Influence of the N-acetylation polymorphism on the metabolism of talampanel: an investigation in fasted and fed subjects genotyped for NAT2 variants.

Talampanel is a 2,3-benzodiazepine-type allosteric (noncompetitive) AMPA-antagonist currently being developed as an orally active, broad-spectrum anticonvulsant. Here, a detailed study of its N-acetylation in humans is presented using plasma concentration data of both TLP and its N-acetyl metabolite obtained from healthy volunteers (n = 28) genotyped for N-acetyltansferase NAT2 isozymes. Plasma samples were obtained for up to 48 h after a single oral dose of 75 mg TLP both in fasted and in fed subjects. A perfect correspondence could be established between the phenotype inferred before the study from genotyping and that determined after the study by using plasma metabolite-to-parent molar ratios confirming that this route of metabolism is indeed mediated by NAT2. Analysis of the data has been performed using both noncompartmental analysis and a custom-built, unified parent-metabolite PK model, which incorporates three different acetylation rates according to the genotype-based classification of each subject as slow, intermediate, or fast acetylator to simultaneously fit plasma levels for both TLP and its metabolite. This suggest that for TLP in humans, (i) N-acetylation represents only a relatively small fraction of its total elimination (about one-fourth in fast acetylators and much less in slow acetylators), (ii) acetylation is about eight-twelve times faster in fast and three-six times faster in intermediate acetylators than in slow acetylators, and (iii) the N-acetyl metabolite is eliminated faster than the parent TLP.

A pharmacokinetic study of JOMO-tech in rats.

A pharmacokinetic study of 99mTc labelled JOMO-tech in rats (after intravenous administration of a dose of 20 microg/kg body weight) was conducted. JOMO-tech is a heterogeneous extract derived from Nocardia opaca cell walls. An excellent fitting of the three-compartmental disposition model was achieved. The first apparent elimination half-life was very short (t1/2alpha = 0.0572 +/- 0.01383 h) followed by longer second apparent elimination half-life (t1/2beta = 0.817 +/- 0.1922 h), whereas at late post-treatment time the third apparent elimination half-life (t1/2gamma = 21.7 +/- 2.1 h) proved to be long. The peak concentration in the blood extrapolated to t = 0 yielded 32.3 +/- 7.54 ngeq/ml, this being approximately 2-fold the amount of that measured in the 5th post-treatment minute (16.84 +/- 1.447 ngeq/ml). It was determined that the main route of excretion was renal. Up to the 48th post-treatment hour, 30.03 +/- 2.788% of the dose was excreted via the urine, and only 6.71 +/- 0.973% was excreted in the feces by the 7 rats evaluated. The amount of radioactivity detected in selected tissue samples (expressed in ngeq JOMO-tech/g wet tissue) decreased in the sequence liver > kidneys > lungs > blood > plasma. In the time period studied, the highest amount of the dose was found in the liver, whereas up to the 3rd post-treatment day a practically equivalent part of the dose was found in the excreta and in the liver.

Whole-body autoradiography and quantitative organ-level distribution study of deramciclane in rats.

The distribution of 3H-labelled deramciclane (EGIS-3886), a new 5-HT2 antagonist with anxiolytic activity, has been investigated by whole-body autoradiography and quantitative organ-level determination after intravenous and oral administration to male and female rats at a dose of 3 mg kg(-1). Pregnant dams were also studied, but by autoradiography only. In the autoradiographic study 32 organs were investigated, while in the quantitative organ-level study the radioactivity in 15 organs were determined. There are no sex differences in the distribution of deramciclane, absorption is rapid, elimination is comparatively fast, no specific organ is targeted, and the accumulation of the compound is very unlikely. Penetration of the blood-brain barrier was complete and extremely fast, a very important feature of a potential anxiolytic drug. There is no penetration of the foetus in pregnant dams. The study demonstrated that deramciclane has advantageous pharmacokinetic properties in rats.

Study of the bone marrow penetration of radioactivity after oral administration of radiolabelled girisopam (EGIS-5810) in mice.

