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The purpose of this work was to investigate the interest of the TEER measurement, a quite simple measure, as an evaluation of toxicity, in function of time and level of drug in the experimental medium, for different anticalcic agents. The extend of the increase and decrease of TEER, was evaluated by measuring the TEERmax and calculating the TEER50 respectively. Three groups of compounds could be therefore considered. The first, characterized by a quite short period of TEER increase, followed by an extremely rapid decrease (high values of TEER50), included perhexiline and prenylamine. The most toxic compounds, the second one, characterized by the smallest variations of TEER, included verapamil and diltiazem. This group was related with the less toxic compounds, the third group, represented by bepridil, characterized by dependant-dose TEERmax and TEER50. Therefore, the TEER measurement appeared as a quite simple approach of comparative toxicity of anticalcic agents.

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The purpose of the study was to go further into the transepithetial transport of bepridil, an anticalcic agent, through monolayer cells Caco-2, using a "dynamic model" including a transfer of inserts with Caco-2 cells into new wells, free of drug, at regular intervals, in order to simulate the blood flux. The state of cells was evaluated by measuring the transepithelial electrical resistance and the transport of bepridil was followed using a gas chromatography/mass spectrometry determination. This study exhibits the importance of the basolateral renewal both on the transport of bepridil and the maintenance of cells in a satisfactory state. Two elimination phases from the cell compartment seem to occur, with basolateral half lives respectively of 12.2 and 25.6 hours, probably linked with two kinds of cellular binding sites. This dynamic model permits the reflection and simulation of the slowness of the in vivo absorption of bepridil in the small intestine.

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The purpose of this work was to study transepithelial transport of bepridil, an anticalcic agent, through monolayer cells Caco-2, using two experimental media with different chemical components. For experimentation, the measure of the transepithelial electrical resistance (TEER) allowed us to evaluate the state of cells; and the quantities of bepridil have been quantified using a gas chromatography/mass spectrometry system. First, when using the medium alone, without bepridil, Caco-2 cell integrity is, at least, maintained for 8 h using both media. However, for 24-h studies, only the DMEMc medium, rich in essential nutriments, allowed cell integrity to be maintained. Then, with bepridil in HBSS medium, the TEER measurement showed a dose-dependent toxic effect of bepridil, whereas in the DMEMc medium, the toxic effect was only found for the highest dose (12 microg). This difference is probably related to the high binding of bepridil to proteins of the DMEMc medium, therefore minimising the concentration of the free compound. The kinetics of bepridil result from two phenomena: first, an immediate passage of a slight part of bepridil through the cell barrier and second, a high retention of most of the bepridil dose in the cell level. The transfer of bepridil from the apical to the basolateral compartment appears quantitatively and kinetically different using DMEMc or HBSS medium. The retention of the compound in the 'filter with Caco-2 cells' compartment is higher in DMEMc medium (60% at 3 microg) than in HBSS medium (46% at 3 microg), and bepridil entering the basolateral compartment is delayed in the DMEMc medium. This study exhibits the importance of the selected medium on results and interpretation of data and the predominance of DMEMc to study the transport of lipophilic compounds highly retained in cells.

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The biotransformation of several analogs of the anti-calcium agent bepridil was studied comparatively in liver cells isolated from one rat. Three types of metabolites were identified by mass spectrometry, resulting from three phase I reactions: hydroxylation, N-debenzylation and pyrrolidine ring opening. The amount of each bepridil analog untransformed after 18 h of incubation depended on its liver toxicity rather than on its concentration in the culture medium. The proportion of phase I metabolites identified remained constant regardless of toxicity. The difference delta c (in %) between the initial concentration of the analog tested and the sum of the concentrations of untransformed material and of identified metabolites decreased with the increasing hepatocyte toxicity. The analogs tested were responsible for the liver toxicity. The presence of substituents in different positions on the N-phenyl moiety increased liver toxicity; ortho-substituted analogs were more toxic than para- or meta-substituted ones.

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Interrelationships that might exist between potency, lipophilicity and cytotoxicity of the chemically diverse calcium antagonist group of drugs have been examined in the present study. The potency of 11 representative calcium antagonists in inhibiting KCl-induced contractions in rabbit isolated aortic rings and their relative lipophilicity was determined using reversed phase HPLC. Their cytotoxicity in rat hepatocyte primary cultures was also determined. Cytotoxicity failed to correlate with potency, except for the highly lipophilic, non-selective, diphenylalkylamines (DPAs), suggesting that cytotoxicity was not caused by blockade of plasmalemmal voltage-operated calcium channels. Cytotoxicity moderately correlated with relative lipophilicity, the most lipophilic drugs also being the most cytotoxic. Relative lipophilicity may partly determine the cytotoxicity and pharmacological potency of Ca++ antagonists in a broad sense, but this correlation was not valid in each individual chemical series. We suggest that the higher cytotoxicity of the DPAs is at least partly due to a greater incorporation of the drugs into the hepatocyte plasmalemma compared to compounds in other chemical classes investigated. Further studies are required to elucidate the particular cytotoxic mechanisms involved.

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A specific and highly sensitive method for the measurement of trazodone in plasma and brain of rat is presented. The compound and the internal standard were extracted from alkalinized samples with hexane and analysed by capillary gas chromatography with nitrogen-selective detection. The method was demonstrated to be accurate and precise. The limits of determination were 2 ng/ml for plasma and 24 ng/g for brain, which makes this procedure suitable for pharmacokinetic analysis.

