Suppression of ischemia-reperfusion injury in murine models by neopterins.

We investigated the effects of D-neopterin (NP) and its reduced form, 5,6,7,8-tetrahydro-D-neopterin (NPH4), in two models of ischemia-reperfusion injury, i.e., ischemic paw edema in mice and gastric ischemia in rats. In ischemic paw edema, iv administration of either NP or NPH4 more potently inhibited the increase of paw thickness after release from ischemia than did administration of superoxide dismutase plus catalase or allopurinol. In gastric ischemia, NP and NPH4 also significantly suppressed the formation of gastric mucosal erosions. Lipid peroxidation in the stomach was increased by ischemia-reperfusion treatment, and the increase was inhibited by the administration of NP or NPH4. The minimum dose of NPH4 required to suppress the gastric ischemic injury in this experiment was 0.3 mg/kg of body weight. These results suggest that neopterin may be effective as a protective agent against ischemia-reperfusion injury, in which active oxygen species are believed to play a major role.

Inhibition of carbon tetrachloride-induced hepatotoxicity by neopterins.

We investigated the inhibitory effects of neopterin (NP) and its reduced form, 5,6,7,8-tetrahydroneopterin (NPH4), on carbon tetrachloride (CCl4)-induced hepatotoxicity. In in vivo experiments, intraperitoneal administration of NP or NPH4 significantly inhibited the elevation of plasma alanine aminotransferase activity induced by CCl4 in mice. In in vitro experiments using cultured rat hepatocytes, CCl4 induced in a manner which was both time- and dose-dependent lactate dehydrogenase release, and the addition of NP or NPH4 to the culture-medium significantly inhibited its release from cells. NPH4, but not NP, reacted directly with a stable radical, 1,1-diphenyl-2-picrylhydrazyl. These results suggest that NP and NPH4 inhibit CCl4-induced hepatotoxicity through different mechanisms.

General pharmacology of the non-sulfhydryl angiotensin converting enzyme inhibitor N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline.

The effects of N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline (AB-47, CAS 120008-53-9), an orally active angiotensin converting enzyme inhibitor, on the central nervous, respiratory and cardiovascular, autonomic systems, isolated smooth muscles and other functions were investigated in various experimental animals. AB-47 had no effect on central nervous, autonomic systems and isolated smooth muscles. AB-47 (10 and 30 micrograms/kg i.v.) significantly lowered femoral blood pressure without affecting respiration and heart rate in anesthetized rats. However, AB-47 had no effect on the contractile tension of mammalian isolated atrium and aorta. AB-47 had no effect on gastrointestinal transit in mice. Very slight injury of gastric mucosa was observed 4 h after the oral administration of AB-47 in rats but AB-47 did not damage the small intestinal mucosa. AB-47 had no effect on the contraction of rat phrenic nerve-diaphragm preparation induced by electrical stimulation. AB-47 did not affect the incidence of acetic acid-induces writhings. AB-47 potentiated carrageenan-induced hind paw edema in rats. The potentiation of edema may be due to an accumulation of bradykinin induced by the inhibition of angiotensin converting enzyme (ACE), because ACE is the identical enzyme with kinase II. The pretreatment of AB-47 for 7 days (1, 3, 10 mg/kg/d p.o.) inhibited the cardiac hypertrophy induced by isoproterenol (isoprenaline). This result suggests that the renin-angiotensin-aldosterone system directly or indirectly participates in the cardiac hypertrophy induced by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of 5,6,7,8-tetrahydroneopterin on adriamycin-induced cardiotoxicity.

The protective effect of the reduced form of neopterin, 5,6,7,8-tetrahydroneopterin (NPH4), against the cardiotoxicity of adriamycin (ADR) was evaluated in mice by assay of serum creatine phosphokinase (CPK) and heart lipid peroxide (i.e., thiobarbituric acid reactive substances, TBARS). The activity of CPK was drastically elevated at around 3 days after a single s.c. injection of ADR and had returned to normal at 6 days. There were no marked changes in the TBARS content of the heart by 6 days, but a significant increase was apparent at about 8 days after ADR treatment. The effect of NPH4 was evaluated in terms of the serum CPK activity at the 4th day and in terms of TBARS content at the 8th day after ADR treatment. The elevation of CPK activity induced by ADR was significantly suppressed by two treatments with NPH4 every day for 4 days, i.e., from day 0 to day 3 after injection of ADR, at doses of 3 mg/kg and 0.3 mg/kg. The increase of TBARS content in the heart at 8 days after ADR treatment was also suppressed at both doses of NPH4. The effect of the timing of NPH4 treatment on the suppression of elevated CPK was examined. The suppressive potency of NPH4 could not be elicited by any treatment schedule that terminated at or before 2 days after injection of ADR. Consecutive treatments from day 0 to day 3 after injection of ADR were effective, as was a simple schedule of only two treatments with NPH4 on day 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of neopterin on NADPH-dependent superoxide-generating oxidase of rat peritoneal macrophages.

