Synthesis and diuretic activity of bicyclic fused heterocycles containing oxime-O-sulfonic acid moiety.

In order to investigate the origin of the loop-type diuretic activity of M17055 (1), several variants (3-9) were designed and synthesized by modifying the quinolinone skeleton, and their diuretic activities were compared with the lead 1 and furosemide in dogs. It was found that the negative charge distribution pattern afforded by the dispositional arrangement of the 4-oxime-O-sulfonic acid and 1-N-acyl carbonyl moiety attached to the tetrahydropyridine ring system is inevitable for the development of the activity, which strongly supports the previously proposed model for the active site of the Na(+)-K(+)-2Cl(-) cotransporter. Also reported is the first synthesis of the dihydrothieno[3,2-b]pyridine-7(4H)-one ring system required in the synthesis of compound 9.

Onset age-dependent variations of three islet specific autoantibodies in Japanese IDDM patients.

The age related incidence rate of insulin-dependent diabetes mellitus shows a bimodal distribution, not only in Caucasians but also in Japanese. To evaluate the onset age-related autoimmune profile at presentation in insulin-dependent diabetes mellitus (IDDM), glutamic acid decarboxylase (GAD) autoantibody, islet cell antibody (ICA), and insulin autoantibody (IAA) were measured in 137 newly diagnosed Japanese IDDM patients with onset ages between 0-29 years. The prevalence of GAD autoantibody was significantly increased from the lowest (32%) in the 0-5 years onset age group to 75% in the 13-19 years onset age group (P < 0.05), whereas the IAA prevalence significantly decreased from the peak (48%) in the 6-12 years onset age group to 10% in the 20-29 years onset age group (P < 0.05). The ICA prevalence was increased from the lowest (32%) in the 0-5 years onset age group to the highest (53%) in the 20-29 years onset age group similar to that for the GAD autoantibody. Such results demonstrate that there was age-related autoimmune characteristics at presentation of IDDM in Japanese as well as in Caucasians.

Cytoprotective effect of ulinastatin on LLC-PK1 cells treated with antimycin A, gentamicin, and cisplatin.

The cytoprotective effect of ulinastatin was studied in LLC-PK1 cells treated with antimycin A, gentamicin, or cisplatin. All of the three agents induced a concentration-dependent increase in the release of lactate dehydrogenase and a decrease in the amount of remaining protein. In the cell injury models treated with 1.5 microM antimycin A, 10 mM gentamicin, and 0.3 mM cisplatin, ulinastatin tended to show a cytoprotective effect at a concentration of 3,000 U/ml and provided a significant protective effect at 10,000 U/ml. LLC-PK1 cells treated with 0.3 mM cisplatin, bovine serum albumin, and alpha1-acid glycoprotein at a concentration of 3.54 mg/ml, which is a comparable protein concentration to that of 10,000 U/ml ulinastatin, showed no protective effect but rather enhanced cell injury. These results suggest that ulinastatin exerts a direct protective effect on LLC-PK1 cells against various renal toxicities.

Effects of 1-(3-bromobenzofuran-2-ylsulfonyl)hydantoin on human aldose reductase examined by a new application of HPLC system for measuring tissue polyol.

M16209 (1-(3-bromobenzofuran-2-ylsulfonyl)hydantoin, CAS 128851-36-5) displayed potent inhibitory effects upon recombinant human aldose reductase (rhAR, IC50 = 0.051 mumol/l). The inhibition of rhAR by M16209 was uncompetitive with respect to both glyceraldehyde and NADPH. The effects of M16209 on human AR were investigated using a new application of HPLC system developed for analysis of tissue polyol. M16209 and epalrestat suppressed galactitol accumulation in human erythrocytes cultured in 25 mmol/l galactose with IC50 values of 1.2 and 2.6 mumol/l, respectively. The new application of HPLC system equipped with an electrochemical detector did not require any derivatization procedure for polyols and enabled simultaneous determination of glucose, galactose, fructose, myo-inositol, galactitol and sorbitol.

Improvement of metabolic disorders and visceral fat obesity by the beta 3-adrenoceptor agonist (R*,R*)-(+/-)-methyl-4-[2-[2-hydroxy-2 -(3-chlorophenyl)ethylamino]propyl]-phenoxyacetate hydrobromide (BRL35135A) in genetically obese rodents.

