Selective inhibition of trypsin by (2R,4R)-4-phenyl-1-[Nalpha-(7- methoxy-2-naphthalenesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid.

Evidence is accumulating indicating that trypsin stimulates divergent cellular reactions through the proteinase-activated receptor, in addition to its role as the digestive enzyme. In this report, we introduce (2R,4R)- 4-phenyl-1-[N(alpha)-(7-methoxy-2-naphthalenesulfonyl)-l-arginyl]- 2-p iperidinecarboxylic acid as a potent and selective trypsin inhibitor. The agent inhibited trypsin competitively with the K(i) value of 0. 1 micrometer. It inhibited thrombin weakly (K(i) = 2 micrometer) and did not inhibit plasmin, plasma kallikrein, urokinase, and mast cell tryptase (K(i) values for these enzymes are >60 micrometer). Comparative studies with several established proteinase inhibitors revealed that the compound was the first small molecular weight trypsin inhibitor without tryptase inhibitory activity. A docking study has provided a plausible explanation for the molecular mechanism of the selective inhibition showing that the agent fits into the active site of trypsin without any severe collision but that it comes into clash at the 4-phenyl group of piperidine ring against the "60-insertion loop" of thrombin and at the 7-methoxy-2-naphthalenesulfonyl group against Gln(98) of tryptase.

Evaluation of the pharmacological actions and pharmacokinetics of BOF-4272, a xanthine oxidase inhibitor, in mouse liver.

BOF-4272 (+/-)-8-(3-methoxy-4-phenylsulphinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4-(1H)-one, a new synthetic anti-hyperuricaemic drug, which has a chiral centre and exists as racemates, is a potent inhibitor of xanthine oxidase/dehydrogenase in the purine catabolism pathways. The present studies using mice demonstrated that BOF-4272 was specifically distributed in the liver, which is the main organ of uric acid production. Therefore, a decrease in uric acid concentration in the liver, rather than the plasma, was identified as a pharmacological action of BOF-4272. The ratio of liver to plasma concentrations of BOF-4272 increased from 2.5 to 6.3 over time, up to 8 h after oral administration. The elimination half-life of BOF-4272 in the liver was 5-1-fold longer than that in the plasma. High concentrations of BOF-4272 were observed in the liver up to 8 h after oral administration. Furthermore, the influx of BOF-4272 into hepatocytes occurred in a temperature-dependent manner. The liver concentrations of uric acid from 1 h to 8 h after the oral administration of BOF-4272 (0.34-0.75 microg (g tissue)(-1)) were significantly lower than those in control animals (5.03-10.96 microg (g tissue)(-1)). BOF-4269 (the sulphide metabolite of BOF-4272) was the only metabolite detected in plasma or faeces after intravenous or oral administration. BOF-4269, which has no inhibitory action on the uric acid biosynthesis system, is generated by the metabolism of BOF-4272 in the intestinal tract. In conclusion, this work using the liver as the target organ has allowed us to identify the pharmacological actions of BOF-4272 in mice. The long-lasting effect of BOF-4272 in reducing levels of hepatic uric acid was consistent with the prolonged high BOF-4272 concentrations in the liver. These results also demonstrate that the mouse is a suitable animal species for evaluating the clinical pharmacology and pharmacokinetics of BOF-4272.

Cyclopropenone-containing cysteine proteinase inhibitors. Synthesis and enzyme inhibitory activities.

By focusing on the amphiphilic properties of cyclopropenone (e.g. a good electrophile and a precursor for a stable 2pi-aromatic hydroxycyclopropenium cation), a new class of cysteine proteinase inhibitors containing a cyclopropenone moiety was designed. For the purpose of the present research, we needed to devise a new method to introduce a peptide-related moiety as a substituent on the cyclopropenone residue. We investigated the reaction of metalated cyclopropenone acetal derivatives (2, R2 = metal) with N-protected alpha-aminoaldehydes 4 to obtain the adduct 5, and succeeded in the preparation of highly potentiated cysteine proteinase inhibitors 8 after several steps transformations. They showed strong inhibitory activities only to cysteine proteinases such as calpain, papain, cathepsin B, and cathepsin L and not to serine (e.g. thrombin and cathepsin G) and aspartic proteinases (e.g. cathepsin D). Kinetic studies indicated that they are competitive inhibitors, and by the examinations of their inhibitory mechanism it became clear that they are reversible inhibitors.