The effect of the oxidized form of neopterin (NP) on the NADPH-dependent superoxide-generating oxidase (NADPH-oxidase) was investigated in both whole-cell and cell-free activation systems by using peritoneal macrophages of rats which had received an intraperitoneal injection of mineral oil. In the whole-cell system, NP remarkably inhibited the generation of superoxides in macrophages stimulated with phorbol myristate acetate (PMA). NP also showed an significant suppression of the activation of superoxide-generating NADPH-oxidase in the cell-free system using solubilized membranes and sodium dodecyl sulfate (SDS) as a stimulant. The 50%-inhibitory concentration (IC50) of NP was about 1 microM in both assay systems. In a kinetic study, competitive inhibition of the NADPH-oxidase by NP was observed in the cell-free system with a calculated inhibition constant (Ki) of 6.50 microM. These findings suggest that NP may play an important role in the suppression of superoxide generation via the inhibition of the NADPH-oxidase in phagocytes.

Protective effects of tetrahydroneopterin against free radical-induced injury.

The therapeutic potency of 5,6,7,8-tetrahydroneopterin (NPH4) was investigated in ischemic paw edema, Adriamycin (ADR)-induced cardiotoxicity, and endotoxin [lipopolysaccharide (LPS)]- and carbon tetrachloride (CCl4)-induced hepatotoxicity in mice. Ischemic paw edema was completely suppressed by pre-administration of NPH4. ADR-induced cardiotoxicity and LPS-induced hepatotoxicity were significantly decreased by post-administration of NPH4. Furthermore, NPH4 ameliorated CCl4-induced hepatotoxicity. These results suggest that NPH4 may be useful for the treatment of free radical, especially superoxide radical, -related tissue injury.

Toxicity studies of the non-sulfhydryl angiotensin converting enzyme inhibitor N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline in rats.

The acute and subacute toxicities of N-[8-amino-1(S)-carboxyoctyl]-L- alanyl-L-proline (AB-47, CAS 120008-53-9), which is a non-sulfhydryl angiotensin converting enzyme inhibitor, were studied in male and female Fischer 344 rats. In the acute study, male and female rats were orally given 5000 mg/kg of AB-47. No rats were dead during the observation period (14 days) after the administration. The LD50 values of AB-47 were more than 5000 mg/kg in both male and female rats. Although only diarrhoea as toxic sign was observed 2 to 7 h after the administration, this sign disappeared within 24 h after the administration. Necropsy at the termination of observations revealed no macroscopic lesions in any rats. In the subacute study, male and female Fischer 344 rats were orally given 40, 200 or 1000 mg/kg/d of AB-47 for 4 weeks. Neither toxic signs nor death due to drug effects were observed at any dosage levels of AB-47. Furthermore, AB-47 did not influence body weight, food consumption, food efficiency, urinalytical and hematological parameters in both male and female rats. The minor changes of serum parameters, which consisted of very slight increases in serum potassium and decreases in serum albumin/globulin ratio, occurred in male rats given 200 and 1000 mg/kg/d of AB-47. Serum urea nitrogen values were elevated in both male and female rats given 1000 mg/kg/d of AB-47. Slight decreases of heart weight and heart weight/body weight ratios were observed at all dosage levels of AB-47.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of neurite-promoting activity and protease-inhibiting function of alpha 2-macroglobulin in culture.

The relationship between the neurite-promoting effect and the protease-inhibiting function of alpha 2-macroglobulin (alpha 2M) was examined in a culture of dissociated neurons from embryonic rat neocortical tissue. The neurite-promoting effect of various protease inhibitors (soybean trypsin inhibitor, alpha 1-antitrypsin, aprotinin, phenylmethanesulfonyl fluoride, leupeptin, antipain, human alpha 2M and purified rat alpha 2M) was investigated. At lower concentrations, only alpha 2M significantly promoted neurite outgrowth. Furthermore, alpha 2M-trypsin complexes, which had lost their protease-inhibiting function and exposed their receptor-recognition site, promoted neurite outgrowth more efficiently than native alpha 2M. These results indicate the possibility that the neurite-promoting effect of alpha 2M is mediated by its receptor-binding activity but is not directly related to its protease-inhibiting function.

Core protein of chondroitin sulfate proteoglycan promotes neurite outgrowth from cultured neocortical neurons.

Chondroitin sulfate proteoglycan (CS-PG) was purified from rat brain and examined for its effect on neurite outgrowth in primary cultures of embryonic rat neocortical neurons. Neurite outgrowth was increased in culture wells coated with CS-PG. The core protein and glycosaminoglycan (GAG) prepared from the CS-PG were also examined for neurite-promoting activity. The activity was observed in culture wells coated with the core protein but not with GAG. These results suggest that CS-PG stimulates neurite outgrowth from the cultured neurons via its core protein.

