Effects of nefiracetam (DM-9384), a pyrrolidone derivative, on brain monoamine systems.

The pyrrolidone cyclic gamma-aminobutyric acid (GABA) derivative nefiracetam [DM-9384; N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide] has been shown to enhance acquisition and ameliorate amnesia in different learning tasks in rodents. In the present study, the effects of nefiracetam on the brain monoamine systems were studied. Male, adult Sprague-Dawley rats were treated with nefiracetam in single doses (0, 1, 3, 10, 30 or 100 mg/kg, p.o.) and analyzed after 1 and 4 hr, or treated by daily doses (0, 1, 3, 10 or 30 mg/kg, p.o.) for 2 weeks. In general, no or only weak effects were observed on tissue monoamine levels, either following acute or 14 days treatment with nefiracetam. Acute administration of intermediate doses of nefiracetam induced minor increases in dopamine (DA) and homovanillic acid (HVA) tissue levels in the striatum, while hypothalamic 3,4-dihydroxyphenylacetic acid (DOPAC) decreased at 1 hr. Noradrenaline (NA) and serotonin (5-HT) levels increased in some regions after higher doses of nefiracetam. Increases in 5-hydroxyindoleacetic acid (5-HIAA) were also seen at 4 hr, but only after the 3 mg/kg dose. Minor decreases of HVA and DOPAC levels were seen in some regions after treatment with various doses of nefiracetam for 14 days, while an increase in 5-HT levels was observed occasionally. Using in vivo microdialysis in freely moving animals, no significant effects on extracellular levels of HVA, DOPAC and 5-HIAA in the striatum or of HVA, DOPAC and NA levels in the dorsal hippocampus were seen after acute administration of nefiracetam. On the other hand, extracellular hippocampal 5-HIAA levels decreased by 20% after the 1 and 3 mg/kg doses. Nefiracetam, in a concentration range of 1 nM to 10 microM, did not affect the in vitro synaptosomal uptake of [3H]NA and [3H]5-HT in the cortex or of [3H]DA in the striatum. Taken together, nefiracetam appears to exert minor, regionally restricted and not dose-dependent effects on the monoamine systems following either acute or repeated administrations in normal rats. A direct or indirect, possible GABA-mediated, influence of nefiracetam may underlie the modest changes seen on monoamines. The cognitive-enhancing action of nefiracetam does not seem to be related to effects on presynaptic monoamine functions.

Inhibitory effect of perindopril, a novel angiotensin-converting enzyme inhibitor, on neointima formation after balloon injury in rats and cholesterol-fed rabbits.

We investigated the effect of perindopril, a novel angiotensin-converting enzyme (ACE) inhibitor, on neointima formation in vessel walls after balloon injury in rats (carotid artery) and cholesterol-fed rabbits (thoracic aorta). Continuous treatment with perindopril significantly reduced neointima formation in both species, as compared with the control group: intima/media (I/M) ratio (rats -62%; p < 0.001; rabbits -25%, p < 0.05); neointima area (rats -65%, p < 0.01; rabbits -24%, p < 0.05). These changes involved reduction of intimal smooth muscle cells (SMC) in rats and of macrophages in rabbits. Furthermore, perindopril also significantly decreased ACE activity in both aortic tissue and serum [11.38 +/- 0.87 vs. 34.93 +/- 6.44 pmol His-Leu (HL)/mg/min (p < 0.01) and 2.79 +/- 0.28 vs. 38.50 +/- 5.41 pmol HL/mg/min (p < 0.001), respectively], aortic contraction evoked by angiotensin I (AI) and mean blood pressure (BP, 84.9 +/- 3.5 vs. 109.3 +/- 3.8 mm Hg, p < 0.001) as compared with control values. These results indicate that perindopril may reduce neointima formation by suppressing the aortic renin-angiotensin system (RAS). These findings indicate that perindopril may be capable of preventing restenotic lesions after angioplasty in humans [corrected].

