ONO-1603, a potential antidementia drug, delays age-induced apoptosis and suppresses overexpression of glyceraldehyde-3-phosphate dehydrogenase in cultured central nervous system neurons.

Primary cultures of rat cerebral cortical cells and cerebellar granule cells die by an apoptotic mechanism after more than 2 weeks in cultures in the absence of medium change and glucose supplement, a process termed age-induced apoptosis of cultured neurons. Our preliminary study has shown that age-induced apoptosis of cerebellar granule cells is protected by pretreatment with tetrahydroaminoacridine (THA), an antidementia drug. In this study, we systematically compared the neuroprotective effects of THA with those of (S)-1-[N-(4-chlorobenzyl)succinamoyl]pyrrolidine-2-carbaldehyde (ONO-1603), a novel prolyl endopeptidase inhibitor and potential antidementia drug. Both ONO-1603 and THA effectively delay age-induced apoptosis of cerebral and cerebellar neurons, as demonstrated morphologically with toluidine blue and fluorescein diacetate/propidium iodide staining or biochemically by DNA laddering analysis on agarose gels. ONO-1603 is about 300 times more potent than THA, with a maximal protective effect at 0.03 and 10 microM, respectively. ONO-1603 shows a wide protective range of 0.03 to 1 microM in contrast to a narrow effective range of 3 to 10 microM for THA. Moreover, ONO-1603 is nontoxic to neurons, even at the high concentration of 100 microM, whereas THA elicits severe neurotoxicity at a dose of >/=30 microM. Both ONO-1603 and THA robustly suppress overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) mRNA and accumulation of GAPDH protein in a particulate fraction of cultured neurons undergoing age-induced apoptosis. Because we documented that GAPDH overexpression participates in neuronal apoptosis induced by various insults, we conclude that the neuroprotective actions of ONO-1603 and THA appear to be mediated by suppression of this protein overexpression.

Overexpression of glyceraldehyde-3-phosphate dehydrogenase is involved in low K+-induced apoptosis but not necrosis of cultured cerebellar granule cells.

We have reported that overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is involved in age-induced apoptosis of the cultured cerebellar granule cells that grow in a depolarizing concentration (25 mM) of KCI. The present study was undertaken to investigate whether GAPDH overexpression also occurs and participates in apoptosis of the cerebellar granule cells that result from switching the culturing conditions from high (25 mM) to low (5 mM) concentrations of KCl. We found that exposure of granule cells to low potassium (K+) for 24 hr induces not only apoptosis but also necrotic damage. The latter is supported by the morphological observations that a subpopulation of neurons showed cell swelling, extensive cytoplasmic vacuolization, damaged mitochondria, and apparently intact nuclei. Treatments with two antisense but not sense oligodeoxyribonucleotides directed against GAPDH attenuated low K+-induced neuronal death by approximately 50%. Morphological inspection revealed that GAPDH antisense oligonucleotides preferentially blocked low K+-induced apoptosis with little or no effect on necrotic damage. Similar to antisense oligonucleotides, actinomycin-D partially inhibited low K+-induced death of granule cells with a predominant effect on apoptosis. In contrast, cycloheximide almost completely blocked low K+-induced neuronal death and seemed to prevent both apoptotic and necrotic damage. The levels of GAPDH mRNA and protein were markedly increased in a time-dependent manner after low K+ exposure. The overexpression of GAPDH mRNA and protein was completely blocked by cycloheximide, actinomycin-D, and its antisense but not sense oligonucleotides. Taken together, these results lend credence to the view that exposure of cerebellar granule cells to low K+ induces both apoptosis and necrosis and that only the apoptotic component involves overexpression of GAPDH.

Simultaneous determination of human neutrophil elastase inhibitor (ONO-5046) and its metabolite in plasma and urine by direct injection column-switching HPLC.

The direct injection method for the simultaneous determination of a human neutrophil elastase inhibitor (ONO-5046) and its metabolite (ONO-EI-601) in plasma and urine has been developed using a column-switching HPLC system. The system was set up with a pre-treatment column, a pre-concentration column and an analytical column which were connected in series via two automatic switching valves. The calibration lines showed a good linearity for concentrations of ONO-5046 and ONO-EI-601 in a range of 156-20000 ng ml-1 in plasma and 1.56-100 micrograms ml-1 in urine, all correlation coefficients being greater than 0.9999. The limit of quantitation of ONO-5046 was 156 ng ml-1 in plasma, that of ONO-EI-601 was 313 ng ml-1 in plasma, and those of ONO-5046 and ONO-EI-601 were 1.56 micrograms ml-1 in urine. The developed method is rapid and sensitive with automated operation, allowing untreated samples to be analysed every 45 min. The application of the present method to the real plasma and urine samples proved to be useful for pharmacokinetic, toxicological and clinical studies.