Hypotensive and hemodynamic effects of the new non-sulfhydryl angiotensin converting enzyme inhibitor N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline.

The hypotensive effects of N-[8-amino-1(S)-carboxyoctyl] -L-alanyl-L-proline (AB-47, CAS 120008-53-9) were examined in normotensive rats and various hypertensive rat models. The hemodynamic effect of AB-47 was also examined in anesthetized spontaneously hypertensive rats (SHR). In 2-kidney, 1-clip renal hypertensive rats (2K, 1C-RHR) and SHR, the single administration of AB-47 (10 mg/kg, p.o.) induced potent and long-lasting hypotensive effects. The repeated administration of AB-47 (1 to 10 mg/kg, p.o.) to SHR for 29 days produced a dose-dependently and sustained hypotensive effect of 20 to 70 mmHg. AB-47 (10 mg/kg, p.o.) had a weak hypotensive effect in DOCA-salt hypertensive rats but no effects in normotensive and 1-kidney, 1-clip renal hypertensive rats (1K, 1C-RHR). AB-47 (3 mg/kg, p.o.) reduced blood pressure in intact SHR but not in bilateral nephrectomized SHR. The single intravenous injection of AB-47 (10 to 100 micrograms/kg) dose-dependently lowered systemic blood pressure, left ventricular systolic pressure (LVSP) and dp/dtmax without affecting heart rate (HR) and these effects of AB-47 were more potent than those of captopril and enalaprilat. These results suggest that AB-47 is a potent and long-lasting hypotensive agent and may be useful for the therapy of both hypertension and congestive heart failure.

Inhibitory activity and protein binding of L-lysine derivatives as angiotensin converting enzyme inhibitors.

Four compounds 1a-4a containing one L-lysine residue in the molecule including a diastereomer mixture of lisinopril (N-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) and N epsilon-carbobenzoxy-L-lysine derivatives 1b-4b of each of the four compounds were synthesized to compare the angiotensin converting enzyme (ACE) inhibitory activities in vitro and in vivo. They all showed high ACE inhibitory activity in vitro (IC50 = 0.14-42 nmol/l). A marked difference, however, was observed in inhibition of the pressor response to angiotensin I between 1a-4a (high activity) and 1b-4b (low activity). The binding of these compounds to human serum proteins in vitro was investigated by means of equilibrium dialysis and ultracentrifugation. Compounds 1b-4b showed higher levels of binding to serum albumin than that of the corresponding compounds 1a-4a, and the percentage of binding ranged from 20.1 to 89.1%. Furthermore, the inhibitory activity of compounds 1b-4b in vitro was decreased by the addition of albumin in a concentration-dependent manner. These results suggested that the difference in the protein binding rate of compounds is one of the important factors influencing the inhibitory activity in vivo.

Alpha 2-macroglobulin is an astroglia-derived neurite-promoting factor for cultured neurons from rat central nervous system.

The neurite promoting factors in the astroglial conditioned medium (As-CM) were characterized by using primary cultures of embryonic rat neocortical neurons. The factors in the As-CM bind to lectins such as wheat germ agglutinin (WGA), suggesting that they contain sugar moieties. When the WGA-bound fractions were applied on a Superose 6 column, the activity was recovered mainly in two fractions, peak I and peak II. The peak II fraction was further purified by Mono Q anion exchange chromatography. A single protein band of 180 kDa was detected in the final Mono Q fraction by sodium dodecylsulfate polyacrylamide gel electrophoresis. The molecular weight coincided with that of alpha 2-macroglobulin (alpha 2M). Western blotting showed that the single protein band was reacted with anti-alpha 2M antibody but not with anti-fibronectin and anti-laminin antisera. The neurite-promoting activity of the Mono Q fraction was inhibited by anti-alpha 2M antibody. Furthermore, commercially available alpha 2M also promotes neurite outgrowth in our assay system. These results strongly suggested that alpha 2M is one of the neurite-promoting factors in the As-CM.

Pharmacological properties of the new non-sulfhydryl angiotensin converting enzyme inhibitor N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline.

AB-47 (N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline) is a non-sulfhydryl angiotensin converting enzyme (ACE) inhibitor with omega-aminoalkyl group. AB-47 was slightly more potent than enalaprilat in inhibiting rabbit lung ACE. The ACE inhibition and bradykinin (BK) potentiation by AB-47 in guinea-pig ileal longitudinal muscle were as potent as with enalaprilat. In conscious rat, AB-47 (i.v.) inhibited angiotensin I (A-I)-induced pressor response and augmented BK-induced depressor response more potently and in a long-lasting manner than enalaprilat. Furthermore, AB-47 exhibited higher selectivity for ACE inhibition than for BK inactivation and higher selectivity than enalaprilat and captopril. The inhibition of A-I-induced pressor response by AB-47 (p.o.) was as potent as that of enalapril. These results suggest that AB-47 is a highly potent, long-lasting and relatively A-I-selective ACE inhibitor.

