Haruo Ohnishi

Effects of urinary trypsin inhibitor on pancreatic enzymes and experimental acute pancreatitis

Therapeutic effect and the mechanism of the action of human urinary trypsin inhibitor (MTI) on experimental acute pancreatitis were studied. MTI significantly increased survival rate of animals with experimental acute pancreatitis induced by the infusion of trypsin or phospholipase A2 into pancreas or by a closed duodenal loop. The efficacy of MTI on these types of pancreatitis were higher than those of aprotinin. Pancreatic enzymes were released from pancreatic slice by trypsin or phospholipase A2, and this release was inhibited by MTI. Further, these pancreatic enzymes caused a secondary release of enzymes from other pancreatic slice, suggesting that these enzymes injured pancreatic tissue and that a chain reaction of pancreatic enzyme activation may play an important role in the pathogenesis of acute pancreatitis. MTI suppressed the secondary enzyme-induced pancreatic injury more strongly than aprotinin. These results suggest that MTI may suppress pathogenesis and development of pancreatitis by inhibiting the chain reaction of pancreatic enzyme activation.

A new approach to the treatment of atherosclerosis and trapidil as an antagonist to platelet-derived growth factor

Abstract It has been proposed that platelet-derived growth factor (PDGF) may be a key event in intimal thickening in the development of atherosclerosis. In the present paper, the inhibitory action on the growth-promoting activity of PDGF (anti-PDGF activity) was examined with the use of an in vitro model. A coronary vasodilator, trapidil, inhibited BALB/c 3T3 cell proliferation promoted by PDGF, but did not inhibit BALB/c 3T3 cell proliferation promoted by fibroblast growth factor and Ca 3 (PO 4 ) 2 , and SV40-transformed 3T3 cell proliferation. The anti-PDGF activity of trapidil was markedly weakened when trapidil was added to medium 2 hr after the addition of PDGF. None of other tested drugs except trapidil showed the anti-PDGF activity. The data indicate that trapidil acts as an antagonist of PDGF, and suggest that trapidil may be effective on the prevention of atherosclerosis.

Gastric antisecretory effect of FRG-8813, a new histamine H2 receptor antagonist, in rats and dogs

FRG-8813, a new histamine H2 receptor antagonist, was examined for antisecretory effects and compared with famotidine and cimetidine in rats and dogs. In pylorus-ligated and lumen-perfused rats, FRG-8813 given i.v. reduced basal gastric acid secretion and the acid secretion evoked by histamine, tetragastrin, bethanechol, and 2-deoxy-D-glucose in a dose-dependent manner. The i.v. antisecretory activity of FRG-8813 was equivalent to or slightly less than that of famotidine and the intraduodenal (i.d.) activity was greater than that of cimetidine. The duration of action of FRG-8813 was substantially longer than that of farmotidine and cimetidine for both i.v. and i.d. routes. The i.v. ED40 values for the histamine- and tetragastrin-evoked responses and the i.v. ED30 value for the bethanechol-evoked response were 0.15, 0.09 and 0.43 mg2kg, respectively. In Heidenhain pouch dogs, when the three H2 antagonists were given i.v. or orally, the relative antisecretory potency of the compounds was similar to that in rats. The long-lasting antisecretory effect of FRG-8813 was also observed, and the i.v. ED50 values for histamine-, tetragastrin- and bethanechol-evoked responses were 0.1, 0.24 and 1.0 mg/kg, respectively. Comparison of the parenteral and enteral potencies indicated that FRG-8813 has a lower bioavailability than famotidine and cimetidine in rats and dogs. These data suggest that FRG-8813 has a potent and long-lasting antisecretory effect with a far greater potency than cimetidine and with a slightly lower potency than famotidine.

The immunomodulatory action of inosiplex in relation to its effects in experimental viral infections.

