Macular hole formation after macular haemorrhage associated with rupture of retinal arterial macroaneurysm.

AIM: To investigate the frequency and risk factors of macular hole (MH) formation after rupture of a retinal arterial macroaneurysm. METHODS: Fifty-six eyes from 56 patients with rupture of a retinal arterial macroaneurysm with or without an MH (MH and non-MH groups, respectively) were reviewed. Frequency and risk factors related to MH formation were assessed, with risk factors including age; sex; distance from the macroaneurysm to the fovea; incidence of haemorrhages involving the macula such as preretinal, subinternal limiting membrane (sub-ILM), subretinal and vitreous; and vitreous surgery. MH formation in these patients was recorded and analysed. RESULTS: Of the 56 eyes reviewed, seven (12.5%) had an MH after rupture of the retinal arterial macroaneurysm. The incidence of subretinal and sub-ILM haemorrhages involving the macula was significantly greater in the MH group than in the non-MH group (p = 0.037 and 0.045, respectively). CONCLUSION: These results suggest that the presence of subretinal and sub-ILM haemorrhages after rupture of a retinal arterial macroaneurysm may contribute to formation of an MH.

Effect of sepimostat mesilate on experimental venous thrombosis in rats.

Sepimostat mesilate (FUT-187: 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl) amino] benzoate dimethane sulfonate) is a newly synthesized serine protease inhibitor. In the present study, the oral administration of FUT-187 inhibited stasis-induced venous thrombosis in rats. We supposed that such effect of this compound was caused by its inhibitory effect on coagulation. However, the dose of FUT-187 that was effective at inhibiting thrombosis (10 and 30 mg/kg, po) had no effect on the plasma recalcification time (PRCT), activated partial thromboplastin time (APTT) and prothrombin time (PT) in rats. Therefore, we investigated the fibrinolytic activity of FUT-187 in rat plasma. The results revealed that rat plasma after FUT-187 administration exhibited increased amidolytic activity for a plasmin-, tissue-type plasminogen activator (t-PA)-, urokinase-type plasminogen activator (u-PA)-, factor Xa-, factor XIa- and factor XIIa-sensitive synthetic peptide substrate. On the other hand, the inhibitory effect of FUT-187 in the thrombosis model was not affected by additional treatment with epsilon-amino-n-caproic acid (EACA), a plasmin-mediated fibrinolysis inhibitor. These results suggest that even if FUT-187 enhanced fibrinolysis, it would be independent of a plasmin-mediated fibrinolytic pathway. To characterize the fibrinolytic activity, which might reduce the thrombus weight in the thrombosis model administered FUT-187, we carried out fibrinogen zymography, and clarified that FUT-187 enhanced the formation of a 20-kDa fibrinolytic fragment. Interestingly, this fragment was not affected by t-PA. Consequently, we consider that the inhibitory effect of FUT-187 on venous thrombosis model is caused by fibrinolysis, which is attributable to the 20-kDa fragment, rather than by inhibition of thrombus formation.

Expression and localization of enzymes of arginine metabolism in the rat eye.

PURPOSE: Production of NO may be regulated by argininosuccinate synthetase and argininosuccinate lyase which recycle citrulline to arginine, and by arginase which hydrolyzes arginine to urea and ornithine. Expression of these and related enzymes in rat eye was studied. METHODS: mRNAs for the enzymes were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Localization of the enzyme proteins and mRNAs was analyzed by immunohistochemistry and in situ hybridization, respectively. RESULTS: In RT-PCR analysis, arginase II (nonhepatic type) mRNA was detected in retina and weakly in cornea, whereas arginase I (hepatic type) mRNA was not detected in any area. mRNAs for argininosuccinate synthetase, argininosuccinate lyase, ornithine aminotransferase and ornithine decarboxylase were present in all areas. In immunohistochemical analysis, arginase II was stained in cornea, epithelium of iris and ciliary process, inner part of neural retina and retinal pigment epithelium. Immunostaining of ornithine aminotransferase resembled that of arginase II. In in situ hybridization, arginase II and ornithine aminotransferase mRNAs were located much as seen with the enzyme proteins. CONCLUSIONS: These results indicate that arginine recycling activity from citrulline is present widely in ocular tissues, whereas expression of arginase II differs among the tissues. We suggest that NO production may be regulated by these enzymes in the cells where NO synthase is colocalized. Colocalization of arginase II and ornithine aminotransferase suggests a role for arginase II in collagen synthesis in the eye.