Effect of argatroban, a selective thrombin inhibitor, on animal models of cerebral thrombosis.

Argatroban, (2R,4R)-4-methyl-1-(N2 [(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-sulfonyl]-L-arginyl)-2-pi peridinecarboxylic acid, a selective and competitive thrombin inhibitor, was examined for effectiveness in three different experimental models of cerebral thrombosis in rats, namely, the four-vessel occlusion model, the middle cerebral artery occlusion model, and the distal middle cerebral artery occlusion model. Argatroban was demonstrated to be effective in these experimental models of thrombosis. Among these models, the distal middle cerebral artery occlusion model was the most similar to clinical cerebral thrombosis with respect to restriction of the infarction to the cerebral cortex and the accompanying stable neurologic deficits. In this model, the thrombus was generated at the Y-shaped bifurcation of the middle cerebral artery by green light irradiation through a cranial window after administration of rose bengal. Argatroban given after thrombus formation by intraperitoneal implantation of an osmotic pressure pump was shown to reduce infarct size and neurologic deficits on day 3 and microthrombi generation on day 1, and to raise the regional cerebral blood flow on day 1, at a plasma level of 0.2 to 0.6 microM supporting its clinical usefulness in the treatment of acute-phase cerebral thrombosis. Argatroban was considered to exert its effects by salvaging neuronal cells in the ischemic penumbra and suppressing extension of the infarction into the penumbra by keeping blood vessels patent, mainly through the inhibition of microthrombogenesis.

Effects of a thrombin inhibitor, argatroban, on ischemic brain damage in the rat distal middle cerebral artery occlusion model.

To elucidate the role of thrombin in brain damage during focal cerebral ischemia, we investigated the effects of a selective thrombin inhibitor, argatroban, on microthrombi formation, regional cerebral blood flow (rCBF), infarct areas and neurological deficits using a rat thrombotic distal middle cerebral artery (dMCA) occlusion model. The rat dMCA was occluded by a platelet-rich thrombus formed after photochemical reaction between rose bengal and green light. One day after dMCA occlusion, the number of microthrombi were counted. In the separate animals, rCBF was measured by using the iodoantipyrine method 1 day after dMCA occlusion. Three days after dMCA occlusion, behavioral tests were performed and the size of the cerebral infarction was determined. In the present study, argatroban was administered i.p. by continuous infusion after dMCA occlusion. Argatroban (0.3 mg/h/rat) significantly (P < .05) decreased the number of microthrombi 1 day after dMCA occlusion. Argatroban (0.1 and 0.3 mg/h/rat) significantly (P < .01) reversed a decrease in rCBF 1 day after dMCA occlusion. Argatroban (0.3 mg/h/rat) also significantly (P < .01) reduced the size of the cerebral infarction. Administration of argatroban (0.1 and 0.3 mg/h/rat) resulted in a significant improvement in neurological deficits 3 days after dMCA occlusion (P < .01 and P < .05, respectively). Argatroban decreased the size of the cerebral infarction and improved neurological deficits in the rat thrombotic dMCA occlusion model. These effects were thought to be due to the improvement of rCBF and to the reduction in secondary thrombus formation after dMCA occlusion.

Inhibition of factor Xa-induced platelet aggregation by a selective thrombin inhibitor, argatroban.

In the present study, a direct selective thrombin inhibitor, argatroban, and an indirect non-selective inhibitor, heparin, were examined for the inhibitory effect on factor Xa-induced platelet aggregation. Platelet aggregation was induced by thrombin or factor Xa+prothrombin in the presence of Ca++ using rabbit gel-filtered platelets. IC50 of argatroban on factor Xa-induced aggregation was 5-7 times higher than that on thrombin-induced aggregation, while IC50 of heparin in the presence of antithrombin III on factor Xa-induced aggregation was 90-200 times higher than that on thrombin-induced aggregation. This finding suggests that argatroban inhibits thrombin generating on the platelet surface more efficiently than heparin, and this may be one of the reasons for the higher efficacy of argatroban in arterial thrombosis as compared with heparin.

Beneficial effects of a new prostacyclin analogue, KP-10614, on acute myocardial infarction in rats.

