Application of LC-MS analysis to the characterisation of the in vitro and in vivo metabolite profiles of RGH-1756 in the rat.

RGH-1756, 1-(2-methoxy-phenyl)-4-(4-[4-(6-imidazol[2,1-b] thiazolyl)-phenoxy]-butyl-4-(14)C)-piperazine dimethane is a novel atypical antipsychotic drug candidate of Gedeon Richter Ltd. The metabolic pathways of the compound have been investigated by profiling the metabolites present in plasma, bile, and faeces samples of rats treated with (14)C-RGH-1756. The metabolites formed in vitro by rat liver microsomes have also been analysed. Good separation of the compounds has been achieved by gradient HPLC method on Zorbax/Bonus RP-C18 column. Radiometry and mass spectrometry have been applied to detect and characterise the metabolites. The metabolite formed by oxidative cleavage of the chain at the carbon atom adjacent to the piperazine nitrogen has been identified as the major plasma metabolite. Glucuronide conjugate of hydroxy-RGH-1756 has been found as one of the main metabolites excreted in the bile where the unchanged compound has not been detected.

An HPLC/UV method for the determination of RGH-1756 in dog and rat plasma.

RGH-1756 (1-(2-methoxy-phenyl)-4-(4-[4-(6-imidazo[2,1-b]-thiazolyl)-phenoxy]-butyl)-piperazine dimethansulphonate) is a novel atypical antipsychotic candidate of Gedeon Richter Ltd. A new HPLC method has been developed and validated for the quantitative determination of RGH-1756 in dog and rat plasma. The compound and the internal standard are extracted from the biological samples by a simple and fast liquid--liquid extraction method, using 1-chlorobutane. The recovery for RGH-1756 is about 90%. The extracts are analyzed by reversed phase HPLC (column: Supelcosil-LC-18-DB 250*4.6 mm, 5 microm, eluent:acetonitrile:methanol:0.2 molar ammonium-acetate 40:25:35, lambda=254 nm). The assay is specific for RGH-1756. The standard curves are linear in the range between 10 and 2000 ng ml(-1). The overall precision (expressed as CV%) and accuracy (expressed as bias%) of quality controls and calibration standards are within 15%. The validated lower limit of quantification is 10 ng/ml. No indications have been found for possible instabilities of RGH-1756 in plasma, in the extraction solvent, or after repeated thawing-freezing cycles. The method has been succesfully applied for the bioavailability studies of RGH-1756 in the two animal species. In these studies results of the inprocess method validation have shown the reliability of the method, too. CV% of quality controls in the rat study has been found between 7.4 and 10.0%, in the dog study between 4.1 and 12.5%. The bias has ranged from 0.4 to 3.8% and from -4.5 to 1.2% in the rat and dog study, respectively.

Role of sodium channel inhibition in neuroprotection: effect of vinpocetine.

Vinpocetine (ethyl apovincaminate) discovered during the late 1960s has successfully been used in the treatment of central nervous system disorders of cerebrovascular origin for decades. The increase in the regional cerebral blood flow in response to vinpocetine administration is well established and strengthened by new diagnostical techniques (transcranial Doppler, near infrared spectroscopy, positron emission tomography). The latest in vitro studies have revealed the effect of the compound on Ca(2+)/calmodulin dependent cyclic guanosine monophosphate-phosphodiesterase 1, voltage-operated Ca(2+) channels, glutamate receptors and voltage dependent Na(+)-channels; the latest being especially relevant to the neuroprotective action of vinpocetine. The good brain penetration profile and heterogenous brain distribution pattern (mainly in the thalamus, basal ganglia and visual cortex) of labelled vinpocetin were demonstrated by positron emission tomography in primates and man. Multicentric, randomized, placebo-controlled clinical studies proved the efficacy of orally administered vinpocetin in patients with organic psychosyndrome. Recently positron emission tomography studies have proved that vinpocetine is able to redistribute regional cerebral blood flow and enhance glucose supply of brain tissue in ischemic post-stroke patients.

High-performance liquid chromatographic method for the determination of RGH-5002 in human, rabbit and rat plasma.

A sensitive, accurate and reproducible high performance liquid chromatography (HPLC) procedure for the analysis of RGH-5002 in biological fluids was developed. After a single step liquid-liquid extraction at pH 9 using n-hexane, RGH-5002 and internal standard were eluted from a Hypersil Si column with 5 mM ammonium acetate-methanol-acetonitrile (0.5:45:50 v/v/v) at 28 degrees C. The method could accurately detect 5 ng ml-1 of RGH-5002.

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.

In vitro degradation of thymopoietin32-35 in human, dog and rat plasma.