Anti-platelet and anti-thrombotic effects of OP-41483.alpha-CD, a prostacyclin analogue, in experimental animals.

The effects of OP-41483.alpha-CD, 5(E)-6,9-deoxa-6,9 alpha-methylene-15-cyclopentyl-16,17,18,19,20-pentanor prostacyclin (PGI2).alpha-cyclodextrin clathrate, on platelet function and experimental thrombosis were studied. In human platelets, OP-41483 inhibited aggregation induced by adenosine diphosphate (ADP) or collagen, promoted disaggregation, and elevated cyclic adenosine monophosphate (cAMP) levels in vitro at the same order of concentrations. The equipotent antiaggregatory activity of OP-41483 to human platelets was observed in monkey platelets in vitro. Furthermore, intravenous administration of OP-41483 to monkeys, unlike PGI2, showed the antiaggregatory effect on platelets but with less effect on blood pressure, suggesting that a differential sensitivity to OP-41483 between platelet function and vascular tone exists in monkeys. In rabbits, OP-41483.alpha-CD attenuated platelet aggregation induced by ADP, collagen and platelet activating factor (PAF), decreased circulating platelet aggregates, and inhibited platelet adhesiveness to de-endothelialized blood vessels. These results suggest that the anti-thrombotic effects of OP-41483 are associated with its potent antiplatelet activities mainly because of the elevation of cAMP levels in the platelets. The potent anti-thrombotic and less hypotensive effects of this compound may be useful for various thrombotic disorders.

Effect of a peptide leukotriene receptor antagonist, ONO-1078, on guinea-pig models of asthma.

Peptide leukotrienes have been suggested to play an important role in bronchial asthma. As antigen-induced bronchoconstrictions, airway hyperreactivity, and pulmonary eosinophil accumulation are characteristics of the pathology of asthma, we investigated the effect of a peptide leukotriene receptor antagonist, ONO-1078, on these responses using guinea-pig models of asthma. Oral administration of ONO-1078 (3 mg/kg) significantly inhibited slow-reacting substance of anaphylaxis-mediated bronchoconstriction induced by i.v. administered ovalbumin. ONO-1078 (30-100 mg/kg), when administered orally both 1 h before and 4 h after ovalbumin challenge, significantly reduced immediate- and late-phase asthmatic responses, with peak responses occurring immediately and 5-11 h after challenge with inhaled ovalbumin. Oral administration of ONO-1078 significantly reduced the airway hyperreactivity (10-30 mg/kg) and the pulmonary eosinophil accumulation (30-100 mg/kg) observed 4 and 24 h after ovalbumin challenge, respectively. These results suggest that ONO-1078 may be of therapeutic use for bronchial asthma.

Inhibitory effect of a peptide leukotriene antagonist ONO-1078 on LTD4- and antigen-induced thromboxane B2 production in guinea pig lungs.

The effect of a peptide leukotriene receptor antagonist ONO-1078 on the production of thromboxane (Tx) B2 induced by leukotriene (LT) D4 and antigen challenge was examined in guinea pig lungs. LTD4 (1-1,000 nM) induced a concentration-dependent production of TxB2 in non-sensitized guinea pig lungs and ovalbumin challenge (0.01-100 micrograms/ml) produced TxB2 and peptide leukotrienes in a concentration-dependent manner in ovalbumin-sensitized guinea pig lungs. ONO-1078 inhibited LTD4 (100 nM)-induced TxB2 production with the IC50 value of 0.24 microM. Furthermore, ONO-1078 inhibited antigen (10 micrograms/ml)-induced TxB2 production with the IC50 value of 0.14 microM without effect on the production of peptide leukotrienes. These results suggest that ONO-1078 may prevent the antigen-induced production of TxB2 through the blockade of the activation of receptors by endogenously generated peptide leukotrienes.

Effect of OP-41483.alpha-CD, a prostacyclin analog, on a clamp-induced endothelial injury in rats.