Expression of citrulline-nitric oxide cycle in lipopolysaccharide and cytokine-stimulated rat astroglioma C6 cells.

Nitric oxide (NO) is involved in many physiological and pathological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS), with citrulline generated as a by-product of the reaction. Thus, citrulline can by recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) via the citrulline-NO cycle. Rat astroglioma C6 cells were treated with bacterial lipopolysaccharide (LPS), interferon-gamma (IFNgamma) and tumor necrosis factor-alpha, and the expression of the enzymes of the citrulline-NO cycle was investigated by RNA blot and immunoblot analyses. NO production from arginine and citrulline was also assessed. iNOS mRNA and protein were induced 6-12 h after stimulation with LPS and cytokines and decreased at 24 h. AS mRNA increased up to 12 h and decreased at 24 h. AS protein increased gradually up to 48 h. On the other hand, AL mRNA remained unchanged by stimulation. NO production from arginine was enhanced by the treatment with LPS and cytokines. NO production was also observed when arginine was replaced by citrulline. These results indicate that NO production is enhanced in LPS- and cytokine-stimulated C6 cells due to induction of iNOS and that the citrulline-arginine recycling is important for NO production.

[Adhesive and sealing effects of TO-193 on tissues and organs in various experimental models].

We studied the adhesive and sealing effects of sheet style fibrin adhesive, TO-193 (TachoComb), on some tissues and organs, comparing them with those of sheet style collagen agent, collagen sponge, Novacol, and Avitene and liquid fibrin adhesive agent, Beriplast P. TO-193 showed more a potent adhesive effect on liver than the sheet style collagen agents and was more potent on bone and skin than the liquid fibrin adhesive agent. Furthermore, TO-193 had a potent sealing effect at the site of incomplete suture immediately after application on a motile organ such as lung and stomach. These effects may be partly attributable to rapid expression of the effect due to the presence of a high concentration of fibrinogen on coverage. Enhancement of fibrin penetrability to the tissues by compression and inhibition of cleavage of coverage by the collagen sponge also may be participating in the effects of TO-193. These results suggest that TO-193 will be a valuable adhesive and sealing agent.

[Hemostatic effect of a novel sheeted fibrin adhesive agent, TO-193, on experimental incision models].

TO-193 (TachoComb) is a new fibrin adhesive agent that consists of a collagen sheet coated with fibrin glue. We compared the hemostatic effect of TO-193 in several experimental models with Beriplast P as a fibrin adhesive agent, Avitene and Novacol as a microfibrillar collagen hemostat, and a sponge-like collagen sheet as the constitutional parts of TO-193. In the in vitro bleeding model in which blood leaks out through cotton cloth, the pressure of TO-193 when blood leakage was observed was higher than those of Beriplast P, Avitene, Novacol and the collagen sheet, indicating that TO-193 possessed a strong adhesive effect on the bleeding surface. On the kidney resection surface, TO-193 showed a more potent adhesive effect than those of Beriplast P and the collagen sheet, suggesting that TO-193 has a potent hemostatic effect. In the liver resection, TO-193 significantly reduced the bleeding volume compared with that of Novacol in normal rats. Furthermore, the bleeding volume of TO-193 was about half that of Beriplast P and equivalent to that of Novacol even in anticoagulant-treated rats. From these data, it is expected that TO-193 would be a valuable hemostatic agent for clinical use since TO-193 possesses a potent adhesive ability on the bleeding resection surface and would certainly stop bleeding in both patients with normal coagulation and those with a low blood coagulation condition.

Pharmacological studies of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzoate dimethanesulfonate. Effects on experimental pancreatitis.

