Cardiovascular activities of the new potent and long-lasting antihypertensive calcium entry blocker (+-)-3-ethyl,5-methyl,2- ([2-(formylamino)-ethyl]- thiomethyl)-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicar box ylate.

BBR 2160 ((+-)3-ethyl,5-methyl,2-([2-(formylamino)-ethyl]- thiomethyl)-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarb oxy late, CAS 118587-22-7) is a new calcium entry blocker (CEB) which completely displaces 3H-nitrendipine from binding sites, is 10 times more potent than amlodipine (A) and equiactive with nifedipine (N). On the rat aorta contracted by 10 mmol/l Ca++, or 45 mmol/l K+, BBR 2160 shows higher CEB activity than N and A, achieving the maximum effect on voltage operated channels-induced contractions in 6 h, while N takes about 2 h. BBR 2160, N and A negatively affect the chronotropism on spontaneously beating, and inotropism on electrically driven guinea pig atria, respectively. In vitro BBR 2160 has marked vasoselectivity. Administered orally to conscious hypertensive rats (SHR) and renal hypertensive dogs (RHD), it caused a dose-dependent reduction in systolic blood pressure with a relatively slow onset, peak effect at 3-6 h and duration over 6 h. BBR 2160 and A have more pronounced activity on SHR than on normotensive rats (NR) (ED20 NR/SHR 3.3 for both compounds), while the antihypertensive and hypotensive activities of N are in the same dose-range (ED20 NR/SHR 1.3). No tolerance develops to the antihypertensive effects of BBR 2160 after five days' dosing up to 3.2 mg/kg in SHR and 1 mg/kg in RHD. In instrumented conscious normotensive dogs BBR 2160, N and A mostly lower diastolic blood pressure and total peripheral resistance, and do not increase total oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of GABA-A and dopamine D-2 brain receptors in dogs with portal-systemic encephalopathy.

The binding characteristics of gamma-aminobutyric acid-A (GABA-A) receptors and the kinetic characteristics of the target enzyme of GABA synthesis in nerve terminals, glutamic acid decarboxylase (GAD), were studied in a dog model of portal-systemic encephalopathy obtained by porta-caval shunt performed in dimethylnitrosamine pretreated animals. Furthermore the properties of dopamine receptors and the levels of catecholamines of encephalopathic dogs were investigated. The mild stage of encephalopathy was characterized by an up-regulation of the inhibitory GABA-A receptors probably related to a decrese of GABA in nerve terminals since GAD was decreased and by a slight decrease of catecholamines and by an increased synthesis of octopamine associated with a decreased affinity of dopamine receptors. In the severe stage there was a selection of high affinity GABA-A receptors with an increased number of benzodiazepine recognition sites which were supersensitive to GABA stimulation, a decreased number of Dopamine D-2 receptors and a marked reduction of catecholamines. These data seem to suggest that the neurological disturbances of experimental portal-systemic encephalopathy might be the result of an imbalance between inhibitory and excitatory systems leading to a prevalence of the first one.

Antihepatotoxic properties of uridine-diphosphoglucose in liver fluke infection. Experimental fascioliasis in the rat.