Nonlinear pharmacokinetics of DQ-2556, a new 3-quaternary ammonium cephalosporin antibiotic, in rats caused by non-Michaelis-Menten type, dose-dependent renal clearance.

The pharmacokinetics and its dose dependency of a new cephalosporin, DQ-2556, were studied in the rat and rabbit. The pharmacokinetics of the compound in the rat was linear up to a dose of 300 mg/kg; however, at a dose of 1200 mg/kg, renal clearance decreased dramatically and the normalized area under the blood concentration-time curve increased remarkably. On the other hand, there were no dose-dependent changes in tissue/serum concentration ratios, serum protein binding, red cell binding, and the distribution of DQ-2556. In the rabbit, the pharmacokinetics of DQ-2556 was linear even up to a dose of 1200 mg/kg and no unusual pharmacokinetic behavior was observed. The renal clearance experiments demonstrated that DQ-2556 is excreted by glomerular filtration. It was also shown that the glomerular filtration rate (GFR) remained constant up to a dose of 1000 mg/kg of DQ-2556 in the rat, whereas the GFR decreased by 95.1% at a dose of 1200 mg/kg. The coincidence of dose relationship and species difference in the disorder of GFR and renal toxicity suggests that the change of pharmacokinetics of DQ-2556 was caused by its renal toxic effects at very high dose.

Effects of nefiracetam on deficits in active avoidance response and hippocampal cholinergic and monoaminergic dysfunctions induced by AF64A in mice.

The effects of nefiracetam [DM-9384; N-(2,6-dimethyl-phenyl)-2-(2-oxo-pyrrolidinyl)acetamide] and of phosphatidylcholine on a step-up active avoidance response, locomotor activities and regional brain cholinergic and monoaminergic neurotransmitters in AF64A-treated mice were investigated. Intracerebroventricular (i.c.v.) injection of AF64A (ethylcholine mustard aziridinium ion; 8 nmol/ventricle) impaired acquisition and retention of the avoidance task, and increased vertical and horizontal locomotor activities. Regional levels of acetylcholine, noradrenaline, 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were significantly decreased and homovanillic acid (HVA) levels were increased in the hippocampus but not in the septum, cerebral cortex or striatum of AF64A-treated animals. Administration of nefiracetam (3 mg/kg, p.o.) twice daily for 9 days to AF64A-treated animals ameliorated the deficit in active avoidance response in addition to attenuating the increase in locomotor activities. In parallel with these behavioural effects, nefiracetam reversed AF64A-induced alterations in the hippocampal profiles of cholinergic and monoaminergic neurotransmitters and their metabolites. In contrast, administration of phosphatidylcholine (30 mg/kg, p.o.) twice daily for 9 days had no significant effect on the deficit in active avoidance response, despite significantly reversing the decrease in acetylcholine levels in the hippocampus. These results indicate that the effects of nefiracetam on AF64A-induced behavioural deficits are probably due to its ability to facilitate both cholinergic and monoaminergic neurotransmitter systems.

Single- and multiple-dose pharmacokinetics of nefiracetam, a new nootropic agent, in healthy volunteers.

The pharmacokinetic profile of nefiracetam (N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide), a new nootropic agent, was studied in healthy Japanese male volunteers. Nefiracetam was administered orally at doses of 10-200 mg in the single-dose studies, and at doses of 200 mg three times a day for seven days in the multiple-dose study. An HPLC method was used to determine the concentrations of nefiracetam in serum, urine and faecal samples. Linear kinetic behaviour was obtained after single oral administration. Serum concentrations of nefiracetam reached maximum values (Cmax) within 2 h for all dosage groups, and declined monophasically after Cmax with half-lives of 3-5 h. The area under the concentration-time curve (AUC infinity) and Cmax were linearly related to the dose. The apparent clearance (CL) values were 94.4-140.3 mL min-1. Urinary excretion of nefiracetam was independent of the administered dose, and less than 10% of the dose was recovered in urine as the unchanged form within 24 h after administration. Renal clearance (CLR) did not change significantly as dose increased from 10 to 1200 mg. Faecal excretion of nefiracetam was less than 0.1% of the dose up to 24 h after a 300 mg oral dose. Food intake delayed the absorption of nefiracetam but did not significantly modify its pharmacokinetics. No clinically significant accumulation of nefiracetam in the body was observed during and after multiple doses.