The effect of inosiplex (Isoprinosine) on viral replication, experimental viral infections and host immune functions has been examined. Inosiplex was found to have a broad spectrum of antiviral activity, inhibiting the RNA viruses, influenza (INFV) and parainfluenza (PIV), as well as the DNA viruses, herpes simplex (HSV) and vaccinia (VACV). However, the antiviral effects were modest when compared to amantadine and adenine arabinoside (ARA-A). Inosiplex in vivo caused a statistically significant increase in survival of treated animals (hamster, mice) infected with RNA or DNA viruses. This effect of inosiplex was apparent in animals which were previously immunosuppressed. Inosiplex, at optimal dose, conferred total protection in treated mice against secondary influenza infection. Since this was accompanied by statistically significant increases in serum anti-hemagglutinin and anti-neuraminidase titers, an effect of inosiplex on host defenses against secondary viral infection was implicated. This effect was further demonstrated by passive transfer of protection by splenocytes from inosiplex-treated donors to untreated recipients. Inosiplex was found to enhance the mitogen- (PHA-, ConA and MLC-) induced blastogenesis of lymphocytes from untreated mice. The LPS response was not affected. Inosiplex added in vitro caused a dose-dependent increase in the primary immune anti-SRBC response in vitro, as determined by direct and indirect PFC; there was also a dose-dependent effect on the secondary in vitro direct and indirect PFC responses. Inosiplex in vivo enhanced the primary immune response to SRBC, as determined by direct PFC assay; this was also the case for immunosuppressed mice. The drug enhanced delayed type hypersensitivity to picryl chloride in the mouse. Macrophage function was also enhanced by inosiplex, as was apparent from phagocytosis of SRBC. Gamma interferon production from murine lymphocytes was augmented by inosiplex in vitro. Treatment with inosiplex had no effect on natural killer cells or on antibody dependent cellular cytotoxicity. Thus, the pronounced effect of inosiplex on secondary viral infections may result through two different mechanisms: a direct antiviral effect and an elevation of multiple parameters of host immunity, which are usually compromised during viral infection. The latter mechanism may be the more important.

Effects of trapidil on thromboxane A2-induced aggregation of platelets, ischemic changes in heart and biosynthesis of thromboxane A2.

Trapidil inhibited the aggregation of rat platelets and the contraction of the isolated aortic strip of rabbit mainly caused by thromboxane A2, and the thromboxane A2 biosynthesis in rabbit platelets. The drug also reduced ischemic changes in ECG, the incidence of myocardial infarction, histopathological changes and a decrease in serum high density lipoprotein cholesterol, and inhibited an increase in plasma thromboxane B2 and a decrease in plasma 6-keto-prostaglandin F1 alpha content in the animals with an experimental ischemic heart injury caused by the injection of thromboxane A2 into the coronary artery. These findings suggest that trapidil is a new type of a therapeutic agent for ischemic heart disease which not only dilates the coronary artery but also inhibits the actions and biosynthesis of thromboxane A2 and may promote the biosynthesis of prostaglandin I2.

Structure of slow-reacting substance of anaphylaxis (SRS-A)

To elucidate the chemical structure of slow-reacting substance of anaphylaxis from rat (SRS-A rat), SRS-A rat were purified by the method of Orange with modification using DEAE-Sephadex A-25 chromatography. Ultraviolet absorption spectrum of purified SRS-A rat indicated the presence of conjugated triene. Arylsulfatase B degradation products and HCl degradation products were subjected to analysis by a gas chromatography and mass spectrometry and a thin layer chromatography. Products obtained by arylsulfatase b catalysis contained 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid. HCl degradation products showed the presence of glycine, glutamic acid and cysteic acid. Furthermore, the analysis of anhydrous hydrazine degradation products of SRS-A rat and of HCl hydrolyzed products of dinitrophenylated SRS-A rat revealed the presence of glycine at C-terminal and glutamine acid at N-terminal. The study of the substrate specificity of arylsulfatase B against various materials including SRS-A rat suggested the presence of sulfone in SRS-A rat. The molecular ion peak of SRS-A rat sodium salt was observed at m/e 680 in field desorption mass spectrum of SRS-A rat. On the basis of these data, we identified the structure of SRS-A rat as [gamma]glutamyl-4(5-hydroxy-7,9,11,14-eicosatetraenoic acid-6-yl)-4,4-dioxyocysteinyl] glycine.