The potential therapeutic value of a new prostacyclin analogue, (4z, 16s)-4,5,18,18,19,19-hexadehydro-16,20-dimethyl-delta 6(9a)- 9-(O)-methano-PGI1 (KP-10614), was studied in acute myocardial infarction in rats. Myocardial infarction was induced by ligation of the left coronary artery and ischemic injury was followed up to 4 h. The infarct size, evaluated by the area unstained by 2,3,5-triphenyltetrazolium chloride, reached 41.1 +/- 1.4% of the left ventricle at 4 h. KP-10614 (3 ng/kg/min x 4 h) reduced the infarct size at 4 h significantly (26.5 +/- 2.9%). At the same dose, KP-10614 inhibited ADP-induced ex vivo platelet aggregation significantly (21.5 +/- 4.0% of the control aggregation), but did not alter the arterial blood pressure or heart rate. To assess the role of platelets in myocardial infarction, circulating platelets were reduced by about 95% with rabbit antiserum to rat platelets. In platelet-depleted rats, the infarct size decreased significantly to 24.1 +/- 4.6% of the left ventricle at 4 h. These results suggest that platelets play an important role in expression of myocardial ischemic injury resulting from coronary artery occlusion in rats, and the ability of KP-10614 to decrease the infarct size appeared to be attributable, at least in part, to the inhibition of platelet aggregation or cellular metabolic effects produced by platelets at the site of tissue injury.

Antithrombotic effect of MCI-9042, a new antiplatelet agent on experimental thrombosis models.

The antithrombotic effect of MCI-9042, (+-)-2-(dimethyl-amino)-1-((o-(m- methoxyphenethyl)phenoxyl]methyl]ethyl hydrogen succinate hydrochloride was investigated in three different experimental thrombosis models in animals. Simultaneous injection of serotonin and collagen into the tail vein in mice induced acute pulmonary thromboembolic death. MCI-9042 reduced the mortality in a dose dependent manner and its ED50 value was 1.9 mg/kg po. Ticlopidine (TCP) which is a positive reference compound as an antithrombotic drug reduced the mortality at doses of 10 mg/kg po and above. Cyproheptadine (CPH) and ketanserin (KTS) which are S2-serotonergic antagonists were also effective on the reduction of mortality. In mesenteric arterial thrombosis induced by electric stimulation in mice, MCI-9042 prolonged the occlusion time resulted from platelet thrombus formation (PD50: 23 mg/kg po). CPH and KTS prolonged the occlusion time as potent as MCI-9042, but TCP prolonged the occlusion time only at the high doses of 100 mg/kg po and above. In experimental arterial thrombosis which generated in implanted polyethylene tubing, MCI-9042 reduced the incidence of thrombus formation in the tubing and its ED50 value was 18 mg/kg po. TCP was also effective in this model with an ED50 of 170 mg/kg po. The present results lead to the consideration that serotonin plays a more important role in thrombus formation than that conjectured formerly, and suggest that MCI-9042 becomes a new kind of antithrombotic drug with S2-serotonergic receptor antagonism.

Effect of (+/-)-2-(dimethylamino)-1-[[o-(m-methoxyphenethyl)phenoxy] methyl]ethyl hydrogen succinate on experimental models of peripheral obstructive disease.

Peripheral obstructive disease in tail or hind limb was experimentally induced by intravenous injection of kappa-carrageenin or intra-arterial injection of sodium laurate in rats, and the suppressive effect of (+/-)-2-(dimethylamino)-1-[[o-(m-methoxyphenethyl)phenoxy] methyl]ethyl hydrogen succinate (MCI-9042, CAS 125926-17-2) on the peripheral obstructive diseases was examined. The injection of kappa-carrageenin induced a thrombotic infarction of the tail vessel in rats. The administration of MCI-9042 dose-dependently suppressed the peripheral infarction with an ED50 value of 16 mg/kg p.o. Ticlopidine, a reference antiplatelet drug, did not affect the peripheral infarction even at 50 mg/kg p.o. Cyproheptadine, however, an S2-serotonergic antagonist, potently suppressed the peripheral infarction with an ED50 of 3.5 mg/kg p.o. After the injection of sodium laurate into the femoral artery in rats peripheral lesions of the paw were generated and extended. MCI-9042 significantly prevented the progression of the disease at doses of 10 mg/kg p.o. and above. When the daily administration of MCI-9042 was started from 1 day after the laurate injection, it was also effective as well as by pretreatment. Ticlopidine was significantly effective only at 100 mg/kg p.o. as pretreatment. From the present study, it is considered that platelet-derived serotonin plays an important role in the development of peripheral obstructive disease and MCI-9042 suppressed the disease through its S2-serotonergic antagonism.