We studied the effect of OP-41483.alpha-CD, a stable prostacyclin analog, on clamp-induced endothelial injury in rats. The injury was assessed by vascular Evans blue leakage and using a scanning electron microscope. OP-41483.alpha-CD significantly reduced the Evans blue leakage at doses of 30 and 100 ng/kg/min. PGE1.CD was also found to show an equipotent inhibitory action on the dye leakage. From scanning electron microscopic observations, a moderate degree of intimal defects, microvillous projections and platelet adhesions at the luminal surface were seen in the specimens from OP-41483.alpha-CD (30 and 100 ng/kg/min) treated rats. Furthermore, OP-41483.alpha-CD, PGE1.CD and Dibutyryl cyclic AMP (DbcAMP) were found to accelerate a proliferation of cultured bovine endothelial cells in a dose-dependent manner in vitro. Taken together, these data indicate that the endothelial regenerative effect of OP-41483.alpha-CD could contribute to healing of clamp-induced endothelial injury and it may be an important therapeutic drug to protect vascular intimal injury.

Effects of OP-41483.alpha-CD, a stable prostacyclin analog, on cultured endothelial cell dysfunction caused by 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) in vitro.

We studied the effect of OP-41483.alpha-CD, a stable prostacyclin analog, on, 15-HPETE induced bovine endothelial cell dysfunction, which was assessed by measuring a number of endothelial cells attached to plastic plates. 15-HPETE decreased the number of attached endothelial cells in a concentration- and time-dependent manner. OP-41483.alpha-CD and PGI2 significantly inhibited 15-HPETE induced dysfunction of the cells at a concentration of more than 10(-9)M. Besides, DDA (10(-6) - 10(-4)M) an adenylate cyclase inhibitor, diminished the inhibitory effect of OP-41483.alpha-CD on 15-HPETE induced cell dysfunction in a concentration-dependent manner. Furthermore, OP-41483.alpha-CD increased cAMP levels in the endothelial cells in the range of 10(-10) to 10(-8)M in a dose-dependent manner. These data suggest that OP-41483.alpha-CD could exert an inhibitory action on 15-HPETE induced endothelial cell dysfunction via partly increasing its effect on the intracellular cAMP level.

In vivo pharmacologic profile of ONO-1078: a potent, selective and orally active peptide leukotriene (LT) antagonist.

We investigated the in vivo antagonistic activity of ONO-1078 against peptide leukotrienes (LTs) in guinea pigs. ONO-1078, when administered p.o. (0.3-3 mg/kg), caused a dose-dependent reduction of LTC4-, LTD4- and LTE4-induced bronchoconstriction, LTD4-induced airway microvascular leakage and LTD4-induced increase in cutaneous vascular permeability. When administered intravenously, ONO-1078 (3-30 micrograms/kg) inhibited these responses approximately 200-600 fold more potently than FPL55712. When guinea pigs were treated with indomethacin to examine the antagonism of ONO-1078 on the direct action against peptide LTs, intravenous (3-30 micrograms/kg) and oral (0.3-3 mg/kg) administration of ONO-1078 also inhibited LTC4- and LTD4-induced bronchoconstriction, and its activity was approximately 300-500 fold more potent than that of FPL55712. ONO-1078 (10 mg/kg, i.v.) had no inhibitory effect on bronchoconstrictions induced by histamine, acetylcholine, serotonin, arachidonic acid, LTB4, prostaglandin (PG) F2 alpha, PGD2, 9 alpha, 11 beta-PGF2, a stable thromboxane A2 mimetic agent and platelet activating factor. Furthermore, oral administration of ONO-1078 (1-10 mg/kg) inhibited slow-reacting substance of anaphylaxis mediated bronchoconstriction induced by antigen in a dose-dependent manner. These results indicate that ONO-1078 is an extremely potent, selective and orally active peptide LT antagonist and that oral administration of ONO-1078 antagonizes not only exogenously administered peptide LTs but also endogenous peptide LTs.

In vitro antagonism of ONO-1078, a newly developed anti-asthma agent, against peptide leukotrienes in isolated guinea pig tissues.