The effects of BRL35135A ((R*,R*)-(+/-)-methyl-4-[2-[2-hydroxy-2 -(3-chlorophenyl)ethylamino]propyl]-phenoxyacetate hydrobromide), a beta 3-adrenoceptor agonist, on visceral and subcutaneous fat weight and metabolic disorders were studied in genetically obese C57BL/KsJ db/db mice and Zucker fa/fa rats. In db/db mice, four weeks of oral administration of BRL35135A (0.5 and 5 mg/kg/day) decreased body weight gain and reduced white fat weight. The rates of reduction of white fat weight were in the order mesenteric fat > retroperitoneal fat > subcutaneous fat. In fa/fa rats, daily administration of BRL35135A (0.05 mg/kg/day)) for 6 weeks reduced the visceral white fat weight/total energy intake ratio, particularly for mesenteric fat, without any clear effect on body weight gain. This tendency of the compound to exert effects on visceral fat was consistent with the findings that the effect of BRL37344 ((R*,R*)-(+/-) -methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl]-phenoxyacet ic acid), an active metabolite of BRL35135A, on the lipolytic activity of isolated adipocytes and the tissue concentration of [14C]BRL37344 in male Wistar rats were each greater in visceral fat than in subcutaneous fat. Moreover, BRL35135A at 0.05 mg/kg/day elevated serum insulin levels and improved hyperglycemia in db/db mice without reducing body weight gain, whereas at doses of 0.5 and 5 mg/kg/day it ameliorated hyperglycemia and hyperlipidemia, and tended to decrease serum insulin levels. In fa/fa rats, BRL35135A (0.005 mg/kg/day) was also effective in improving hyperinsulinemia, glucose intolerance, and hypertriglyceridemia without any effect on body weight gain or fat distribution. These findings suggest that the improvement of metabolic disorders by BRL35135A may be due to improvement in insulin resistance as well as reduction of visceral fat weight.

Stereoselective action of (R*,R*)-(+/-)-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylamino] propyl]-phenoxyacetic acid (BRL37344) on beta-adrenoceptors and metabolic chiral inversion.

Stereoisomers of BRL37344 ((R*,R*)-(+/-)-methyl-4-[2-[2-hydroxy-2 -(3-chlorophenyl)ethylamino]propyl]-phenoxyacetic acid), a beta 3-adrenoceptor agonist, were synthesized and separated with good resolution by derivatization with 1-anthroyl cyanide prior to chiral HPLC. Agonist effects on rat right atria, guinea pig trachea, and rat brown adipocytes were due principally to the (RR) isomer, while other isomers (SS, RS, and SR) were much less potent or inactive. Since the racemate (RR +/- SS) was half as potent as the (RR) isomer in all specimens tested, the (SS) isomer does not appear to have antagonistic effects. When [14C](RR)BRL35135A ((R*,R*)-(+/-)-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propy l] -phenoxyacetate hydrobromide), the HBr salt of the methyl ester of BRL37344, was administered orally to male Wistar rats, both the (RR) and (SR) isomers of [14C]BRL37344 were detected in plasma, while only the (SS) isomer of [14C]BRL37344 was detected after [14C](SS)BRL35135A administration. These findings indicate that there is clear stereoselectivity in the effects of BRL37344 on beta-adrenoceptors, and that stereoselective chiral inversion from the RR isomer to the SR isomer occurs in rats.

Uptake of human urinary trypsin inhibitor by the kidney epithelial cell line, LLC-PK1.

We investigated the uptake of human urinary trypsin inhibitor (UTI) by the kidney epithelial cells, LLC-PK1. Indirect immunogold techniques with an electron microscope demonstrated the localization of UTI within the cells after an incubation during which UTI was added to the apical side. Immunoreactivities were found in endocytic vesicles, vacuoles and lysosomes. Subsequently, we tried to characterize the property of the uptake of UTI using the fluorescein isothiocyanate-labelled UTI (FITC-UTI). FITC-UTI uptake was decreased by an incubation with an excess of unlabelled UTI and showed concentration-dependent saturation. This process was markedly suppressed during the incubation at 4 degrees C. The uptake was significantly lessened with 2,4-dinitrophenol and antimycin A, inhibitors of oxidative phosphorylation, and colchicine, a microtubule-depolymerizing agent. These results indicate that exogenous UTI is internalized by LLC-PK1 cells through an endocytic pathway. From uptake studies, it is suggested that an adsorptive process is partially involved in the mechanisms of endocytosis.

Effects of M16209, a new antihyperglycemic agent, on insulin sensitivity in vivo: euglycemic clamp studies in rats.