MCI-9042, a new antiplatelet agent is a selective S2-serotonergic receptor antagonist.

MCI-9042, (+/-)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3- (dimethylamino)-2-propyl hydrogen succinate hydrochloride inhibited platelet aggregation induced by collagen and secondary aggregation by ADP or epinephrine at 10(-6) M level in platelets of various species. The antiplatelet effect of MCI-9042 was potentiated in aggregation induced by a combination of serotonin with collagen. IC50 value of human platelet aggregation by the serotonin plus collagen was 1.0 x 10(-7) M. MCI-9042 inhibited serotonin release accompanied with collagen-induced platelet aggregation, while it did not affect serotonin uptake into platelet. MCI-9042 also potently inhibited the S2-serotonergic receptor-mediated contraction of rat caudal artery by serotonin in a competitive manner with a Ki value of 1.79 x 10(-8) M, while S1 receptor- or adrenergic receptor-mediated vasoconstriction was inhibited more weakly. Platelet adhesiveness, c-AMP level in platelets and the conversion of arachidonic acid to thromboxane A2 were not influenced by MCI-9042. These results suggest that MCI-9042 is a selective S2-serotonergic receptor antagonist, exhibiting the inhibition of S2-serotonergic potentiated platelet aggregation and the suppression of blood vessel constriction mediated by S2-serotonergic receptor.

Sequence analysis of nutA gene encoding membrane-bound Cl(-)-dependent 5'-nucleotidase of Vibrio parahaemolyticus.

The membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus is unique in requiring Cl- for activity. We cloned the nutA gene encoding the 5'-nucleotidase and sequenced it. It contained an open reading frame consisting of 1,680 nucleotides capable of encoding a protein of 560 amino acid residues. The first 21 amino acid residues of the N-terminal portion of this protein seem to be a signal peptide. The rest of the polypeptide (539 residues) is hydrophilic, and its molecular weight was calculated to be 60,008, which is in good agreement with the value of 63 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the 5'-nucleotidase derived from the cloned nutA gene. We tried to determine the amino acid sequence of the N-terminal portion of the purified enzyme. However, the N-terminal residue seemed to be blocked. As this 5'-nucleotidase can be solubilized from membrane vesicles with detergent, it may be a lipoprotein. The amino acid sequence around the possible cleavage site of the 5'-nucleotidase had homology with the sequences of the cleavage sites of the lipoproteins of Escherichia coli and other bacteria. The amino acid sequence had high (about 60%) homology with the sequence of periplasmic 5'-nucleotidase (uridine diphosphate sugar hydrolase, the product of the ushA gene) of E. coli. It also contained regions that showed some homology with the nucleotide binding sites of many nucleotide binding proteins.

Antithrombotic effects of KP-10614, a novel and stable prostacyclin (PGI2) analog.

A chemically stable prostacyclin analog, KP-10614 [(4z,16s)-4,5,18,18,19,19-hexadehydro-16,20-dimethyl-delta 6(9;alpha)-9(o)- methano-PGI1], has been compared with two other prostacyclin derivatives (Iloprost and TEI-7165) and one prostaglandin E1 derivative (OP-1206) with respect to ADP-induced in vitro aggregation of human platelets and ex vivo platelet aggregation in rats and dogs, given by bolus injection and i.v. infusion. These compounds were also tested on the systemic arterial blood pressure of rats and dogs. KP-10614 was the most potent inhibitor of in vitro platelet aggregation induced by ADP with IC50 of 1 nM among the compounds studied in this report, and it also showed ex vivo effectiveness at doses much lower than the other three compounds. KP-10614 was also orally active. At oral doses of 25, 50 and 100 micrograms/kg, this new compound caused a dose-dependent inhibition of ex vivo platelet aggregation in rats, whereas the other three compounds were effective only at 500 micrograms/kg or more. In addition, KP-10614 showed definite antithrombotic effects at a dose range of 0.1 to 1 microgram/kg i.v. in various thrombosis models in which platelet aggregation was mainly involved. These results indicate that KP-10614 possesses therapeutic potential in thrombotic diseases.

Syntheses and platelet aggregation inhibitory and antithrombotic properties of [2-[(omega-aminoalkoxy)phenyl]ethyl]benzenes.