Antithrombotic activity and the mechanism of action of trapidil (Rocornal

Antithrombotic activity and the mechanism of action of trapidil were investigated, as compared with those of aspirin and dipyridamole. Trapidil at oral doses of 30 and 100 mg/kg inhibited arterial thrombosis in rats, while aspirin and dipyridamole at doses up to 300 mg/kg showed only a mild activity. This action may be explained by the fact that trapidil at concentrations ranging from 139 microM to 251 microM exerted 50% inhibition of platelet aggregation induced by ADP, arachidonic acid, thrombin or thromboxane A2 mixture, inhibition of platelet release reaction induced by ADP, arachidonic acid or thrombin, disaggregatory effect on aggregated rabbit platelets by arachidonic acid and potentiating action on antiaggregatory action of prostacyclin in vitro. In vitro actions of trapidil were apparently different from those of aspirin and dipyridamole. Trapidil also showed inhibition of platelet phosphodiesterase activity and thromboxane synthetase activity. Trapidil was expected to be an effective antithrombotic agent. The antithrombotic action of trapidil may be mediated by the inhibition of platelet function which is characterized by the inhibition of both thromboxane synthetase and phosphodiesterase activities, and by the potentiation of the antiaggregatory action of prostacyclin.

Reduction of Myocardial Infarct Size by Trapidil in Anesthetized Dogs

Summary We studied the effect of trapidil on acute experimental myocardial infarction in anesthetized, open-chest dogs. The size of myocardial infarction 8 h after ligation of the left anterior descending coronary artery was determined by planimetry of myocardial slices stained by the nitroblue tetrazolium method. Systemic hemodynamic variables, epicardial ST-segment elevation, activity of serum creatine phosphokinase (CPK), and changes of myocardial blood flow in the ischemic area were measured. Infusion of 3 mg/kg trapidil reduced the size of infarction and ameliorated the infarction-induced deterioration of systemic hemodynamic variables, such as the decrease in left ventricular dP/dt, aortic blood flow, and regional endomyocardial blood flow in the ischemic area. This dose of trapidil also suppressed ST elevation and significantly inhibited the increase in activity of serum CPK. Hyaluronidase also reduced the size of infarction significantly. These results suggest that trapidil alters the course of acute myocardial infarction favorably, presumably by increasing regional endomyocardial blood flow.

Pepsin and autoimmune disease mouse

The effects of pepsin on autoimmune glomerulonephritis of MRL/1 mice were investigated. Intravenous administration of pepsin significantly improved survival rate and suppressed progressive increase in urinary excretion of protein and various histopathological changes in kidney. Increased serum levels of immune complex and anti-DNA antibody in MRL/1 mice were decreased by pepsin. Pepsin ameliorated abnormalities in lymphocyte subsets and lymphocyte functions. The fact that pepsin ameliorated abnormalities in immune function may contribute to the preventive effects of pepsin against pathogenesis and progress of immune complex nephritis.

ヒト腸上皮細胞株Caco-2のacyl-CoA:cholesterol acyltransferase(ACAT)活性とコレステロールエステル分泌に対するACAT阻害薬,F-1394の作用

The present study was conducted to investigate the inhibitory effect of F-1394, a potent and selective inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), on incorporation of 14C-oleic acid into cholesteryl ester in cultured Caco-2 cells, a human intestinal cell line, and compare its effect to those of other ACAT inhibitors and hypolipidemic agents. The cholesterol esterification in Caco-2 cells was strongly inhibited by F-1394 in a concentration-dependent manner with the estimated IC50 value of 71 nM. In contrast, the estimated IC50 values of the other ACAT inhibitors such as YM-17E, CI-976, CL-277,082 and DL-melinamide are 121 nM, 702 nM, 21.5 microM and 20.9 microM, respectively. Simvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, also inhibited the ACAT activity in Caco-2 cells with an IC50 value of 22.5 microM, whereas pravastatin Na, probucol and clofibrate did not affect the activity. Furthermore, F-1394 at a concentration of 100 nM inhibited the basolateral secretion of cholesteryl ester by 90% from differentiated Caco-2 cells that were cultured on a membrane filter. These results demonstrate that F-1394 strongly inhibits human intestinal ACAT activity and basolateral secretion of cholesterol from Caco-2 cells. Therefore, F-1394 may have a therapeutic potential for dietary hyperlipidemic subjects.