The effects of FUT-187 (6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzoate dimethanesulfonate, CAS 103926-82-5), a novel synthetic protease inhibitor, were examined in experimental rat and canine models of pancreatitis. 1. FUT-187 significantly increased the survival of rats with trypsin- and phospholipase A2-induced pancreatitis in a dose-dependent manner (10-100 mg/kg, p.o.). 2. FUT-187 decreased plasma enzymatic activity reflecting the degree of pancreatitis in rats with ethionine-induced pancreatitis, and showed a tendency to ameliorate histopathological changes in the pancreas (10-100 mg/kg p.o.). 3. FUT-187 (10 mg/kg) produced an obvious improvement of various biochemical parameters of pancreatitis and also reduced histopathological changes in the pancreas in animals with experimental pancreatitis produced by the closed duodenal loop method. In addition, FUT-187 significantly increased the survival of dogs when given by direct administration into the lumen of the closed duodenal loop. The therapeutic effects of FUT-187 in experimental pancreatitis were nearly equal in most instances to those of camostat mesilate. Thus, FUT-187 would appear to be an effective new agent for the treatment of pancreatitis.

[The effects of FUT-175 (nafamostat mesilate) on blood coagulation and experimental disseminated intravascular coagulation (DIC)].

FUT-175 is a newly synthesized serine protease inhibitor. In the present study, we investigated the effects of FUT-175 on blood coagulation and experimental DIC. The effects on coagulation were examined in vitro by measuring the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) of rat plasma in the presence of FUT-175. FUT-175 exhibited remarkable anticoagulative effects to prolong APTT at a plasma concentration of 3 x 10(-7) M, PT at 1 x 10(-5) M and TT at 3 x 10(-5) M. The anticoagulative effect of FUT-175 at 1 x 10(-6) M on APTT was almost similar to that of heparin at 0.3 U/ml or that of gabexate mesilate at 1 x 10(-3) M. Experimental DIC was induced by a four-hr sustained intravenous infusion of endotoxin. FUT-175 was administered intraperitoneally prior to the injection of endotoxin or infused intravenously with endotoxin. As a result, the prolongation of APTT and PT, the decreases of fibrinogen level, platelet counts and complement level, and the increase of FDP were remarkably improved by FUT-175. Furthermore, glomerular fibrin deposits were reduced by the infusion of FUT-175. These results indicate that FUT-175, having a potent inhibitory effect on blood coagulation, is clinically applicable to therapy for DIC.

Effects of FUT-175, a novel synthetic protease inhibitor, on the development of adjuvant arthritis in rats and some biological reactions dependent on complement activation.

1. The effects of FUT-175 on the development of adjuvant arthritis in rats were studied and compared with those of indomethacin. FUT-175 inhibited both primary and secondary paw lesions in the adjuvant arthritic rats when it was administered orally on a daily basis from the day before through 18th day after adjuvant injection. 2. In addition, FUT-175 inhibited the increase in hemolytic complement in adjuvant arthritic rats in a dose-dependent manner. 3. Indomethacin also showed an inhibitory effect on the development of arthritic lesion, but had no effect on the increase in hemolytic complement in the adjuvant arthritis in rats. 4. Furthermore, FUT-175 inhibited the activities of various proteases in vitro, and then strongly inhibited complement-mediated hemolysis via the classical and alternative pathways, while indomethacin had no effect on them. 5. These results suggest that the anti-inflammatory activity of FUT-175 may differ from indomethacin in the mechanisms of action and, at least in part, due to the anti-complement activity.

Inhibitory effects of a novel synthetic protease inhibitor, FUT-175, on the paw edema in rats and zymosan-induced complement activation in vitro.

FUT-175 inhibited the zymosan-induced rat paw edema in a dose-dependent manner, while indomethacin exhibited no significant activities in this model. FUT-175 also inhibited the decrease in hemolytic complement (CH50) induced by zymosan in vitro, and indomethacin was inactive. These results suggest that FUT-175 has potent in vitro and in vivo inhibitory activity against the activation of the complement system induced by zymosan.

[Pharmacological studies of FUT-175, nafamstat mesilate. IV. Effects on coagulation, platelets and fibrinolysis].