A series of [2-[(omega-aminoalkoxy)phenyl]ethyl]benzene derivatives were synthesized and evaluated for their ability to inhibit collagen-induced platelet aggregation in vitro and to protect experimental thrombosis in mice. The results showed that the compounds were in vitro inhibitors of collagen-induced platelet aggregation. Most of them were also effective in the mouse antithrombotic assay. The compounds were found to be potent antagonists to S2 serotonergic receptor, and good correlation (r = 0.85) between their S2 serotonergic receptor antagonism and their potency as platelet antiaggregatory drugs was observed. Among the compounds studied, mono[2-(dimethylamino)-1-[[2-[2-(3- methoxyphenyl)ethyl]phenoxy]methyl]ethyl] succinate hydrochloride (12b, MCI-9042) was selected for further pharmacological and toxicological evaluation.

Cloning and expression of the 5'-nucleotidase gene of Vibrio parahaemolyticus in Escherichia coli and overproduction of the enzyme.

The gene encoding the membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus was cloned and expressed in Escherichia coli. Cells of E. coli harboring a plasmid, pNUT5, which carries the 5'-nucleotidase gene were able to grow on ATP as the sole source of carbon, although the original cells were not. The 5'-nucleotidase activity was detected in whole cells of E. coli harboring pNUT5 and in membrane vesicles prepared from these cells. Most properties of the 5'-nucleotidase produced in E. coli, that is, its requirements for Cl- and Mg2+, substrate specificity, and inhibition by Zn2+, were similar to those observed in V. parahaemolyticus, but some alterations in properties were observed: The 5'-nucleotidase was partially inducible in V. parahaemolyticus, but its expression in E. coli was completely constitutive. The specific activity of the 5'-nucleotidase in membrane vesicles of E. coli harboring the plasmid was 30 times that observed in whole cells, whereas the specific activities in membrane vesicles and in whole cells of V. parahaemolyticus were almost the same. A new, dense band of protein with an apparent molecular mass of 63 kDa was detected when membrane proteins of E. coli harboring the plasmid were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

N alpha-dansyl-L-arginine 4-phenylpiperidine amide. A potent and selective inhibitor of horse serum cholinesterase.

The relationship between chemical modifications of arginine derivatives and inhibitory activity to horse serum cholinesterase (BuChE) was investigated. It provided a new insight into the topography of the active site of BuChE. 1) BuChE has the hydrophobic binding pocket, the depth of which corresponds to the length of ethylpiperidine. 2) In the opposite side to the hydrophobic binding pocket, BuChE has a certain entity which repulses carboxyl group at the 2-position of piperidine of L-arginine piperidine amide. 3) The P site of BuChE can allow 4-propyl and 4-phenyl group attached to piperidine. Comparison of the results with those of thrombin and trypsin clearly revealed similarities and dissimilarities among BuChE, trypsin, and thrombin in the active site topography, and hence, we introduce a new selective inhibitor for BuChE, N alpha-dansyl-L-arginine 4-phenylpiperidine amide. It inhibits BuChE strongly (Ki = 0.016 microM), whereas it inhibits trypsin, thrombin, plasmin, and glandular kallikrein only weakly and shows actually no inhibition on acetylcholinesterase from the human erythrocyte. In addition, the new inhibitor becomes highly fluorescent when bound with BuChE, indicating that the compound is an ideal probe of the interactions of BuChE as well as a titrant of it.

Effect of a synthetic thrombin inhibitor MCI-9038 on experimental models of disseminated intravascular coagulation in rabbits.

We examined the effect of a synthetic thrombin inhibitor, MCI-9038, on two experimental animal models of disseminated intravascular coagulation (DIC). In a model that DIC induced by the intravenous infusion of thrombin, MCI-9038 suppressed the decrease of platelet count by about 50% at a dose of 0.2 micrograms/kg/min and almost completely at 2 micrograms/kg/min. When MCI-9038 was administered orally, the suppressive effect was also observed. Heparin suppressed the platelet count decrease by about 50% at 1 unit/kg/min. In another model of DIC induced by lactic acid and tissue thromboplastin infusion, MCI-9038 prevented the decrease of platelet count and the consumption of coagulation factors. The suppression effect by about 50% on these changes was observed at a dose of 3.16 micrograms/kg/min. Thromboelastogram pattern indicating the consumption coagulopathy in control experiments was normalized by the MCI-9038 administration. Heparin suppressed the decrease of fibrinogen content as effectively as MCI-9038, but it was less effective on the platelet count decrease. From these results, it was concluded that MCI-9038 might be useful for the treatment of DIC.

