[General pharmacological studies on 5,8,11,14,17-eicosapentaenoic acid ethyl ester (EPA-E)].

EPA-E, even at 3,000 mg/kg, p.o., did not affect the general behaviors, spontaneous locomotor activities, pentobarbital hypnosis and body temperature; and it did not elicit anticonvulsant, analgesic and muscle relaxant actions. It had no influence on spontaneous EEG activities, even at 3,000 mg/kg, i.d. EPA-E at concentrations up to 10(-4) M, did not affect the tonus or agonist-induced contraction of the isolated ileum, trachea, fundus and vas deferens. EPA-E had no influence on the spontaneous movement of isolated ileum or uterus. EPA-E did not affect the nictitating membrane contraction and intestinal propulsive motility, and it did not damage gastric mucosa nor elicit antiulcer action. EPA-E at 1,000 mg/kg were without effect on gastric secretory volume (SV), total acidity (TA) and pepsin activities (PA). However, EPA-E at 3,000 mg/kg significantly decreased SV and TA without significantly decreasing PA. EPA-E caused no changes in the respiration, blood pressure, heart rate and ECG at the doses up to 3,000 mg/kg; and it did not affect the heart rate and contractile force on the isolated atria at concentrations up to 10(-4) M. The intracutaneous injection of 2.0% EPA-E produced neither anesthetic nor irritative action. EPA-E did not elicit hemolytic action at 10(-4) M. EPA-E, even at 3,000 mg/kg, did not affect the neuro-muscular transmission, urine volume, urinary excretion of electrolytes and carrageenin edema. These results suggested that EPA-E has no noticeable effects on the central nervous, autonomic nervous, respiratory and cardiovascular systems and so on.

Mechanisms involved in the effect of M6434 on experimental hemorrhagic shock: I. Effects on myocardial contractility and venous return.

To elucidate the mechanisms of protective effect of M6434 on experimental shock, the authors examined the effects of this compound on the survival time and hemodynamic changes in severely hemorrhagic-shocked dogs. We also examined the effects of M6434 on contractile tension of isolated canine ventricular strips and on venous return in dogs with cardiopulmonary bypass in normal and shock state. Intravenous infusion of M6434 at 10 micrograms/kg/min prolonged survival and maintained mean arterial pressure, cardiac output, and first derivative of left ventricular pressure at higher levels than those in the control group, whereas dopamine (10 micrograms/kg/min) did not significantly affect survival time and hemodynamic parameters. M6434 did not change contractile tension in electrically stimulated canine ventricular strips. M6434 (20 micrograms/kg/min) increased the venous return of dogs with cardiopulmonary bypass in both shock and normal state. Phenylephrine (20 micrograms/kg/min) slightly increased venous return in normal state, but not during shock. Dopamine had no effect at 20 micrograms/kg/min, but it increased venous return in both states at 50 micrograms/kg/min. These results suggest that M6434 may improve the hemodynamic derangement in severe hemorrhagic shock through decreasing venous blood pooling.

Mechanisms involved in the effect of M6434 on experimental hemorrhagic shock: II. Effects on energy metabolism and organ blood flow.

Effects of M6434 on survival time and hepatic energy metabolism of hemorrhagic-shocked rats were examined. Effects of the compound on rat mitochondrial respiration and regional blood flow in hemorrhagic-shocked rats were also studied to clarify the mechanisms of the antishock effects. Intravenous infusion of M6434 (3 or 10 micrograms/kg/min) prolonged the survival time of hemorrhagic-shocked rats. M6434 at 10 micrograms/kg/min significantly suppressed the decline of adenosine triphosphate contents and energy charge of the liver, shifted the blood flow distribution from skin and skeletal muscles to vital organs such as the liver and the heart, and also increased cardiac output in hemorrhagic-shocked rats. The mitochondrial respiration was unaffected by M6434 in vitro (10(-6)-10(-5) M). These data suggest that mechanisms of the beneficial effect of M6434 in hemorrhagic-shocked rats may not be based on the direct activation of energy metabolism, but rather on the redistribution of organ blood flow as well as an increase in cardiac output.