Girisopam (EGIS-5810) is a potent anxiolytic compound. Recent in vitro studies with the substance, in Chinese hamster ovary cells, indicated dose-dependent mutagenic activity. At the same time, in ex vivo bone marrow micronucleus tests performed after treating CFLP mice with extreme oral doses (875, 1300 and 1750 mg/kg) no mutagenic activity could be observed at any of the dose-levels. On the basis of the above results, it seemed reasonable to study the absorption and distribution of radioactivity and particularly its bone marrow penetration after administering tritiated and 14C-labelled girisopam at the same doses as those applied in the micronucleus test. The animals were sacrificed 30 minutes, 2 and 24 hours after treatment and the radioactivity content of blood, plasma and bone marrow was determined. For whole body autoradiography studies, the animals were sacrificed at the same time points, however they were treated with tritium-labelled girisopam. The results indicated that the absorption of radioactivity from the gastro-intestinal tract of the animals started immediately. The samples collected had well measurable radioactivity even 30 minutes after treatment. At the same time, it was also evident, that, in spite of the high doses, the absolute amount of radioactivity was rather low. At both dose-levels, the radioactivity concentration was the highest in samples collected 24 hours after treatment. This results indicated extremely delayed absorption. The radioactivity level of bone marrow was practically the same as that measured in blood. The samples of animals treated with the high-dose had higher radioactivity content, however the increase was not linearly proportional to the dose. Disproportionality can probably be explained by delayed absorption. The whole body autoradiography was in good agreement with the results of quantitative determinations. This results confirmed the observations obtained by ex vivo micronucleus test. The radioactivity penetrated in the bone marrow resulting a long time exposure of the radioactivity without any mutagenic effect.

Distribution and elimination of RGH-5002 in rats.

In the present study distribution and elimination of RGH-5002--a new centrally acting muscle relaxant--were investigated in rats by using 14C-labelled compound. Whole-body autoradiography and quantitative determination of the radioactivity in various organs following single and repeated oral administration of [14C]RGH-5002 demonstrated extensive distribution of the drug with high levels in the gastrointestinal tract, kidneys, liver, endocrine and exocrine glands and lungs. Minimal accumulation was observed after repeated (8 days) administration. The same distribution characteristics were observed in both sexes. In pregnant rats radioactivity appeared in the placenta and fetal tissues. Elimination was investigated by measuring radioactivity in 24 h fractions of urine and faeces after single dose administration of the drug. The larger portion of radioactivity was excreted in the urine (81.67 +/- 1.61% of the dose). The faecal recovery was 11.12 +/- 1.19% of the administered dose. Approximately 80% of the excreted radioactivity was recovered within the first 24 hours.

Pharmacokinetic study on a new antiischaemic agent (BRLP-42).

In the present study the pharmacokinetics of BRLP-42--a new antiischaemic agent--was investigated in dogs and rats. Plasma concentrations were measured by HPLC. After intravenous application the curves can be characterized by a two-compartment open pharmacokinetic model. The central volume of distribution (Vcentr.) is large (1.07 +/- 0.14 l/kg in dogs and 2.74 l/kg in rats), the first elimination half-life (t1/2 alpha) is 5.47 +/- 1.67 min in dogs and 13.7 min in rats. These facts indicate rapid and large tissue distribution. The excretion and/or metabolic elimination of BRLP-42 resulted in short second elimination half-life (t1/2 beta = 41.45 +/- 2.34 min in dogs and 43.8 min in rats). After oral application high individual variability can be seen. This fact may be due to the different rate and/or extent of absorption process. The plasma level curves can be characterized by a one-compartment open pharmacokinetic model. The absorption seems to conceal the distribution phase of the kinetic curve. The absorption half-life was short (t1/2a = 17.36 +/- 5.90 min in dogs and 2.7 min in rats). The bioavailability was 40 +/- 8% in dogs and 28% in rats. The elimination half-life (t1/2e = 28.77 +/- 0.88 min in dogs and 30.1 min in rats) is connected dominantly with metabolic elimination and/or excretion of BRLP-42. In the cases of intravenous as well as oral administrations the plasma concentrations decreased under the limit of quantitation by 4-6 hours in dogs and 4 hours in rats after treatments.

Amphetamine-metabolites of deprenyl involved in protection against neurotoxicity induced by MPTP and 2'-methyl-MPTP.