The effects of M16209 (1-(3-bromobenzo[b]furan-2-ylsulfonyl)hydantoin) on the in vivo insulin sensitivity of rats were studied by euglycemic clamp methods after 1 week of administration (10 or 100 mg/kg/d). M16209 increased both the glucose infusion rate (GIR) and metabolic clearance rate (MCR) of 3-[3H]-glucose, but did not suppress hepatic glucose output. M16209 also increased the [3H]-2-deoxyglucose utilization rate, rate of incorporation of [14C]-glucose into glycogen, and glycolytic flux in the soleus and red gastrocnemius muscles, but not in the extensor digitorum lungus and white gastrocnemius muscles. M16209 affected neither the [3H]-2-deoxyglucose utilization rate nor the rate of incorporation of [14C]-glucose into lipids in epididymal adipose tissue. In the soleus muscle, M16209 decreased glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) content, but did not affect fructose-1,6-bisphosphate (F-1,6-BP) content. Moreover, M16209 increased glycogen synthase-I activity and fructose-2,6-bisphosphate (F-2,6-BP) content in the soleus muscle. These results suggest that M16209 increases insulin-stimulated glucose uptake in peripheral tissues, particularly oxidative muscles, through potentiation of insulin action on glycogen synthesis and glycolysis. Glycogen synthase and phosphofructokinase (PFK) appear to be major targets of the action of M16209.

Acceleration of glycolysis in erythrocytes by the antidiabetic agent M16209.

The effects of M16209 (1-(3-bromobenzofuran-2-ylsulfonyl)hydantoin), an antidiabetic agent and aldose reductase inhibitor, on glycolysis were studied in rat and human erythrocytes in vitro. M16209 increased lactate production from glucose when incubated with rat and human erythrocytes, and also increased glucose consumption in rat erythrocytes. The rates of production of lactate in rat erythrocytes treated with M16209 at 10, 25 and 50 microM were 113, 118 and 123%, respectively, of those in vehicle treated cells. Sorbinil (aldose reductase inhibitor), tolbutamide (sulfonylurea), and buformine (biguanide) did not increase lactate production in rat erythrocytes when tested at 50 microM. On the other hand, M16209 did not affect lactate production from D-glyceraldehyde in rat erythrocytes. At 100 microM the agent decreased both glucose-6-phosphate and fructose-6-phosphate in rat erythrocytes, and increased fructose-1,6-bisphosphate; at 10 microM it also increased 6-phosphofructokinase activity in rat hemolysates. These findings suggest that M16209 accelerates glycolysis in erythrocytes via activation of 6-phosphofructokinase.

Amelioration of insulin resistance in genetically obese rodents by M16209, a new antidiabetic agent.

Improvement of metabolic disorders by M16209 (1-(3-bromobenzofuran-2-ylsulfonyl)hydantoin), an antidiabetic agent, was studied in genetically obese Zucker fa/fa rats and C57BL/6J ob/ob mice. In fa/fa rats oral administration of M16209 (30 and 100 mg/kg/day) for 7 days dose dependently improved hyperinsulinemia without affecting body weight. Oral glucose loading (2 g glucose/kg body weight) after 10 days of administration to fa/fa rats revealed that M16209 significantly improved glucose tolerance both 30 and 60 min after glucose loading, but did not affect preload serum glucose levels. At one day after 13 days of administration of M16209, the serum levels of triglyceride, total cholesterol and free fatty acid were clearly lower in treated fa/fa rats than those in untreated rats. In C57BL/6J ob/ob mice, M16209 given for 28 days at doses of 30 and 100 mg/kg/day improved hyperinsulinemia, hyperglycemia and hypercholesterolemia without affecting body weight. In a hyperinsulinemic euglycemic clamp study in fa/fa rats, administration of M16209 for 7 days at a dose of 100 mg/kg/day significantly normalized the decreased metabolic clearance rate but did not show any effect on the augmented hepatic glucose output. These findings demonstrate that improvement of metabolic disorders in genetically obese rodents by M16209 is due to amelioration of insulin resistance in peripheral tissues.

Protective action of ulinastatin against cisplatin nephrotoxicity in mice and its effect on the lysosomal fragility.

The development of azotemia after cisplatin injection in mice was inhibited by ulinastatin treatment in a dose-dependent manner. Reduction in creatinine clearance and elevation in fractional excretion of sodium in mice receiving cisplatin was ameliorated by ulinastatin administration. Epithelial necrosis and hyaline cast formation in the proximal tubule were also suppressed. Ulinastatin showed no influence on the kidney platinum level after cisplatin injection. In LLC-PK1 cells, addition of ulinastatin to the incubation medium markedly reduced the release of N-acetyl-beta-D-glucosaminidase, on of the lysosomal enzymes, during hypotonic treatment only when cells were damaged with cisplatin. On the other hand, ulinastatin showed no effect on the elevation of malondialdehyde concentration in the murine kidney cortical slices after the treatment with cisplatin. These results indicate that ulinastatin has a protective effect against cisplatin nephrotoxicity, and its prevention of the increase in lysosomal fragility is a probable mechanism involved in the renal protection.