The antihepatotoxic properties of uridine-diphosphoglucose (UDPG, Toxepasi) have been evaluated in a well-established model of liver damage, the liver fluke infection (experimental fascioliasis in the rat), which causes a dramatic loss of the microsomal drug-metabolizing monooxygenase (MFO) and glucuronosyltransferase (GT) enzyme systems as a consequence of peroxidative damage to microsomal membrane lipids. Administration of 100 mg/kg UDPG i.p. to the infested rat for the entire course of the infection (40 days) positively affects the parameters reflecting the integrity of the liver cell (serum glutamate-pyruvate, GPT and glutamate-oxaloacetate, GOT, transaminases) and the detoxifying capacity of the liver (cytochrome P-450, cytochrome b5, cytochrome P-450-dependent p-nitroanisole O-demethylase and aniline hydroxylase activities, and the p-nitrophenol glucuronidation) and greatly reduces the lipid peroxidative phenomen in membranes from whole liver (tissue malonic dialdehyde content) and in membranes of the microsomal fraction (conjugated diene absorption). As a consequence of this, the total lipid and phospholipid contents of the liver are restored, there is minimal loss of latency of GT enzyme(s), cytochrome P-450 conversion to cytochrome P-420 is fairly negligible and total liver glutathione content is also restored. Therefore, UDPG restores liver function by protecting the endoplasmic reticulum membranes from the oxidative stress resulting from activation of the CN-insensitive respiratory burst of the phagocytic cells consequent to Fasciola hepatica invasion, migration and growth. It is very likely that UDPG acts as an effective antilipoperoxidative agent through both direct (as demonstrated by our in vitro experiments) and indirect mechanisms (stimulation of the glycolytic pathway, and hence of the reducing equivalents----glutathione----vitamin E supply).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological evaluation of substituted benzo[h]cinnolinones and 3H-benzo[6,7]cyclohepta[1,2-c]pyridazinones: higher homologues of the antihypertensive and antithrombotic 5H-indeno[1,2-c]pyridazinones.

Several substituted benzo[h]cinnolinones 3 and 3H-benzo[6,7]cyclohepta[1,2-c]pyridazinones 4, which were designed as less rigid congeners of 5H-indeno[1,2-c]pyridazinones 2, were synthesized and tested as antihypertensive, inotropic, antithrombotic, antiinflammatory, and antiulcer agents. While the seven-membered ring derivatives displayed only antithrombotic properties, which were comparable to that of acetylsalicylic acid, most of the benzo[h]cinnolinones exhibited significant antihypertensive, inotropic, and antithrombotic properties. In this respect, the 8-amino (3b) and 8-acetylamino (3c) together with the 4,4a-dehydro analogue of 3c (11) were found to possess the most potent and long-lasting antihypertensive activity. In particular, the dextro isomer of 3c was more active than the racemic form, with lower tachycardiac effects. Unlike the lower homologues 2, none of the compounds showed significant antiinflammatory or antiulcer activity.

Participation of lipid peroxidation in the loss of hepatic drug-metabolizing activities in experimental fascioliasis in the rat.

Fascioliasis causes a dramatic decrease in drug-metabolizing ability of the hepatic monooxygenase (MFO) and glucuronosyltransferase (GT) enzyme systems in the rat. The present study was undertaken to determine whether lipid peroxidation is involved in the enzymatic loss. Peroxidative damage of membrane lipids (as assessed by the tissue content of malonic dialdehyde, MDA, and the diene conjugation absorption in microsomal membranes) was found to occur over the entire course of the liver infection (concomitant to a decrease in glutathione levels), and to different degrees in relation to the various steps of the parasite cycle. The onset (MDA six times the controls; delta E 1% = 1.55 at the 20th day) coincides with the beginning of the loss of MFO (-30%) and GT (-20% at the 20th day), and peaks between the 30th and 40th day (MDA eight times the controls; delta E 1% = 1.96), when the loss in the enzyme activities is maximal (MFO - 60/70%; GT - 65/95%). There was a strict correlation at all the observation times between the extent of lipid peroxidation and the decrease in drug metabolizing ability: this supports the view that lipid peroxidation is the major agent in the impairment of MFO and GT enzyme activities, and very likely in the initiation of the pathological degeneration of the liver tissue. As evidenced by histological examination, the phagocytic response of the liver tissue to the parasite invasion and growth leads to oxidative stress, which is the causative agent in the initiation and development of lipid peroxidation.

Dose-response decrease in plasma tryptophan and in brain tryptophan and serotonin after tryptophan-free amino acid mixtures in rats.

Rats fasted 15 hours were treated p.o. with increasing amounts (660 and 1320 mg/kg body weight) of a mixture containing a fixed proportion of seven essential amino acids (L-phenylalanine 13.6%, L-leucine 6.0%, L-isoleucine 12.1%, L-methionine 12.1%, L-lysine 30.3%, L-threonine 10.6%, L-valine 15.2%) and lacking tryptophan. The mixtures produced a dose-response decrease of free (by 34% after the lower dose and by 58% after the higher dose of the mixture) and total (by 10 and 31%) plasma tryptophan and of brain tryptophan (by 38 and 65%), serotonin (by 17 and 41%) and 5-hydroxyindole acetic acid (by 21 and 49%). The mechanisms of these changes are discussed.