Bactericidal activity of M14659 enhanced in low-iron environments.

The bactericidal activity of M14659 against Escherichia coli in low-iron environments was investigated and compared with that of ceftriaxone and ceftazidime. The bactericidal activity of M14659 against E. coli in Mueller-Hinton broth was enhanced 30- to 20,000-fold by addition of transferrin, which is an iron-binding protein, whereas the activity of ceftriaxone or ceftazidime was much less strongly affected. This enhancement by transferrin was completely inhibited by saturating the iron-binding capacity of transferrin with FeCl3. M14659 was taken up markedly into bacterial cells in the presence of transferrin, and its uptake was inhibited by the protonophore dinitrophenol, which inhibits active-transport systems coupled to an energized membrane such as the iron transport systems of E. coli. The bactericidal activity of M14659, which chelates Fe3+, was also enhanced in the presence of other iron-binding compounds such as lactoferrin and alpha,alpha'-dipyridyl or in iron-deficient Mueller-Hinton broth (Fe3+ concentration, less than 2 nM) supplemented with FeCl3 at 0.1 to 1.0 microM, but not in unsupplemented iron-deficient Mueller-Hinton broth. The E. coli used in this study was confirmed to derepress iron transport systems in the presence of transferrin, lactoferrin, and alpha,alpha'-dipyridyl and in the iron-deficient Mueller-Hinton broth supplemented with FeCl3 at 0 to 1.0 microM. M14659 also showed an excellent antibacterial activity in vitro against other gram-negative bacteria in the low-iron environments. These findings indicate that M14659 may be actively taken up with Fe3+ into bacterial cells, probably through the iron transport systems under conditions of low iron and, thus, kills bacteria effectively.

Metabolic disposition of ethyl eicosapentaenoate and its metabolites in rats and dogs.

When orally administered to rats, 14C-labelled ethyl eicosapentaenoate (14C-EPA-E) was hydrolyzed and, in the lymph, incorporated mainly into triglycerides (TG) in chylomicrons. In plasma and other tissues, eicosapentaenoic acid (EPA) and its metabolites, such as docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), were detected in TG and phospholipid fractions. In plasma, EPA and its metabolites were found to be integrated into lipoproteins. Tissue distribution of these metabolites showed characteristic patterns from one tissue to another, as did their compositional distribution in lipids. EPA, DPA and DHA were found to be metabolized via beta-oxidation in in vitro experiments with mitochondrial fraction.

Antibacterial and pharmacokinetic properties of M14659, a new injectable semisynthetic cephalosporin.

In vitro and in vivo antibacterial activities and pharmacokinetics of M14659 were investigated. In vitro activity of M14659 was superior to that of ceftazidime against Staphylococcus aureus. Against Gram-negative bacteria except Pseudomonas aeruginosa, its activity was almost equal to that of ceftazidime. M14659 was more active against P. aeruginosa including multi-drug resistant strains than cefsulodin, cefoperazone or ceftazidime. Affinities of M14659 for penicillin-binding proteins (PBPs) of Escherichia coli and P. aeruginosa were 2 to 14 times higher for PBP-1A and PBP-1B than found for ceftazidime, and almost the same for PBP-3. In vivo activity of M14659 against S. aureus was superior to that of cefamandole, cefotaxime or ceftazidime. Against Gram-negative bacteria including P. aeruginosa, M14659 was 2 to 220 times more active than cefotaxime or ceftazidime. Plasma half-life of M14659 in mice was about 3 times longer than that of ceftazidime. M14659 administered intravenously to mice was mainly excreted in urine without metabolism, and its recovery rate was almost equal to that of ceftazidime.

[Effects of ulinastatin on experimental arthritis].

Effects of the urinary enzyme inhibitor ulinastatin on experimental arthritis were investigated. Ulinastatin at the dose of 30,000 units/kg/day, i.v., significantly restored the swelling of hind paw, inflammatory score and bone damage in adjuvant arthritic rats. Intraarticular administration of ulinastatin at 3000 units/site x 3, significantly suppressed the articular swelling and the elevated inflammatory parameters in the synovial fluid of carrageenin-induced arthritic rabbits. Moreover, ulinastatin at the dose of 50000 units/kg/day, i.v., significantly prevented the elevation of serum rheumatoid factor and articular lesions in MRL/l mice. In addition, ulinastatin significantly inhibited human granulocyte elastase and cathepsin G. These findings indicate that ulinastatin may be a useful therapeutic agent for arthritis.