Metabolic disposition of DQ-2556, a new cephalosporin, in rats, rabbits, dogs, and monkeys.

The metabolic disposition of DQ-2556 was studied in rats, rabbits, dogs, and monkeys after an intravenous administration of 20 mg of 14C-labeled drug per kg of body weight. The serum data were analyzed by the two-compartment open model. The mean half-lives for the drug in serum at excretion phase were 18.1, 54.4, 21.8, and 63.6 min in rats, rabbits, dogs, and monkeys, respectively. The volume of distribution and total body clearance ranged from 0.18 to 0.30 liter/kg and 0.065 to 0.45 liter/h/kg, respectively. This compound was distributed to the tissues rapidly and well, especially to the kidney, trachea, liver, thyroid, skin, and lung. Tissue concentrations declined rapidly in a few hours and then very slowly. However, no accumulation was observed in any tissues. The results of a protein-binding study by ultracentrifugation indicated that DQ-2556 was 20 to 30% bound to serum proteins in animals and its affinity was low. Almost 90% of the administered radioactivity was excreted into urine in all species. Biliary excretion in rats was 3.1% of the dose. Nearly 70% of the dose or more was excreted into urine as unchanged drug, and the amounts of urinary metabolites were small except in rabbits, in which substantial amounts of polar metabolites were detected.

Species-related stereoselective disposition of ofloxacin in the rat, dog and monkey.

1. The stereoselective disposition of ofloxacin (OFLX) was studied in rats, dogs and monkeys after oral administration of racemic OFLX. 2. In rats serum concentrations of (R)-(+)-OFLX were much greater than those of (S)-(-)-OFLX, which is the active form of OFLX. In monkeys, by contrast, serum concentrations of (S)-(-)-OFLX predominated over (R)-(+)-OFLX levels. In dogs there were no differences in AUC or Cmax between the enantiomers. Thus, there exists a species-related difference in the stereoselective disposition of OFLX. 3. In rats the stereoselective differences were mainly due to stereoselective glucuronidation; OFLX is hardly metabolized in dogs, monkeys and humans. 4. In monkeys the AUC of (S)-(-)-OFLX was increased by co-administration of the (R)-(+)-form, indicating that the stereoselectivity of OFLX disposition in monkeys may be caused by competition between the enantiomers for renal excretion, especially for renal tubular secretion.

Effects of DM-9384, a pyrrolidone derivative, on ischemia-induced changes in the central monoamine systems.

Alterations in brain tissue levels of monoamines and monoamine metabolites were studied in gerbils 60 min after cerebral ischemia induced by 10 min carotid ligation after pretreatment with the antiischemic drug DM-9384 (1, 3, 10, 30 mg/kg, PO). The DA levels decreased in striatum after the ischemia, while cortical and hippocampal DA levels increased. The DOPAC levels increased in cortex, but were essentially unaffected in other regions. The HVA levels increased in all forebrain regions studied. NA levels decreased in hippocampus and superior colliculus, while a general increase in MHPG levels was seen. Decreases in 5-HT levels were seen in all forebrain regions except cortex. The 10 mg/kg and 30 mg/kg doses of DM-9384 counteracted the decrease in striatal 5-HT and hypothalamic MHPG/NA ratio, respectively. Thus pretreatment with DM-9384 exerted minor protective effects on the alterations induced in monoamine systems by transient forebrain ischemia.

Stereoselective glucuronidation of ofloxacin in rat liver microsomes.