Protective effect of human ulinastatin against gentamicin-induced acute renal failure in rats.

We investigated the protective effect of human ulinastatin against gentamicin-induced acute renal failure in rats. Gentamicin sulfate was subcutaneously injected at a dose of 200 mg/kg for 5 consecutive days. After 3 days administration of gentamicin, a slight decrease in renal function was observed, as well as granulovascular degeneration in the proximal tubular cells as a change in the renal histology. After 5 days administration of gentamicin, a remarkable increase in plasma concentration of creatinine (from 0.27 +/- 0.02 to 1.17 +/- 0.18 mg/dL) and urea nitrogen (from 17.8 +/- 0.6 to 48.8 +/- 5.1 mg/dL) and a significant decrease in creatinine clearance (from 0.64 +/- 0.08 to 0.20 +/- 0.03 mL.100 g-1.min-1) were observed. In addition, an apparent increase in urinary excretion of N-acetyl-beta-D-glucosaminidase and albumin was detected. In the renal histology, proximal tubular necrosis and desquamation of the epithelial cells in the cortex were observed. Furthermore, hyaline cast formation was frequently observed in the outer stripe of the outer medulla. Ulinastatin at doses of 100,000 or 300,000 U/kg was coadministered intraperitoneally just after each gentamicin injection. Ulinastatin treatment showed a dose-dependent suppression of gentamicin-induced biochemical alterations and histological changes. After 5 days treatment with 300,000 U.kg-1.day-1 of ulinastatin, the magnitude of gentamicin-induced changes in renal function was significantly lessened, by 45-80%. The score for proximal tubular injuries and the rate of hyaline cast formation were also significantly lower in the same group of animals than those in the group treated with gentamicin alone. In the in vitro study, ulinastatin at 10-300 U/mL showed a concentration-dependent suppression on the fragility of the lysosomal membrane isolated from rat kidney cortex during hypotonic treatment. These results indicate that human ulinastatin has a prominent protective effect on gentamicin-induced acute renal failure in rats, and the lysosomal membrane stabilizing effect is possibly involved as a mechanism of this action.

Serratia marcescens lung abscess in a child with autoimmune neutropenia.

Though Serratia marcescens is widely known to be the cause of serious infections in immunocompromised hosts, a lung abscess caused by S. marcescens is very rare. A 5 year old boy who had previously been diagnosed with autoimmune neutropenia was admitted because of fever and cough. In spite of treatment with some antibiotics, he developed a lung abscess. Aspiration of the pleural fluid revealed that S. marcescens was the pathogen of the disease. In the present case, there were feasible risk factors for the development of Serratia lung abscess namely neutropenia, chronic gingivitis at the time, and treatment with cyclosporin A. There are no reported cases of autoimmune neutropenia which developed into S. marcescens lung abscess in the literature as far as we can determine.

Antihyperglycemic effects of M16209, a novel aldose reductase inhibitor, in normal and diabetic rats.

The effect of a single oral administration of M16209 (1-(3-bromobenzo[b]furan-2-yl-sulfonyl)hydantoin), a novel aldose reductase inhibitor, on serum glucose was investigated. In normal rats, M16209 (100 mg/kg) had a weak hypoglycemic effect but markedly stimulated the disappearance of serum glucose in intravenous glucose tolerance tests. In diabetic rats, M16209 (100 mg/kg) significantly suppressed the hyperglycemia of streptozotocin-induced, mildly diabetic rats and stimulated serum glucose disappearance in neonatally streptozotocin-induced, non-insulin-dependent diabetes mellitus (NIDDM) rats in glucose tolerance tests. Additionally, M16209 augmented insulin secretion in glucose-loaded, normal and NIDDM rats and restored the reduced serum insulin in streptozotocin-induced, mildly diabetic rats. M16209, however, showed no hypoglycemic effect in severely diabetic rats. In contrast, gliclazide, a sulfonylurea, showed a much more potent hypoglycemic effect in normal rats than in mildly diabetic rats. These results suggest that M16209 suppresses hypoglycemia through augmentation of glucose-stimulated insulin secretion. The antihyperglycemic activity of M16209, combined with its potent aldose reductase inhibiting activity, is expected to be beneficial in the treatment of diabetic complications.