Protective effect of reduced glutathione against cisplatin-induced renal and systemic toxicity and its influence on the therapeutic activity of the antitumor drug.

Reduced glutathione has been shown to be an effective protector against cisplatin-induced nephrotoxicity of potential clinical value, since it does not reduce antitumor activity of the cytotoxic drug. This paper extends previous observations on the protective potential of reduced glutathione against cisplatin-induced nephrotoxicity, in different rodent models. Following i.v. administration, glutathione protection against cisplatin-induced nephrotoxicity was found to be critically dependent on timing of thiol administration. Whereas the sulfhydryl compound provided almost complete protection in CD rats, the protective effect against toxic renal damage was only partial in mice of different strains. In spite of the modest protection against kidney toxicity, glutathione reduced lethal toxicity in the mouse. Under the same experimental conditions at protective dose levels, the tripeptide thiol did not interfere with the antitumor effectiveness of cisplatin, in any of the tumor models examined. The kidney content of non-protein sulfhydryls of CD rats produced by the effective dose of glutathione was markedly higher than that found in the mouse treated with the same dose. This finding is consistent with a differential protection provided by glutathione against cisplatin-induced renal toxicity in these species.

Carboxylic metabolites of tiadenol as "proximate" inducers of hepatic peroxisomal beta-oxidation activity.

Chronic exposure of rats to the hypolipidemic agent tiadenol causes a dramatic dose-dependent increase of peroxisomal beta-oxidation activity. To elucidate which metabolite of the drug is the "proximate" inducer (tiadenol is eliminated completely in metabolized form after acute administration) we investigated the qualitative and quantitative metabolic profile of the drug at different doses (50, 150, 300 mg/Kg in two-weeks chronically treated rats, in parallel to that of a model compound, tiadenol-disulfoxide, a weak inducer of palmitoyl-CoA oxidation activity. No changes in the biodisposition of tiadenol (and tiadenol-disulfoxide) were found following chronic treatment for all the doses tested. For both the compounds a strict correlation was evidenced between the extent of formation of carboxylic metabolites and their inductive potencies on peroxisomal beta-oxidation activity. This indicates that tiadenol carboxylic metabolites act as the enzymatic effectors.

Decrease in plasma tryptophan after tryptophan-free amino acid mixtures in man.

Male healthy subjects, fasting 12 hours, ingested increasing amounts of a mixture containing a fixed proportion of seven essential amino acids (L-isoleucine 11.5%, L-leucine 18.0%, L-lysine 13.1%, L-methionine 18.0%, L-phenylalanine 18.0%, L-threonine 8.2%, L-valine 13.1%) and lacking tryptophan. The diets produced a rapid fall in plasma tryptophan which was proportional to the total amount of the amino acids ingested. Following the highest dose administered (36.6 g) plasma tryptophan fell to a minimum of about 35% the initial level and remained markedly reduced at 6 hours after treatment. The mechanism of this decrease and its potential clinical relevance are discussed.

Anti-convulsant effects by reduced glutathione and related aminoacids in rats treated with isoniazid.

Rats were treated with different doses of isoniazid (INH) causing convulsions. Lethal dose (DL50) and effective convulsant dose (ED50) were calculated. Reduced glutathione (GSH) and related aminoacids were administered to rats receiving INH: the latency and duration of convulsions were recorded; cerebral gamma-aminobutyric acid (GABA) concentrations were determined in rats receiving INH and an association of GSH and INH. GSH and its related aminoacids as cysteine and glycine greatly decreased the duration of INH-induced seizures, while glutamic acid did not protect against convulsions caused by INH. Furthermore, INH causes a decrease in cerebral GABA levels to about half and GSH repeated pretreatment did, however, not prevent the INH induced decline of GABA content: hence, the anticonvulsant effect of GSH can not be ascribed to the restoration of normal levels of anti-epilectically acting GABA, but can be attributed to cysteine and glycine, aminoacids linked to GSH.