The stereoselective glucuronidation of ofloxacin [(+/-)-OFLX], a new quinolone antibacterial agent, was studied in vitro using rat liver microsomes. OFLX glucuronidation exhibited Michaelis-Menten kinetics in rat liver microsomes. Stereoselective glucuronidation of the optical enantiomers occurred. S-(-)-OFLX glucuronide was produced 7-fold more than R-(+)-OFLX glucuronide with little or no difference in the values of KM of the enantiomers. The value of Vmax/KM for the glucuronide conjugate of S-(-)-OFLX was 8-fold greater than for the conjugate of R-(+)-OFLX. These results demonstrate that OFLX undergoes stereoselective glucuronidation in vitro. Moreover, we studied the in vivo interaction between enantiomers of OFLX in rats to clarify the effects of R-(+)-OFLX on the metabolism and disposition of S-(-)-OFLX. When the racemate [(+/-)-OFLX (20 mg/kg)] or single enantiomer [S-(-)-OFLX (10 mg/kg)] is administered iv to the rat, the serum concentrations of S-(-)-OFLX were higher after racemate administration than those after enantiomer administration, although the dose of S-(-)-OFLX was identical in both cases. These results indicate that R-(+)-OFLX may compete with S-(-)-OFLX in the in vivo glucuronidation. Furthermore, the results of the enantiomeric inhibition study showed that R-(+)-OFLX competitively inhibited S-(-)-OFLX glucuronidation in vitro with a Ki value of 2.92 mM.

Biotransformation of a new pyrrolidinone cognition-enhancing agent: isolation and identification of metabolites in human urine.

1. The metabolites of N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide (DMPPA; MH-1), in the urine of human volunteers have been investigated. 2. Ten metabolites together with the unchanged drug (MH-1) were isolated by h.p.l.c. and identified by n.m.r. and mass spectrometry as: three metabolites hydroxylated in the pyrrolidine ring of MH-1 (MH-2, MH-3 and MH-4), three metabolites hydroxylated in the dimethylphenyl ring of MH-1 (MH-6, MH-7 and MH-8), N-[(2,6-dimethylphenylcarbamoyl)methyl]-4-hydroxybutyrylamide++ + (MH-5), N-[(2,6-dimethylphenyl-carbamoyl)methyl]succinamic acid (MH-9), the 3-O-sulphate of MH-6 (MH-10) and the 3-O-sulphate of N-(2,6-dimethyl-3-hydroxyphenyl)-2-(5-hydroxy-2-oxo-1-pyrrolidinyl)aceta mide (MH-11). 3. DMPPA was extensively metabolized. The principal metabolic transformations were hydroxylation of the pyrrolidine ring at the C5 carbon followed by oxidative C-N cleavage, and hydroxylation of the phenyl ring followed by sulphate conjugation.

Simple method for determination of the cephalosporin DQ-2556 in biological fluids by high-performance liquid chromatography.

A sensitive method for the determination of DQ-2556 by high-performance liquid chromatography was established. The limits of detection for serum and urine were 0.1 and 2 micrograms/ml, respectively. Two clean-up procedures for serum samples were developed. In the first, deproteinization with 10% trichloroacetic acid was used and the recovery was 68.5%. In the other, ultrafiltration under acidic conditions was employed and the recovery was 85.1%. The former procedure is economical but complicated, whereas the latter is simple and labour-saving, but a special ultrafiltration tube is required. This situation offers a flexible choice, depending on the conditions of the laboratory.

Stereoselective metabolic disposition of enantiomers of ofloxacin in rats.