Inhibitory effects of FUT-175 (nafamstat mesilate) on coagulation, platelets and fibrinolysis were examined. FUT-175 prolonged activated partial thromboplastin time, thrombin time and prothrombin time in rabbit plasma. FUT-175 prolonged these coagulation times in human plasma at lower concentration than in rabbit plasma. FUT-175 inhibited platelet aggregation induced by a variety of aggregation agents in rabbit platelet-rich plasma (PRP). In human PRP, FUT-175 inhibited platelet aggregation induced by a variety of aggregation agents at lower concentration than in rabbit PRP. Lipopolysaccharide induced a dose-dependent platelet aggregation in dog PRP. FUT-175 showed an inhibitory effect on this aggregation. FUT-175 inhibited clot retraction in rabbit plasma. The fibrinolysis activity was measured on fibrinolysis of rabbit plasma activated by urokinase. FUT-175 prolonged this fibrinolysis time. Inhibitory effects on coagulation and fibrinolysis were also found ex vivo. FUT-175 prolonged bleeding time in mice. These results indicate that FUT-175 has potent inhibitory effects on coagulation, platelets and fibrinolysis.

[Pharmacological studies of FUT-175, nafamstat mesilate. III. Anti-inflammatory activities of FUT-175].

Anti-inflammatory effects of FUT-175 (nafamstat mesilate), a new synthetic serine protease inhibitor, on various types of experimental inflammation were investigated in vivo and in vitro, in comparison with non-steroidal anti-inflammatory drugs (NSAID). The in vivo studies showed that FUT-175 has the abilities to inhibit almost all types of inflammatory reactions employed in the present study. In particular, being evaluated on the basis of the effect of indomethacin, FUT-175 exhibited relatively higher potencies against some reactions such as zymosan-induced increase of vascular permeability, scald paw edema, zymosan-induced granuloma-pouch, the Arthus reaction and acetic acid-induced writhing in which the complement system or the kallikrein-kinin system are considered to play an important role. The in vitro studies showed that FUT-175 is quite different from NSAID, that is, FUT-175 had no effects on heat-induced erythrocyte-lysis and heat-induced denaturation of bovine serum albumin. FUT-175 also had no effect on chemotaxis of polymorphonuclear leucocytes, but inhibited the production of chemotactic factor by antigen-antibody reaction. These above results suggested that FUT-175 has a different mode of action from NSAID and that serine protease inhibiting activities of this compound might play an important role in its anti-inflammatory effect.

Pharmacological studies of FUT-175, nafamstat mesilate. I. Inhibition of protease activity in in vitro and in vivo experiments.

FUT-175, 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate (nafamstat mesilate), a novel synthetic protease-inhibiting agent, was studied to determine its in vitro effects against various proteases and other enzymes, as well as to determine its in vivo protease inhibitory effects. FUT-175 was found to inhibit, in an intense, specific and reversible way, the enzyme activities of trypsin, C1r, C1s, thrombin, kallikrein and plasmin with IC50 values of the order of 10(-6)-10(-8) M. FUT-175 also inhibited complement-mediated hemolysis, including both classical and alternative pathways, sites of inhibition being on C1r and C1s as evidenced by the intermediate-cell technique. In animal model reactions in which the complement system is known to be involved as pathogenetic factors, e.g., Forssman shock, Forssman cutaneous vasculitis, zymosan-induced paw edema, endotoxin shock and local Shwartzman reaction, FUT-175 was highly effective in that, for example, intravenous dosing at 3 mg/kg could completely protect guinea pigs from the lethal Forssman shock. FUT-175 was also found to be effective in trypsin-induced shock in mice, in lethality due to thrombin-thrombosis in mice and in kinin formation in the inflammatory process in rats.

Immunological studies of pityriasis rosea (Gibert).

In order to elucidate the immunological mechanism in the pathogenesis of pityriasis rosea, immunofluorescent studies were performed on sera obtained from forty patients with this disease. Antibodies against the cytoplasm of normal human epidermal cells were demonstrated in the sera of all patients. The antibody titer showed a tendency to increase within 3 weeks after onset of secondary eruptions and then to decrease gradually until the period of recovery. The immunoglobulin class was determined to be IgM. Furthermore, by the direct immunofluorescent technique, deposits of IgM in the epidermal cells of skin lesions were demonstrated in 3 of 6 herald lesions and in 1 of 4 secondary eruptions. It is suggested that anti-cytoplasmic antibodies produced by some unknown cause may induce the development of secondary eruptions of this disease.