Pharmacokinetics of isosorbide mononitrate in a new sustained release oral form in comparison with a conventional formulation.

40 mg of isosorbide mononitrate (isosorbide-5-mononitrate, IS5MN) in conventional (Ismo 20) and sustained release formulations (Ismo Diffutab) were administered to 5 male healthy volunteers. The administration of the sustained release formulation was repeated for seven days in order to evaluate the possible occurrence of accumulation. Pharmacokinetic data showed that the sustained release formulation reached significantly lower and delayed mean peak plasma levels compared with the conventional formulation, respectively 452.8 +/- 67.8 ng/ml and 706.7 +/- 57.3 (mean +/- SE) (p less than 0.05). Peak times were 4.6 +/- 0.2 and 2.4 +/- 0.2 (p less than 0.005) h, respectively. A longer plasma half-life together with larger AUC for the sustained release formulation was also observed. The pharmacokinetic parameters of the sustained release formulation are consistent with a therapeutic usefulness and suggest further clinical evaluation.

Experimental studies on pharmacology, metabolism and toxicology with tiadenol-disulfoxide. Dissociation of lipid lowering effects and the induction of peroxisomal and microsomal drug-metabolizing enzymes.

The hypolipidemic activity of tiadenol-disulfoxide, the major metabolite of 1,10-bis(hydroxyethylthio)decane (tiadenol, Eulip) in man and in the rat was assessed in various experimental models versus the corresponding activity of tiadenol. Tiadenol-disulfoxide in the normolipidemic rats lowers total serum cholesterol and serum and liver triglycerides in an extent comparable to that of the reference compound. Likewise, it is equally effective as tiadenol in preventing Triton-induced hyperlipidemia and Nath diet induced hypercholesterolemia; in addition tiadenol-disulfoxide is slightly more effective than tiadenol in increasing HDL-cholesterol in hypercholesterolemic rats. At hypolipidemic doses the compound causes no hepatomegaly, no induction of peroxisomal catalase and palmitoyl-CoA oxidase activities, no smooth endoplasmic reticulum proliferation and no induction of microsomal cytochrome P-450 and of cytochrome P-450 dependent enzyme activities: aminopyrine (aminophenazone) N-demethylase, aniline hydroxylase, zoxazolamine hydroxylase and hexobarbital oxidase. At the suprapharmacological dose of 300 mg/kg tiadenol-disulfoxide, if compared to the reference compound, shows a generally lower order of toxicity on these hepatic parameters. Orally administered tiadenol-disulfoxide is well absorbed by the gastrointestinal tract and is eliminated in urine at 45% of the dose in unchanged form, and the remaining being: glucuron-conjugated tiadenol-disulfoxide (10%), S-oxidized metabolites (15%) and sulfoxidized carboxylic metabolites (15%). The compound is well tolerated both in mice and rats. The results of this comparative study demonstrate that: 1. tiadenol-disulfoxide is a substance with promising hypolipidemic properties; 2. tiadenol-disulfoxide is largely responsible for the hypolipidemic activity of tiadenol; 3. hepatomegaly consequent to tiadenol administration is the consequence of the response of the liver cell to the increased functional demand of the mixed function oxidase (MFO) system involved in the metabolism of the drug; 4. peroxisomal enzyme activities induction observed with both drugs at non-pharmacological doses does not play any role in their hypolipidemic action and is not associated with hepatomegaly.

Analysis of tiadenol in human plasma by capillary gas chromatography with electron capture detection.

A sensitive and specific method for the quantitative determination of tiadenol in human plasma is described. After addition of the internal standard, both compounds were quantitatively extracted into chloroform and then derivatized with heptafluorobutyric anhydride (the structures of both derivatives were confirmed by electron impact mass spectrometry). Quantitation was achieved by capillary gas chromatography, using a (63) Ni-electron capture detector. Linearity was observed in the concentration range 5-100 ng ml(-1) and the minimum concentration of tiadenol detectable in plasma was 2.0 ng ml(-1). The method was successfully applied to plasma specimens collected from healthy human volunteers following a single oral administration of 800 mg of tiadenol.