1. Stereoselective metabolic disposition of ofloxacin (OFLX) was studied in rats after oral administration of S-(-)-14C-OFLX and R-(+)-14C-OFLX at a dose of 20 mg/kg. 2. Radioactivity of the S-(-)-isomer was eliminated from blood much faster than that of the R-(+)-isomer. Marked differences in pharmacokinetic parameters exist between the enantiomers; the half life and AUC values of R-(+)-OFLX were greater than those of S-(-)-OFLX. Enantiomeric differences were also seen in the excretion of radioactivity, especially in biliary excretion. 3. 31.3 and 7.4% dose were excreted in the 8 h bile as ester glucuronides after oral administration of S-(-)- and R-(+)-OFLX, respectively. The enantiomeric difference in biliary excretion may be caused by stereoselective glucuronidation of S-(-)-OFLX to the ester glucuronide. 4. The metabolite pattern in serum and urine showed that the ester glucuronide of S-(-)-OFLX was more predominant than that of R-(+)-OFLX. 5. The stereoselective ester glucuronidation of the S-(-)-isomer in rats may induce significant differences in the pharmacokinetic parameters of S-(-)- and R-(+)-OFLX.

Determination of tranexamic acid in human serum by high-performance liquid chromatography using selective pre-column derivatization with phenyl isothiocyanate.

A high-performance liquid chromatographic method for determination of tranexamic acid in human serum using a selective derivatization has been developed. Tranexamic acid in the sample was allowed to react with phenyl isothiocyanate to form the phenylthiocarbamoyl derivative. Interfering alpha-amino acids in the sample were eliminated by selective derivatization to phenylthiohydantoin derivatives by acid treatment of the phenylthiocarbamoyl derivatives followed by solvent extraction. Then, the sample was analysed by conventional high-performance liquid chromatography with ultraviolet detection. The limit of detection of this method for serum sample was 0.2 micrograms/ml at a signal-to-noise ratio of 2. This method gave results comparable with those obtained by amino acid analysis (regression line: y = 0.4531x-0.02596, r = 0.9998, n = 21).

High-performance liquid chromatographic determination of a new nootropic, N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide, in human serum and urine.

A high-performance liquid chromatographic method for the determination of a novel nootropic agent, N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide (DM-9384, I), in human serum and urine has been developed. Compound I and the internal standard were extracted with chloroform from alkalinized serum and urine, and the organic layer was evaporated to dryness. The residue was chromatographed on a Nucleosil 7C18 reversed-phase column using 1/15 M potassium dihydrogen-phosphate-acetonitrile (7:3, v/v) as a mobile phase. Quantitation was achieved by monitoring the ultraviolet absorbance at 210 nm. The response was linear (0-2114.0 ng/ml) and the detection limits were 30 ng/ml for serum samples and 50 ng/ml for urine samples. The utility of the assay was demonstrated by determining compound I in serum and urine samples from three healthy male subjects receiving an oral dose of 30 mg of the drug. This method is satisfactorily sensitive and accurate, and is applicable for pharmacokinetic studies of I in humans.

Effect of new quinolones on drug-metabolizing enzyme system of rat hepatic microsomes.

Effects of repeated oral administration of new quinolones, ofloxacin, enoxacin and norfloxacin, once daily for 7 days, on the drug-metabolizing enzyme system of rat hepatic microsomes were studied in comparison with that of phenobarbital, a potent inducer of cytochromes P-450. Treatment of phenobarbital at the oral dose of 120 mg/kg induced significant increases in the contents of cytochrome P-450, cytochrome b5 and NADPH-cytochrome P-450 reductase and in the activity of ethoxycoumarin O-deethylase, and significant decreases in the activities of benzphetamine N-demethylase and aniline hydroxylase. However, ofloxacin, enoxacin and norfloxacin at the oral dose levels of 80 and 320 mg/kg showed no significant effect on the content of each constituent of the drug-metabolizing enzyme system, and the three enzyme activities. Thus, it is concluded that new quinolones including ofloxacin have no ability to induce a cytochrome-P-450-dependent monooxygenase system.

Lack of effect of ofloxacin on theophylline pharmacokinetics in rats.