Synthesis and angiotensin converting enzyme inhibitory activity of N-carboxymethyldipeptides with omega-(4-piperidyl)alkyl group.

The synthesis of a series of novel, potent angiotensin converting enzyme (ACE) inhibitors containing 1(S)-carboxy-omega-(4-piperidyl)alkyl group at the N-terminal of the dipeptide is described. These 1-carboxy-omega-(4-piperidyl)alkyl derivatives possess greater or equivalent in vitro potency and in vivo efficacy than captopril and enalapril. The length (n) of the carbon chain in the omega-(4-piperidyl)alkyl moiety was varied from two to six to investigate the optimal structure for long-acting ACE inhibitors. 1-[N-[1(S)-Carboxy-6-(4- piperidyl)hexyl]-L-alanyl]-(2a,3a beta, 7a beta)-octahydro- 1H-indole-2-carboxylic acid (9b), the most potent member of the series, had an in vivo area under the curve (AUC) of 685, which was calculated by the inhibition of angiotensin I-induced pressor response vs. time curves (0 to 8 h) after p.o. administration.

Synthesis and angiotensin converting enzyme inhibitory activity of N-carboxymethyldipeptides with an omega-aminoalkyl group.

A series of novel L-alanyl- and L-lysyl-L-proline derivatives having an omega-amino-1-carboxyalkyl group was prepared, and assayed for their inhibitory activity against angiotensin converting enzyme (ACE). The dicarboxylic acids possesing S,S,S configuration showed potent in vitro ACE inhibitory activity with IC50 values of 0.68-1.4 nmol/l. The length of the carbon chain in the omega-aminoalkyl moiety was varied from 6 to 9 to investigate the optimal structure for long-acting ACE inhibitors. The most prolonged activity in vivo was observed with N-[8-amino-1(s)-carboxyoctyl]-L-alanyl-L-proline upon i.v. and p.o.

[Regulation of neuronal development by astroglia].

Accumulating lines of evidence indicated that glial cells play important roles in regulating the neuronal development. It has been reported by a number of authors that astroglia promote the survival of neurons and the neurite outgrowth by several diffusible factors and membrane-associated factors. In the present article, we have reviewed the astroglia-derived bioactive substances which possibly affect the neuronal development in the central nervous system.

Synthesis and angiotension converting enzyme inhibitory activity of L-lysyl-N-substituted glycine derivatives.

The synthesis and biological activity of novel L-lysyl-N-substituted glycine derivatives which exhibit in vitro and in vivo angiotensin converting enzyme (ACE) inhibition, are described. Particularly, N-[N alpha-(1-carboxy-3-phenylpropyl)-L-lysyl]-N-(4-phenylcyclohexyl)glycine (11e), N-[N alpha-(1-carboxy-3-cyclopentylpropyl)-N epsilon-carbobenzoxy-L- lysyl]-N-cyclopentyl glycine (9h) and N-[N alpha-[1(S)-carboxy-3-cyclohexylpropyl]-L-lysyl]-N-cyclopentyl glycine (19a) showed a strong inhibitory activity at IC50 as low as 0.65, 0.64 and 0.11 nmol/l, respectively. The most potent activity in vivo was observed with the compound 19a after i.v. administration in the rat.

Purification of human plasma lecithin:cholesterol acyltransferase and its specificity towards the acyl acceptor.

A simple and convenient method for the purification of human plasma lecithin-cholesterol acyltransferase was developed. The method involves the adsorption of the enzyme from diluted human plasma on DEAE-Sephadex, treatment with 1-butanol in the presence of (NH4)2SO4, DEAE-Sephadex chromatography, treatment with dextran sulfate in the presence of Ca2+, and hydroxyapatite chromatography. The enzyme purified showed a single main band by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In addition, the enzyme obtained was stable for more than four weeks, when it was kept at 4 degrees C under N2 in a buffer of low ionic strength. The purified enzyme was used to study its specificity toward the acyl acceptor. This specificity was found to be broad in that not only sterols but also long chain primary alcohols exhibited considerable acceptor activity. Furthermore, in agreement with our previous observations with crude enzyme (Piran, U. and Nishida, T. (1976) J. Biochem. (Tokyo) 80, 887-889), the purified enzyme was found to be capable of hydrolyzing the ester linkage at the carbon-2 position of phosphatidylcholine. The transesterification, as well as the hydrolytic reaction, required the presence of the cofactor polypeptide, apolipoprotein A-I.