Effects of M16209 on insulin secretion in isolated, perfused pancreases of normal and diabetic rats.

We investigated the stimulatory effect of M16209 (1-(3-bromobenzo[b]furan-2-yl-sulfonyl)hydantoin), a novel aldose reductase inhibitor, on insulin secretion using isolated, perfused pancreases of rats. In the pancreases from normal rats, M16209 (100 microM) greatly augmented glucose-stimulated insulin secretion, but showed no effect on unstimulated insulin secretion at 2.8 mM glucose. In contrast, gliclazide (10 microM), a sulfonylurea, strongly enhanced both glucose-stimulated and unstimulated insulin secretion. Sorbinil and epalrestat, potent aldose reductase inhibitors, had no stimulatory effect on insulin secretion. M16209 (100 microM) improved appreciably the decreased insulin response to 22.2 mM glucose and enhanced slightly unstimulated insulin secretion in the pancreases of rats with neonatally streptozotocin-induced, non-insulin-dependent diabetes mellitus (NIDDM). Gliclazide (10 microM), however, failed to affect the pancreases of NIDDM rats. Furthermore, M16209 showed no appreciable effect on ATP-sensitive K(+)-channels in pancreatic beta-cells. These results suggest that M16209, unlike sulfonylureas, selectively enhances glucose-stimulated insulin secretion in both normal and NIDDM rats through a direct action on the pancreas. The site of action remains unknown, but the inhibition of aldose reductase or the ATP-sensitive K+ channels is unlikely to be involved.

[Effects of ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E) on elasticity and endothelium-dependent relaxation of the aorta in high cholesterol diet-fed rabbits].

We studied the elasticity and endothelium-dependent relaxation (EDR) of the aorta in 1% cholesterol diet (HCD)-fed rabbits. Furthermore, the effects of ethyl all-cis-5,8,11,14, 17-icosapentaenoate (EPA-E) were examined in this model of atherosclerosis. After 12 weeks of feeding with HCD, the animals showed increase in plasma total cholesterol level, formation of atherosclerotic plaque, decrease in aortic elasticity and impairment of EDR to acetylcholine (ACh). The levels of aortic elasticity in HCD-fed rabbits administered orally with EPA-E (300 mg/kg for 12 weeks) were almost the same as those of rabbits fed a normal diet, although EPA-E showed no effects on the plasma total cholesterol level and formation of atherosclerotic plaque in HCD-fed rabbits. On EDR in response to ACh and cyclic GMP formation in the HCD-fed rabbit aorta, EPA-E improved the impairment of these parameters, but not significantly. Therefore, EPA-E had little effect on the endothelium in this model of atherosclerosis, although EPA-E improved the decrease in the aortic elasticity. Because the levels of aortic elasticity showed no significant correlation with the magnitude of EDR to ACh or the size of atherosclerotic plaque, the decrease of aortic elasticity in this model of atherosclerosis was thought to have little relation to the dysfunction of the endothelium.

Beneficial effect of a novel diuretic, M17055, on blood pressure and cardiovascular hypertrophy in spontaneously hypertensive rats.

We investigated the effects of a novel diuretic, M17055, on blood pressure and cardiovascular hypertrophy in spontaneously hypertensive rats (SHR). M17055 was orally administered once a day for 24 consecutive days to 14-week-old male SHR. M17055 at doses of 1.25, 2.5 and 5 mg/kg/day exerted a dose-related diuretic and antihypertensive effect during the treatment. The weight of the left ventricle normalized by body weight on the following day of the last dosage was significantly (P < 0.01) reduced by M17055 at doses of 2.5 and 5 mg/kg/day in a dose-dependent manner. The effect of M17055 on cardiac hypertrophy was more potent (P < 0.01) than that of captopril, when the comparison was performed at the doses of M17055 and captopril inducing the same extent of blood-pressure decrement. Vascular hypertrophy was evaluated by the media/lumen ratio (M/L) in the thoracic aorta and the first branch of the superior mesenteric artery. In the aorta, M/L was slightly, but not significantly, decreased by M17055 at doses of 2.5 and 5 mg/kg/day, whereas it was decreased significantly (P < 0.01) by captopril. In the mesenteric artery, the ratio was significantly (P < 0.05) reduced by M17055 at a dose of 5 mg/kg/day. These results suggest that M17055 possesses beneficial properties for the clinical treatment of hypertension.