Conformationally restricted congeners of hypotensive and platelet aggregation inhibitors: 6-aryl-5-methyl-4,5-dihydro-3(2H)-pyridazinones derived from 5H-indeno[1,2-c]pyridazine.

A number of 7-amino and 7-acylamino substituted 4,4a-dihydro-5H-indeno[1,2-c]pyridazin-3-ones have been synthesized as rigid congeners of hypotensive 6-aryl-5-methyl-4,5-dihydro-3(2H)-pyridazinones and tested as antihypertensive, antithrombotic, antiulcer, and antiinflammatory agents. Unlike the previously described 7-cyano derivative, which displayed only antiinflammatory action, the new series exhibited significant antihypertensive and antithrombotic properties. In this respect, the 7-amino (2b) and the 7-acetylamino (2c) derivatives were found to be the most potent and long lasting in reducing the blood pressure in spontaneously hypertensive rats and in protecting mice from the induction of thrombosis. These compounds, as well as the 7-(2-chloropropionyl) derivative 2d, also exhibited antiinflammatory activity; in addition, 2c,d were highly effective in inhibiting indomethacin-induced ulcers in the rat.

Comparison of reduced glutathione with 2-mercaptoethane sulfonate to prevent cyclophosphamide-induced urotoxicity.

Since the usefulness of high-dose cyclophosphamide is often limited by hemorrhagic cystitis, and the use of sulfhydryl-containing compounds prevents this side effect, this study was carried out to compare the uroprotective efficacy of 2-mercaptoethane sulfonate (MESNA) with that of reduced glutathione. In experimentally cyclophosphamide-induced urotoxicity in mice, the protective efficacy and potency of these thiols were similar, since both agents afforded complete protection in the same dose range. This finding suggests that these compounds are suitable sources of urinary thiols. Although the rationale for the clinical use of these protectors is similar, glutathione may have therapeutic advantages over MESNA because of a wider margin of safety and multiple protective actions displayed by the tripeptide thiol.

Metabolism of the hypolipidemic agent tiadenol in man and in the rat.

The metabolism of the hypolipidemic agent 1,10-bis(hydroxyethylthio)decane (tiadenol, Eulip) has been studied in vivo in man and in the rat and in vitro in the rat. Following oral administration, in both species tiadenol was completely absorbed, extensively metabolized by the liver and more than 95% of the dose was eliminated in this form via kidneys within 48 h. Insignificant was the excretion of the unchanged drug in urine (approximately 1%) as well as that of its metabolites in the feces. 8 metabolites were isolated from human or rat urine and their structures were elucidated by means of electron impact, field desorption and positive and negative fast atom bombardment mass spectrometry. Both in man and in the rat the main metabolic pathway was the oxidation of the thioether sulfur, followed by oxidation or conjugation of the primary alcohol group(s). The urinary excretion of S-oxidized metabolites and sulfoxidized carboxylic metabolites accounted for 75% of the dose and that of S-oxidized conjugated metabolites for 20%. Rat in vitro studies showed that hepatic microsomal cytochrome P-450-dependent monooxygenase catalyzes the S-oxidative pathway, which governs the in vivo elimination of the drug in both species. Thus cytochrome P-450 is the key enzyme in the hepatic detoxification of tiadenol.

Determination of bezafibrate in human plasma and urine by high-performance liquid chromatography.

A selective and time-saving high-performance liquid chromatographic method to assess bezafibrate plasma and urine levels is described. Bezafibrate is extracted from plasma matrix using diethyl ether, after acidification with hydrochloric acid. The urine samples are directly analysed, after dilution with the mobile phase. The method is used to assess bezafibrate plasma and urine levels in man, after administration of therapeutic doses of bezafibrate. The results obtained are in agreement with previously published data.