Similarity and dissimilarity in the stereogeometry of the active sites of thrombin, trypsin, plasmin and glandular kallikrein.

The relationship between chemical modifications of arginine derivatives and inhibitory activity to trypsin, plasmin and glandular kallikrein was investigated comparing with that of thrombin and concluded as follows: The hydrophobic binding pocket, which has been reported previously to be stereogeometrically very similar in trypsin and thrombin, corresponded to the length of ethylpiperidine. Concerning the site (termed the P site) next to the hydrophobic binding pocket, there were large differences in stereogeometry between trypsin and thrombin; the binding site of trypsin extended further to allow propyl and phenyl group attached to piperidine, while that of thrombin would be much narrower and unable to allow them. The P sites of plasmin and glandular kallikrein resembled that of trypsin in being able to allow phenyl group. To substantialize the hydrophobic binding pocket and the P site, a (2R, 4R)-MQPA-trypsin complex model was generated using the results of X-ray crystallography of (2R, 4R)-MQPA and BPTI-trypsin complex by calculation to minimize van der Waals contacts, and it was of great use for understanding the geometry of the active sites of trypsin, thrombin, plasmin and glandular kallikrein.

Effect of a selective thrombin inhibitor MCI-9038 on fibrinolysis in vitro and in vivo.

The effect of a selective thrombin inhibitor, (2R, 4R)-4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)sulfonyl]- L-arginyl]-2-piperidinecarboxylic acid (MCI-9038), on the fibrinolysis induced by t-PA and u-PA was studied in vitro and in vivo. MCI-9038 remarkably reduced the lysis time of the plasma clot generated by the addition of calcium chloride to the plasma at the concentration ranging from 0.01 to 0.3 microM. Heparin also reduced the plasma clot lysis time with a lower effect than MCI-9038. The fibrin crosslinkage in the plasma clot was inhibited by MCI-9038 or heparin. MCI-9038 potently inhibited the factor XIIIa generation from factor XIII by thrombin. The effect on the in vivo thrombolysis was studied on the arterial thrombosis generated by the endothelial cell injury of the rabbit carotid artery by acetic acid. t-PA dissolved the thrombi with the infusion at 0.96 mg/kg over 2 h without a significant activation of a systemic fibrinolysis. u-PA dissolved the thrombi with the infusion at 180,000 and 360,000 IU/kg over 2 h. At a dose of 0.48 mg/kg t-PA or 90,000 IU/kg u-PA, the thrombi were not dissolved, but the combined use of MCI-9038 at 1.2 mg/kg over 2 h effectively dissolved the thrombi. Thus, combination of MCI-9038 with plasminogen activators accelerated thrombolysis of an experimental thrombosis in rabbits.

[Effects of 4-(o-benzylphenoxy)-N-methylbutylamine hydrochloride (MCI-2016, bifemelane hydrochloride) on coagulation, fibrinolysis, hemolysis, hemorheological properties and platelet aggregation].

MCI-2016 showed little influence on coagulation (APTT) and fibrinolysis (plasma clot lysis activated by urokinase) at doses (concentrations) as high as 300 mg/kg, p.o. or 8.6 X 10(-4) M. Hemolytic action of MCI-2016 was only observed at the concentrations above 2 mM. The drug also showed no influence on blood glucose level (30-300 mg/kg, p.o.). Effects of MCI-2016 on hemorheological properties were studied either in vitro or ex vivo. Above the doses (concentrations) of 100 mg/kg, p.o. and 10 microM, MCI-2016 suppressed the mechanical hemolysis and accelerated the membrane filtration rate. These effects of MCI-2016 were superior to those of cinepazide, Ca-hopantenate, meclofenoxate and pentoxyfylline. MCI-2016 also inhibited platelet aggregation induced by collagen with the IC 50 of 35 to 60 microM (rabbit and human platelets). Secondary aggregations of ADP and epinephrine were also inhibited by MCI-2016. As for reference drugs, bencyclane showed inhibitory patterns similar to MCI-2016. Other drugs examined exhibited little effect. In summary, it may be suggested that MCI-2016 exhibits beneficial influences in the clinical fields of cerebrovascular diseases.