TP4 (Arg-Lys-Asp-Val) is a synthetic immunomodulatory tetrapeptide of Chemical Works of Gedeon Richter Ltd. The aim of this study was to give comparative data on the in vitro degradation of 14C-TP4 in plasma of different species at two TP4 concentrations to allow suitable extrapolation of preclinical data to human system. The results show that the degradation was very fast in each plasma and varied by the species and concentrations studied. In rat, dog and human plasma for initial concentration of 80 micrograms/ml and 800 micrograms/ml the apparent half-life values proved to be 1.4, 1.8, 2.7 and 3.9, 6.3, 13.6 minutes, respectively. The most susceptible breakdown point was the peptide bond between Arg and Lys. The correlation between TP4 degradation and arginine formation was demonstrated by a straight line with a correlation coefficient of 0.99565 and a slope close to -1.

Pharmacokinetic study of bisaramil in man.

Six healthy volunteers received an oral dose of 100 mg and an intravenous dose of 35 mg of bisaramil in a cross over study. Plasma concentrations were measured by HPLC. Bisaramil was eliminated from the plasma with a half life of 8.6 +/- 1.8 h and 9.0 +/- 4.1 h after iv. and oral administration, respectively. The mean total plasma clearance and volume of distribution were found to be 70 +/- 13.1 l/h and 864 +/- 204 l, respectively. The calculated oral bioavailability of bisaramil in tablets amounted to 56 +/- 20%.

The influence of gastrointestinal transit on drug absorption in healthy volunteers.

1. The effect of variability of gastric emptying and oro-caecal transit on the absorption of a multicomponent solution of frusemide, atenolol, hydrochlorthiazide and salicylic acid has been studied in six healthy subjects. Each subject was studied on five separate occasions: three times under basal conditions, once following metoclopramide and once following codeine pretreatment in an attempt to speed and slow transit respectively. 2. Inter-subject variability of gastric emptying, oro-caecal transit and the rate and extent of drug absorption was considerable. 3. The absorption of salicylic acid appeared rate-limited by gastric emptying but the rate and extent of frusemide, atenolol and hydrochlorthiazide absorption were unrelated to measures of gastric emptying or oro-caecal transit. 4. Codeine phosphate caused a two-fold delay in oro-caecal transit but did not influence gastric emptying while metoclopramide had no significant effect on either function. 5. Metoclopramide and codeine had no significant effect on the rate or extent of absorption of any of the study drugs. 6. Within the limits of this experiment, oro-caecal transit time did not appear to be an important determinant of frusemide, atenolol, hydrochlorothiazide or salicylic acid absorption. Other factors must account for the observed variability in drug absorption.

Effects of a non-absorbable osmotic load on drug absorption in healthy volunteers.

1. We have studied the effects of a non-absorbable osmotic load on the absorption of a multicomponent solution of frusemide, atenolol, hydrochlorothiazide and salicylic acid in six healthy volunteers. 2. Each subject was studied on up to four separate occasions. The drugs were administered in one of four solutions: a) a mannitol/electrolyte solution, b) a double-strength mannitol/electrolyte solution, c) a glucose/electrolyte solution and d) water. Lactulose or sulphasalazine were added as oro-caecal transit markers. Lactulose was included in the mannitol- and glucose-based solutions, adding a further non-absorbable osmotic load, and sulphasalazine was added to the water, adding little osmotic load. 3. The absorption of atenolol and hydrochlorothiazide was two- to three-times less from all lactulose-containing solutions than from the sulphasalazine-containing solution. The absorption of frusemide and salicylic acid was similar from all four solutions. 4. The largest non-absorbable osmotic load impaired the absorption of atenolol and hydrochlorothiazide most and the incorporation of glucose only partly restored absorption. 5. These results suggest that transmucosal water movement is an important determinant of atenolol and hydrochlorothiazide absorption but is less relevant for the absorption of frusemide and salicylic acid. Furthermore, these data demonstrate a previously unrecognised interaction between a commonly prescribed laxative--lactulose, and atenolol and hydrochlorothiazide.

High-performance liquid chromatographic method for the determination of RGH-5702 in plasma samples.

A quick and selective high-performance liquid chromatographic method has been developed for the determination of RGH-5702 in plasma samples. A simple one-step extraction is used followed by reversed-phase chromatography and UV detection. This method allowed the separation of the compound and internal standard within 7 minutes. Validation of the method was performed prior to the assay of samples and continued throughout the study. Acceptable accuracy and precision was achieved at all concentrations investigated. The quantitation limit was 20 ng/ml using 1 ml of plasma. The method has been applied to the analysis of plasma samples from toxicokinetic studies in dogs.