[Effects of human urinary trypsin inhibitor against operative stress].

Effects of human urinary trypsin inhibitor (MTI) against operative stress were investigated. Laparotomy in a mouse caused suppression of immunological functions such as phagocytic activity and antibody formation, followed by loss of resistivity to bacterial infection and acceleration of growth of concealed tumor. The operation also caused damages to the body such as enhancement of protein catabolism and suppression of renal function, followed by increase of blood urea nitrogen, increase of protease activity in skeletal muscle and suppression of PSP clearance. Since MTI wholly ameliorated these undesirable conditions of the body caused by operative stress, it was suggested that MTI has an effect on maintaining the homeostasis of the living body as well as the ability to inhibit trypsin.

The effects of pepsin on the natural progression of autoimmunity in glomerulonephritis in the NZB/W F1 mouse.

The effects of pepsin on autoimmune glomerulonephritis of New Zealand Black and White F1 hybrid (NZB/W F1) mice were investigated. Intravenous pepsin significantly improved survival rate and suppressed progressive increase in urinary protein and histopathological changes in kidney. Increased serum levels of immune complexes, anti-DNA antibody and natural thymocytotoxic autoantibody were decreased and abnormalities in lymphocyte subsets were ameliorated by pepsin. Pepsin suppressed autoantibody production and enhanced antibody production against xenogeneic substance in these mice. The fact that pepsin ameliorates abnormalities in immune function may contribute to the preventive effects of pepsin against pathogenesis and progression of immune complex nephritis.

Mechanism of action of AC-1370 on phagocyte functions.

The mechanism of action of a new semisynthetic cephalosporin AC-1370 on phagocyte functions was investigated. AC-1370 enhanced phagocytic functions of macrophages and neutrophils. AC-1370 bound to 27.4% of mouse peritoneal resident cells. Most of the AC-1370-binding cells were macrophages, and few neutrophils bound AC-1370. Culture supernatant of mouse macrophages cultured with AC-1370 significantly augmented phagocytic functions of mouse neutrophils. This activity of the culture supernatant of AC-1370-stimulated macrophages was abolished by digestion with trypsin but not by heat treatment at 56 degrees C for 30 min. The mechanism of the activation of phagocyte functions by AC-1370 is proposed as follows. First, AC-1370 binds to macrophages and causes their activation. Second, trypsin-sensitive and heat-stable soluble factor(s) is released from these macrophages. And finally, neutrophil functions are activated by the factor(s).

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.

Effects of AC-1370, a new semisynthetic cephalosporin, on phagocyte functions.

The effects of a new semisynthetic cephalosporin, AC-1370, on phagocyte functions were compared with those of cefoperazone. AC-1370 augmented phagocytosis by mouse macrophages in vitro and in vivo, by mouse neutrophils in vivo, and by human neutrophils in vitro. Cefoperazone suppressed phagocytosis by mouse macrophages and neutrophils. Random migration and chemotaxis of mouse and human neutrophils were increased by the addition of AC-1370. Nitroblue tetrazolium reduction by human neutrophils was enhanced by the addition of AC-1370. Intracellular killing of bacteria by macrophages was also enhanced by AC-1370. Further, bactericidal effects of AC-1370 against Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae were augmented when they were each cultured with mouse or human leukocytes. These results suggest that AC-1370 is an unique beta-lactam antibiotic which has a potentiating effect on phagocyte functions as well as a bactericidal effect.

[Therapeutic effects of human urinary trypsin inhibitor on acute experimental pancreatitis].