The ability of 1-deprenyl to protect against the parkinsonian effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been attributed to the inhibition of conversion of MPTP to MPP+ (1-methyl-4-phenylpyridinium) catalyzed by MAO-B. We report here that deprenyl-treatment in mice has an additional neuroprotective element associated with the rapid metabolization of 1-deprenyl to 1-methamphetamine and 1-amphetamine. 1-Methamphetamine and 1-amphetamine inhibit MPP(+)-uptake into striatal synaptosomes prepared from rats. Post-treatment by 1-deprenyl, 1-methamphetamine, 1-amphetamine (at times when MPTP is no longer present in the striatum of mice) protects against neurotoxicity in C57BL mice by blocking the uptake of MPP+ into dopaminergic neurons, and even against the neurotoxicity induced by 2'CH3-MPTP, which is partly bioactivated by MAO-A. These findings may have clinical implications since deprenyl has recently been found to delay the progression of Parkinson's disease.

Methamphetamine protects against MPTP neurotoxicity in C57BL mice.

Methamphetamine (5 mg/kg) administered 30 min prior to each injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (3 x 30 mg/kg, at 24 h intervals) prevents the reduction of striatal levels of dopamine and its metabolites in C57BL mice. Methamphetamine and amphetamine inhibit the uptake of 1-methyl-4-phenylpyridinium (MPP+) by striatal synaptosomes of rats. A 30-min post-treatment with methamphetamine or amphetamine also prevents the MPTP-induced dopamine depletion, suggesting that their protective effect is related to the blockade of MPP+ uptake into dopaminergic neurons. Since amphetamine and methamphetamine are themselves neurotoxins at higher doses, this work demonstrated the protection against the actions of one neurotoxin by the administration of another.

[Pharmacokinetic studies of epervudine in rats using HPLC]. / Epervudine farmakokinetikai vizsgálata HPLC módszerrel patkányokon.

Pharmacokinetic properties of active substance of Hevizos ointment (Epervudine) were studied in rats after intravenous, oral and dermal applications. The animals received 10 mg/kg of Epervudine intravenously and orally. For checking of dermal absorption 220 mg of ointment (containing 0.8% of Epervudine) was applied. An HPLC method was developed for determination of Epervudine concentrations in serum. The method meets the requirements of precision and accuracy of the kinetic measurements (i.e. CV% is less than 20%) within the concentration range of 50-10,000 ng/ml. The mean serum concentration-time curve after i.v. administration can be characterized by a two-compartment open pharmacokinetic model. The first and second elimination half-lives (t1/2 alpha and t1/2 beta) are 0.14 and 0.31 hours, respectively. These values indicate fast distribution and elimination of compound studied. In the case of oral administration the absorption process conceals the fast distribution, so the mean serum concentration-time curve can be characterized by a one-compartment open pharmacokinetic model. The absorption of Epervudine starts practically prompt. The absorption half-life (t1/2a) is 0.11 hours. Highest serum concentrations were measured from 20 to 90 minutes after treatment. The elimination half-life (t1/2e) is 1.53 hours. The ratio of areas under serum level curves following oral and intravenous administration proves a good bioavailability (90%) of Epervudine. After dermal application Epervudine does not absorb.

Chromogen-substrate assay--as a tool for monitoring a new thrombin inhibitor.

The N-methyl-D-phenylalanyl-L-prolyl-arginine-aldehyde sulfate tripeptide-aldehyde (GYKI-14766) is an anticoagulant with specific thrombin inhibitor action. The molecule proved to be effective in rabbits, rats and dogs upon i.v. administration. Chromogen-substrate assay was developed for monitoring of biologically active tripeptide-inhibitor GYKI-14766 in plasma. The assay based on the inhibition of the active center of the thrombin enzyme, so it is suitable also for the assay of all those active metabolites which inhibit thrombin by a mechanism similar to the active parent compound. The chromogen substrate assay was performed in a range of 0.625-10 micrograms/ml GYKI-14766 in dog plasma. The assay was employed in pharmacokinetic study in dogs after i.v. administration. The data obtained in the chromogen-substrate assay were analyzed according to a one-compartment model. The major parameters of the plasma level studies were: D/V = 8.6 microEqv/ml t1/2 = 30.8 min AUC = 380 min microEqv/ml.

Pharmacokinetic and metabolism studies on girisopam by chromatographic and spectrometric methods in humans.