We evaluated the antagonist activity of ONO-1078 against peptide leukotrienes (LTs) by a radioligand binding assay and functional experiments in guinea pigs. In the radioligand binding assay, ONO-1078 inhibited [3H]LTD4 and [3H]LTE4 bindings to lung membranes (Ki = 0.99 and 0.63 nM, respectively) and was 2,000- to 3,000-fold more potent than FPL55712. Antagonism of ONO-1078 against [3H]LTC4 binding (Ki = 5640 nM) was approximately twofold more potent than that of FPL55712. The antagonism of ONO-1078 against [3H]LTD4 binding was competitive. In functional experiments, ONO-1078 showed competitive antagonism against the LTC4- and LTD4-induced contractions of guinea pig trachea and lung parenchymal strips with a pA2 range of 7.70 to 10.71 and was approximately 400- to 3,300-fold more potent than FPL55712. Interestingly, in the presence of an inhibitor of the bioconversion of LTC4 to LTD4, ONO-1078 also antagonized the LTC4-induced contraction of guinea pig trachea (pA2 = 7.78). ONO-1078 significantly reversed the LTD4-induced prolonged contraction without effect on the KCl- and BaCl2-induced contractions of guinea pig trachea. Furthermore, ONO-1078 antagonized the antigen-induced SRS-A mediated contraction of guinea pig trachea. On the other hand, ONO-1078 showed no antagonism against histamine, acetylcholine, 5-hydroxytryptamine, prostaglandin D2 and U-46619. In addition, ONO-1078 showed little or no effect on the activities of cyclooxygenase, 5-lipoxygenase and thromboxane synthetase. These in vitro studies indicate that ONO-1078 is a highly potent, selective and competitive antagonist of peptide leukotrienes that acts with higher affinity at LTD4 and LTE4 receptors than LTC4 receptors.

Inhibitory effect of OP-41483.alpha-CD, a prostacyclin analog, on peripheral vascular lesion models in rats.

The effect of a chemically stable prostacyclin analog, OP-41483 alpha-cyclodextrin clathrate (OP-41483.alpha-CD), on vascular lesions, platelet aggregation and blood pressure were examined and compared with those of prostaglandin E1 alpha-cyclodextrin clathrate (PGE1.CD) in in vivo rat models. 1) In the laurate (1 mg/leg, i.a.)-induced arterial thrombotic model, OP-41483.alpha-CD (1 microgram/kg/min, i.v.) prevented the progression of femoral arterial vascular lesions and enhanced the development of collaterals in the femoral artery. PGE1.CD did not inhibit the progression of vascular damages. 2) In the model of vasoconstriction induced by epinephrine (0.05 mg/tail, s.c.) and ergotamine (2 mg/kg, s.c.), OP-41483.alpha-CD and PGE1.CD, at 1 microgram/kg/min, inhibited the progress of of tail gangrene and lessened the decrease in tail cutaneous blood flow. 3) OP-41483.alpha-CD (1 microgram/kg/min) suppressed the ADP (0.1 mg/kg/min, i.v.)-induced decrease in the number of circulating platelets without affecting the change in blood pressure. In contrast, PGE1.CD (3 micrograms/kg/min) inhibited ADP-induced thrombocytopenia with a decrease in blood pressure. These results indicate that OP-41483.alpha-CD has antiplatelet and cutaneous blood flow improving activities that are greater than its hypotensive effect and may be of therapeutic potential in peripheral vascular diseases.

Effect of a peptide leukotriene antagonist, ONO-1078 on antigen-induced airway microvascular leakage in actively sensitized guinea pigs.

We examined the effect of ONO-1078, a peptide leukotriene antagonist, on antigen-induced airway microvascular leakage in ovalbumin-sensitized guinea pigs. When guinea pigs were pretreated with mepyramine, ovalbumin challenge increased vascular permeability to Evans blue dye in trachea, main bronchi and intrapulmonary airways. Oral administration of ONO-1078 significantly reduced microvascular leakage in intrapulmonary airways at doses more than 3 mg/kg, but not in trachea. Moreover, oral administration of ONO-1078 significantly reduced SRS-A mediated microvascular leakage into all airway tissues and was more effective in intrapulmonary airways at 3 mg/kg. Simultaneously, ONO-1078 also inhibited SRS-A mediated bronchoconstriction. On the other hand, azelastine (10 mg/kg, p.o.), an anti-asthma agent, failed to inhibit microvascular leakage into the airways. These results suggest that peptide leukotrienes may be important mediators of airway microvascular leakage, and that the inhibitory effect of ONO-1078 on antigen-induced airway microvascular leakage in addition to the blockade of bronchoconstriction may have therapeutic implications for bronchial asthma.

Arginine-vasopressin fragment 4-9 stimulates the acetylcholine release in hippocampus of freely-moving rats.

