in an oral glucose tolerance test (OGTT). We therefore initiated an optimization study based on 3a aimed at improving the antagonistic activity and mean residence time (MRT), resulting in the identification of 2-cyclopropyl-5-methoxybiphenyl derivative 3k. However, 3k did not show a sufficient persistent glucose-lowering effect in an OGTT; moreover, hERG inhibition was observed. Hence, further optimization study of the biphenyl moiety of compound 3k, focused on improving the pharmacokinetic (PK) profile and hERG inhibition, was conducted. Consequently, the introduction of a chlorine atom at the 6-position on the biphenyl moiety addressed a putative metabolic soft spot and increased the dihedral angle of the biphenyl moiety, leading to the discovery of 3p with an improved PK profile and hERG inhibition. Furthermore, 3p successfully exhibited a persistent glucose-lowering effect in an OGTT at a dose of 3mg/kg.

P2Y2 receptor-Gq/11 signaling at lipid rafts is required for UTP-induced cell migration in NG 108-15 cells.

Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y(2) receptors are known to couple with G(q) family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca(2+) ([Ca(2+)](i)) levels. In the present study, we investigated the involvement of lipid rafts in P2Y(2) receptor-mediated signaling and cell migration in NG 108-15 cells. When NG 108-15 cell lysates were fractionated by sucrose density gradient centrifugation, Galpha(q/11) and a part of P2Y(2) receptors were distributed in a fraction where the lipid raft markers, cholesterol, flotillin-1, and ganglioside GM1 were abundant. Methyl-beta-cyclodextrin (CD) disrupted not only lipid raft markers but also Galpha(q/11) and P2Y(2) receptors in this fraction. In the presence of CD, P2Y(2) receptor-mediated phosphoinositide hydrolysis and [Ca(2+)](i) elevation were inhibited. It is noteworthy that UTP-induced cell migration was inhibited by CD or the G(q/11)-selective inhibitor YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16, 22-hexamethyl-15-methylene-2,5,8,11,14,17,-20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. Moreover CD and YM254890 completely inhibited Rho-A activation. Downstream of Rho-A signaling, stress fiber formation and phosphorylation of cofilin were also inhibited by CD or YM254890. However, UTP-induced phosphorylation of cofilin was not affected by the expression of p115-regulator of G protein signaling, which inhibits the G(12/13) signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by the G(q/11) signaling pathway. These results suggest that lipid rafts are critical for P2Y(2) receptor-mediated G(q/11)-PLC-Ca(2+) signaling and this cascade is important for cell migration in NG 108-15 cells.

Mastoparan inhibits beta-adrenoceptor-G(s) signaling by changing the localization of Galpha(s) in lipid rafts.

Mastoparan, a wasp venom toxin, has various pharmacological activities, the mechanisms of which are still unknown. To clarify the action of mastoparan on G protein-coupled receptor-mediated signaling, we previously examined the effect of mastoparan on G(q)-mediated signaling and demonstrated that mastoparan binds to gangliosides causing a decrease in Galpha(q/11) content in lipid rafts, and resulting in the inhibition of G(q)-mediated phosphoinositide hydrolysis (Sugama et al., Mol. Pharmacol., 68, 1466, 2005). In the present study, we examined the effect of mastoparan on beta-adrenoceptor-G(s) signaling in 1321N1 human astrocytoma cells. Mastoparan inhibited isoproterenol-induced elevation of cyclic AMP in a concentration-dependent manner. Although mastoparan is known to be an activator of G(i), pertussis toxin only slightly attenuated mastoparan-induced inhibition of cyclic AMP elevation, suggesting that a major part of the inhibition of cyclic AMP elevation induced by mastoparan is not mediated by Galpha(i). By contrast, mastoparan-induced inhibition of cyclic AMP elevation was clearly attenuated by preincubation of the cells with ganglioside mixtures. Moreover, mastoparan changed the localization of Galpha(s) in lipid rafts without disrupting the structure of lipid rafts. Fluorescent staining analysis showed that mastoparan released GFP-Galpha(s) from plasma membranes into the cytosol. These results suggest that the mastoparan-induced suppression of cyclic AMP elevation is mainly caused by changing the localization of Galpha(s) in lipid rafts into a compartment in the cellular interior where it is not available to activate adenylyl cyclase.

Mastoparan changes the cellular localization of Galphaq/11 and Gbeta through its binding to ganglioside in lipid rafts.

Although it is known that mastoparan, a wasp venom toxin, directly activates Gi/o, mastoparan-induced biological responses are not always explained by this mechanism. For instance, we have demonstrated previously that mastoparan suppressed phosphoinositide hydrolysis induced by carbachol in human astrocytoma cells (FEBS Lett 206:91-94, 1990). In the present study, we examined whether mastoparan affected phosphoinositide hydrolysis by interacting with lipid rafts in PC-12 cells. Mastoparan inhibited UTP-induced increase in [Ca2+]i and phosphoinositide hydrolysis in a concentration-dependent manner. UTP-induced phosphoinositide hydrolysis occurred in lipid rafts, because methyl-beta-cyclodextrin, a disrupting regent of lipid rafts, inhibited the hydrolysis. Mastoparan changed the localization of Galphaq/11 and Gbeta together with cholesterol from lipid rafts to nonraft fractions or cytosol. These changes were inhibited by ganglioside mixtures, suggesting that mastoparan interacts with gangliosides in lipid rafts. In fact, ganglioside mixtures and neuraminidase, but not sialic acid, attenuated the inhibitory effect of mastoparan on phosphoinositide hydrolysis. Furthermore, fluorescence intensity of tyrosine residue of [Tyr3]mastoparan was potentiated by ganglioside mixtures, suggesting the direct binding of mastoparan to gangliosides. Mastoparan caused cytotoxicity of PC-12 cells in a concentration-dependent manner, determined by LDH release. The mastoparan-induced cytotoxicity was significantly inhibited by neuraminidase or gangliosides. The order of inhibitory potency of gangliosides was GT1b approximately GD1b > GD1a > GM1 >> GQ1b, but asialo-GM1 and sialic acid were inactive. These results suggest that mastoparan initially binds to gangliosides in lipid rafts and then it inhibits phosphoinositide hydrolysis by changing the localization of Galphaq/11 and Gbeta in lipid rafts.

Sugar-sensitive thin films composed of concanavalin A and glycogen.

Sugar-sensitive thin films were prepared by a layer-by-layer deposition of concanavalin A (Con A) and glycogen on the surface of a quartz slide and their sugar-induced decomposition was studied. The Con A/glycogen multilayer films can be decomposed by exposing them to sugar solutions (D-glucose, D-mannose, methyl-alpha-D-glucose and methyl-alpha-D-mannose), as a result of displacement of sugar residues of glycogen from the binding sites of Con A by the free sugar added in the solution. The rate of decomposition significantly depended on the type of sugar and its concentration.