Effect of ofloxacin, a new quinolone antibacterial agent, on the pharmacokinetics of theophylline was studied in rats in comparison with that of enoxacin and cimetidine. Ofloxacin by pretreatment with five oral doses of 50 mg/kg did not increase serum concentrations of theophylline (5 mg/kg, i.v. single) and showed no significant effect on total body clearance, serum half-life (T1/2) and AUC of theophylline, while enoxacin by the same pretreatment increased significantly serum theophylline concentrations and resulted in significant effect on all the pharmacokinetic parameters. Coadministration of ofloxacin (80 mg/kg, p.o. twice) did not induce a significant effect on the pharmacokinetic parameters of theophylline at repeated doses (50 mg/kg, i.v., twice daily for 3 days). On the contrary, coadministration of enoxacin and cimetidine at the same dose as ofloxacin remarkably increased serum concentrations of theophylline at the same repeated doses, and caused a significant decrease in clearance and an increase in T1/2 and AUC. The three drugs had no influence on rat serum protein binding of theophylline. Ofloxacin exhibited a weak inhibitory effect on rat hepatic microsomal cytochrome P-450-dependent monooxygenases, whereas enoxacin and cimetidine induced a significant inhibition of the enzymes. Thus, it is concluded that ofloxacin has no significant effect on the pharmacokinetics of theophylline in rats, and that enoxacin raises serum theophylline concentrations and results in a significant effect on the theophylline pharmacokinetics by inhibition of the hepatic microsomal monooxygenases in rats.

Isolation and identification of metabolites of ofloxacin in rats, dogs and monkeys.

Metabolites of (+/-)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H -pyrido [1,2,3-de][1,4]benzoxazine-6-carboxylic acid (ofloxacin) in excreta of rats, dogs and monkeys after oral administration of 14C-ofloxacin (20 mg/kg) were isolated and identified. Three metabolites of ofloxacin were detected in the excreta of all three species, and identified by t.l.c., u.v., n.m.r. and mass spectrometry as follows: M-1, ester glucuronide of ofloxacin; M-2, unchanged ofloxacin; M-3, (+/-)-9-fluoro-2,3-dihydro-3-methyl-10-(1-piperazinyl)-7-oxo-7H-pyrido [1,2,3-de][1,4]benzoxazine-6-carboxylic acid (desmethyl ofloxacin); M-4, (+/-)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H -pyrido [1,2,3-de][1,4]-benzoxazine-6-carboxylic acid piperazine-4-oxide (ofloxacin N-oxide). It is concluded that ofloxacin is metabolized by O-acyl glucuronidation, N-demethylation and N-oxidation.

Chemotherapeutic efficacy of ofloxacin on renal and subcutaneous infection models with Staphylococcus aureus in mice.

Ofloxacin, a new pyridone-carboxylic acid derivative, was evaluated in descending nephritis and subcutaneous abscess models with Staphylococcus aureus in mice in comparison with norfloxacin. Descending nephritis was produced by intravenous injection of S. aureus 39 (MIC 0.78 microgram/ml for ofloxacin and 3.13 micrograms/ml for norfloxacin). Subcutaneous abscess was established by subcutaneous injection of soft agar containing S. aureus 56230 (MIC 0.39 microgram/ml for ofloxacin and 1.56 micrograms/ml for norfloxacin). Three days after infection, the lesions of both models were characterized by purulent inflammation accompanied with massive infiltration of neutrophils and bacterial multiplication. The animals were treated twice a day orally with each compound for 4 consecutive days, and subjected to bacteriological examination 18 h later. In the renal model, the 50% effective doses calculated on the basis of clearance of bacteria from kidneys were 38.4 mg/kg for ofloxacin and greater than 100 mg/kg for norfloxacin. In the subcutaneous model, the 50% effective doses based upon 90% reduction of viable bacteria as compared with untreated controls were 25.2 mg/kg for ofloxacin and greater than 100 mg/kg for norfloxacin. The excellent efficacies of ofloxacin in both infection models are attributed to its high oral absorbability and tissue distribution.