We examined the effects of arginine-vasopressin (AVP) C-terminal fragment 4-9, which facilitates learning and memory, on the extracellular acetylcholine (ACh) release in hippocampus of freely-moving rats using the microdialysis technique. Following administration of AVP4-9, p-Glu-Asn-Cys[Cys]-Pro-Arg-Gly-NH2, through the dialysis probe into the hippocampus, ACh levels in dialysates from the hippocampus increased markedly in dose and time dependent manner at 2-2.5 and 2.5-3 hr. AVP1-9, the parent peptide, has a similar enhancing effect on ACh release as AVP4-9. Stimulated ACh release by AVP4-9 was significantly inhibited by V1-selective receptor antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)-tyrosine]AVP), but not by V2-selective antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-D-Ile, 4-Ile]AVP). From these observations, it is demonstrated that AVP4-9 stimulates the ACh release in rat hippocampus via mediating V1-like vasopressin receptors.

ONO-3144, a new anti-inflammatory drug and its possible mechanism of action.

Anti-inflammatory activities of ONO-3144 have been studied in various experimental models. This compound showed an inhibitory effect on increased vascular permeability and acute inflammation. In the carrageenin test the activity of ONO-3144 was comparable to that of indomethacin (IM), while in the dextran, albumin, yeast and scald edema test it was more potent than that of IM. Unlike IM, phenylbutazone (PB) and tiaramide (TI), ONO-3144 showed marked inhibitory effects on H2O2-induced hemolysis and lipid peroxidation. In the prostaglandin (PG) biosynthesis series, ONO-3144 did not inhibit cyclooxygenase activity but stimulated PG hydroperoxidase activity, facilitated conversion to PGH2 and also inhibited thromboxane (Tx) synthetase. The findings suggest that ONO-3144 is potentially a new type of anti-inflammatory drug. Possible sites of action of ONO-3144 are discussed.

[Effect of [ethyl p-(6-guanidinohexanoyloxy)benzoate] methanesulfonate (FOY) on kinin formation and fibrinolysis activity].

Inibitory effects of [Ethyl p-(6-guanidinohexanoyloxy)benzoate] methanesulfonate (FOY) on kinin formation (in vitro and in vivo) and the fibrinolytic activity (in vivo) were examined and compared with otherinhibitors. Inhibitory effect on kinin forming activity (in vitro) of various enzymes was measured in the guinea pig ileum. FOY and Trasylol inhibited the kinin forming activities of trypsin, pancreas kallikrein and plasma kallikrein. Soybean trypsin inhibitor inhibited kinin like substance was formed in the perfusate when the rat's paw was heated at 46 degrees C. FOY and T-asylol added to the perfusion fluid produced a potent inhibition of the formation of bradykinin-like substance. When administered i.v., FOY and Trasylol did not inhibit the formation of bradykinin-like substance. In the dog, activation of plasmin in the circulatory blood and increase of hemorrhagic tendency were caused by the i.v. administration of human serum plus streptokinase. Such responses were inhibited with a previous i.v. infusion of FOY and t-AMCHA. From the above findings, it may be concluded that FOY has inhibitory effects on kinin formation and fibrinolytic activity.

[Pharmacological action of [ethyl p(6-guanidinohexanoyloxy)benzoate] methanesulfonate (FOY)].

General pharmacological effects of [Ethyl p-(6-guanidinohexanoyloxy)benzoate] methanesulfonate (FOY), a new antiproteolytic agent, were studied and the following results were obtained. Acute administration of large doses of FOY in conscious dogs and rabbits caused a decrease in spontaneous motility, ataxia, cyanosis, collapse, mydriasis, and respiratory paralysis. The agent had no effect on the central nervous system and exhibited hypotensive effects in dogs in doses of more than 1 mg/kg. Hypotensive responses were not inhibited by treatment with atropine or hexamethonium. FOY had no effects on ECG in the rabbit at doses of less than 30 mg/kg and at doses from 10(-6) to 10(-4)g/ml, distinctly reduced the amplitude of the spontaneous and rhythmic contractions of the isolated rabbit ileum, guinea-pig ileum and rat uterus preparation. The contractile response to nerve stimulation, noradrenaline and barium was suppressed in isolated guinea-pig vas deferens. FOY had no effects on the twitch response of gastrocnemius muscle to sciatic nerve stimulation in rats. The drug caused local irritant effects in rabbits and rats.