Girisopam possesses selective anxiolytic action without muscle relaxant and anticonvulsive activity. After a 100-mg oral dose of 14C-labelled girisopam to seven male subjects, the mean recovery of 14C radioactivity was 51% in urine and 33% in faeces. A high-performance liquid chromatographic method has been developed for studying girisopam in single-dose pharmacokinetic studies. The serum extract was chromatographed on a normal-phase column using a mobile phase of hexane-ethanol-diethyl ether (66:9:25, v/v) and ultraviolet detection at 235 nm. The recovery was 60% and the detection limit was 3 ng/ml, using 1 ml of serum. After a 20-min delay, girisopam is rapidly absorbed. After reaching a mean serum level of 178 ng/ml at a mean time of 2.0 h, the serum concentration of girisopam decreased with a mean elimination half-time of 22.2 h. The metabolites were separated by high-performance liquid chromatography, radio thin-layer chromatography and gas chromatography. Their structures were determined by liquid chromatography-mass spectrometry, mass spectrometry and gas chromatography-mass spectrometry. Their chemical structures were confirmed by comparison with synthesized reference compounds. The major urinary metabolites were 7-demethylgirisopam (I), 4'-hydroxygirisopam (II) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in conjugated form, and 4-carboxy-4-demethylgirisopam (V), a compound with an open-chain structure (VII) and traces of 4-demethyl-4-oxogirisopam (VIII) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in non-conjugated form. The metabolic profile in the serum consisted predominantly of the glucuronides of I, II and III. The non-conjugated metabolites were the metabolite with the open-chain structure (VII), III and V. Besides the parent compound, the faeces sample contained conjugates of I and II.

Radioimmunoassay for a new phenothiazine derivative and its application.

Fluphenazine-4-chlorophenoxy-isobutyrate ester, a new phenothiazine derivative was synthesized in the Institute for Drug Research Budapest. Radioimmunoassay was developed for the therapeutic monitoring of the drug level after intramuscular depot injection. The fluphenazine hapten was coupled to BSA by mixed-anhydride method. Antisera were produced to this conjugation in New-Zealand white rabbits and were tested for the antibody-titer. The specificity was tested by the cross-reaction with phenothiazine-analogues and other psychotropics. Strong cross-reaction was found with compounds possessing piperazine in side chain (trifluoperazine, perphenazine), but other psychotropic drugs did not react. Tritium-labelled trifluoperazine (spec. activity: 3.5 TBq/mmol) was used as a tracer in the radioimmunoassay. The detection limit was 75 pg with a CV of < 5% in 50 microliters plasma sample (equivalent to 1.5 ng/ml concentration) and a standard curve in the 3 ng/ml-50 ng/ml GYKI-22441 concentration range showed a CV of < 10%. Preliminary pharmacokinetic study was performed in Beagle dogs after intramuscular depot injection with GYKI-22441 in sesame oil in a dose of 0.1 mg/kg. The GYKI-22441 concentration of the plasma samples were measured by the RIA method during a 28-day interval after the treatment and was evaluated by the MultiCalc Immunoassay Data Management program (Pharmacia).

A new psychoactive 5H-2,3-benzodiazepine with a unique spectrum of activity.

The neuropharmacological effects of 1-(4-amino-phenyl)-4-methyl-7,8-dimethoxy-5H-2,3-benzodiazepine (GYKI 52 322) were investigated and compared with those of chlordiazepoxide and chlorpromazine. This novel 2,3-benzodiazepine displays neuroleptic activity in the apomorphine-climbing (ED50 = 1.15 mg/kg i.p.) and swim-induced grooming (ED50 = 6.9 mg/kg i.p.) tests in mice and it inhibits the conditioned avoidance response in rats (ED50 = 8.2 mg/kg i.p. and 9.8 mg/kg p.o.). However, it does not antagonize apomorphine-evoked vomiting in dogs; or stereotypy, hypermotility and turning in rats even at as high a dose as 50 mg/kg i.p. On the other hand it is active in the hole board test in mice (MED (minimal effective dose) = 0.5 mg/kg i.p.) and in the lick conflict assay in rats (MED = 5 mg/kg i.p.), indicating anxiolytic property. It shows antiaggressive effect in the fighting mice test (ED50 = 8.1 mg/kg p.o.) and the carbachol-rage procedure in cats (active at 10 mg/kg i.p.) According to the biochemical findings, this compound does not bind to the central dopamine receptors (IC50 greater than 10(-4) mol/l), but it shows affinity to the 5-HT1 receptors (IC50 = 7.1 x 10(-6) mol/l) and inhibits brain cAMP-phosphodiesterase (IC50 = 2.4 x 10(-5) mol/l). The substance causes no elevation of dopamine turnover and serum prolactin level suggesting fewer side effects. So the term "atypical neuroleptic agent" is proposed to characterize this molecule.