Therapeutic effects of human urinary trypsin inhibitor (MTI) on acute pancreatitis were examined. MTI potently inhibited not only proteases such as trypsin or alpha-chymotrypsin, but also inhibited lipase or creatine phosphokinase which are considered to be related to pancreatitis. Although gabexate mesilate (gabexate) and aprotinin also strongly inhibited trypsin, their inhibition spectra against pancreatic enzymes were narrower and aprotinin also strongly inhibited trypsin, their inhibition against pancreatic enzymes were narrower than MTI. MTI inhibited proteases released from pancreatic slice by trypsin more potently than gabexate or aprotinin. The therapeutic effects of MTI on experimental acute trypsin-induced pancreatitis in dogs or rats were stronger than those of gabexate or aprotinin. These results suggest that MTI may suppress pathogenesis and development of pancreatitis in several ways, for example, by directly inhibiting trypsin and by inhibiting tissue-damaging enzymes released from the pancreas by stimulation with trypsin.

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.

Mechanism of host defense suppression induced by viral infection: mode of action of inosiplex as an antiviral agent.

The mechanism of influenza virus (INFV)-induced immunosuppression and the mode of inosiplex action against INFV infection were studied. INFV suppressed both anti-lipopolysaccharide and anti-sheep erythrocyte antibody production in mice. INFV infection caused viral mRNA synthesis and increased total RNA synthesis in lymphocytes, but total mRNA synthesis was decreased. The translational ability of INFV-infected lymphocytes was also suppressed. Thus, INFV seemed to cause suppression of both mRNA synthesis and the translational ability of lymphocytes, resulting in suppression of lymphocyte functions. Inosiplex potentiated antibody production against sheep erythrocytes but not against lipopolysaccharide in normal and INFV-infected mice. Adamantanamine did not produce such a potentiating effect. The lymphocytes obtained from INFV-immunized and inosiplex-treated mice conferred resistance against INFV infection. This resistance was partially inhibited by anti-Thy 1.2 antibody treatment of the lymphocytes. In an adoptive cell transfer system, inosiplex treatment of T-cell donors potentiated antibody production when a non-immunosuppressive carrier (human serum albumin) was used. When an immunosuppressive carrier (INFV) was used, inosiplex treatment of either B-cell donors or T-cell donors increased antibody production. Direct introduction of inosiplex into lymphocytes by a cell fusion technique stimulated anti-sheep erythrocyte antibody production more effectively than the addition of inosiplex to cultures. Inosiplex increased total RNA and total mRNA syntheses in phytohemagglutinin-treated lymphocytes. In INFV-infected lymphocytes, inosiplex decreased syntheses of total RNA, total mRNA, and viral mRNA and restored translational ability. From these results, we concluded that inosiplex penetrates into lymphocytes and suppresses viral RNA synthesis and that it supports lymphocyte functions by promoting RNA synthesis and translational ability, both of which are necessary for hosts.

Anti-inflammatory properties of a newly synthesized compound, 6-chloro-4-oxyimino-1-phenyl-1,2,3,4-tetrahydroquinoline (M-7074).

Anti-inflammatory properties of a newly synthetized compound, 6-chloro-4-oximino-1-phenyl-1,2,3,4-tetrahydroquinoline (M-7074), have been investigated. Anti-edema activities of M-7074 were more potent than those of phenylbutazone in carrageenin, bradykinin and mustard edema tests in rats. M-7074 showed an inhibitory effect on adjuvant arthritis, especially on the secondary inflammatory lesions in rats. Inhibitory effect of M-7074 on cotton-pellet granuloma formation was all but equal to that of phenylbutazone in rats. M-7074 also showed inhibitory effects on ultraviolet erythema in guinea-pigs and increased vascular permeability in mice, moderate analgesic activity in rats and mice, and antipyretic activity in rats. Furthermore, inhibitory effects of M-7074 on prostaglandin biosynthesis in guinea-pig lung homogenate and arachidonic acid-induced aggregation of rabbit platelets were fairly equal to those of indomethacin. However, M-7074 showed no effect on humoral nor cellular immunity in mice. M-7074 possessed no ulcerogenic activity in rats and mice, and LD50 value of M-7074 was 8.01 g/kg, p.o. in mice. These data indicate that M-7074 is a novel anti-inflammatory agent with large margin of safety.