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1. Beagle dogs were treated orally (10 mg/kg) with para-chloro-, para-fluoro- and para-methyl-phenylpiperazine derivatives, and urine was collected for 72 h after treatment. 2. Metabolites were extracted, converted into trimethylsilyl (TMS) derivatives and examined by g.l.c.-mass spectrometry. 3. The metabolites fall into two main groups, N-desphenylated metabolites, which result from N-desphenylation, and N-phenyl metabolites. 4. Two kinds of hydroxylated metabolites were found. Some lost the original para substituent (Cl, F or CH3); others retained it. 5. These results are consistent with the NIH shift reaction.

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CERM 3517 (mociprazine), a new anti-emetic compound, was administered orally to six beagle dogs at 10 mg/kg b.i.d. for four days. Unconjugated urinary metabolites were identified by GC-MS analysis against synthesized reference compounds, after solvent extraction, purification by TLC and concentration. Twenty one metabolites were identified indicating the following biotransformations: N-dephenylation followed by reactions on the exposed secondary amine such as methylation acetylation; and parahydroxylation on the phenyl ring, and monohyrdoxylation on the cyclohexyl ring in different positions. The parahydroxylation on the phenyl ring was confirmed by NMR analysis. Some reactions on the secondary amine were unexpected, such as N-formylation. N-dephenylation and N-formylation were confirmed not to be artifacts. The role of the para-hydroxyl intermediate was proved to be essential for the N-dephenylation after intravenous administration of meta- and para-hydroxylated derivatives of CERM 3517 to five beagle dogs.

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CERM 3517 (mociprazine), a new antiemetic compound, was administered orally at 10 mg/kg twice a day for 4 days to six Beagle dogs in order to identify the major metabolite. Mass spectrometric comparison of this metabolite and a synthesized reference compound (CERM 4082) showed that both had identical structures. The metabolite originated from cleavage of the aromatic moiety. After iv administration of CERM 3517 (0.9 mg/kg) and CERM 4082 (0.6 mg/kg) to five beagle dogs, 13% and 56% of the dose, respectively, were eliminated in the urine as CERM 4082 compound. It can be calculated that at least 25% of CERM 3517 was biotransformed by N-dephenylation.

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Using radiolabelled bepridil, equilibrium dialysis experiments and direct ligand-binding studies were conducted to characterize the binding of bepridil to some human serum proteins, and to blood cells and platelets, respectively. Bepridil was bound to serum albumin with K = 26 +/- 1.6 X 10(3) mol-1 X 1, n = 1.07 +/- 0.04; to alpha 1-acid glycoprotein with K = 442 +/- 68 X 10(3) mol-1 X 1, n = 0.90 +/- 0.04; and with a lesser extent to lipoproteins and gamma-globulins. Bepridil, propranolol, quinidine and erythromycin were found to share the same site on alpha 1-acid glycoprotein. The binding of bepridil to both RBC and WBC was nonspecific with the following parameters: NK = 10.55 +/- 0.10 per 5 X 10(6) RBC/mm3 and NK = 0.34 +/- 0.01 per 10(3) WBC/mm3, whereas the binding to platelets was saturable with N = 10.97 +/- 1.06 mumol/l per 108 +/- 10(3) platelets/mm3 and K = 40 +/- 8 X 10(3) mol-1 X 1. The binding parameters were used to simulate the distribution of bepridil between serum proteins, blood cells and platelets over the therapeutic range and showed that serum albumin, alpha 1-acid glycoprotein and RBC were the major binding components.

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Bepridil, an antianginal agent similar in some of its properties to the calcium antagonists, causes a prolonged dose-dependent bradycardia in the dog. This study was undertaken to assess this effect in man. Seven volunteers were administered bepridil in 15 days' sequences separated by a three week interval. The doses were 200, 300 and 600 mg daily respectively. A 24 hour continuous ECG was recorded by the Holter method before each drug sequence and on the fifteenth days. The results were submitted for computerised analysis. The plasma concentrations of bepridil were measured on the 15th day before the ingestion of the morning dose, the half life of the product being about 3 days. After 15 days' therapy of bepridil 200 mg the plasma concentration was 0,23 +/- 0,04 microgram/ml and there was no change in heart rate. At the end of the 300 mg/day sequence, the plasma concentrations reached 0,39 +/- 0.07 microgram/ml and the heart rate fell significantly by 7,9 +/- 2,6%. After 600 mg daily; the mean plasma concentration was 0,82 +/- 0,10 microgram/ml and the fall in heart rate was 10,7 +/- 2,3%. There was an excellent correlation between the plasma concentration and the reduction in heart rate. This reduction is caused by an indirect effect decreasing the effects of adrenergic stimulation without apparent inhibition of the beta receptors and a direct effect decreasing the slope of diastolic depolarisation.

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An assay has been developed to determine the anti-ischaemic drug bepridil (as its free base) in human plasma. The assay procedure comprises n-hexane extraction from basic plasma and gas chromatography using nitrogen-selective detection. An analogue of bepridil is used as internal standard. The accuracy and precision of the assay is determined by repeated analyses of drug-free plasma samples spiked with 5, 10, 20, 100, 400 and 1000 ng of bepridil per ml of plasma. The accuracy, defined as the relative difference between the mean bepridil concentration found and the true value, was 8% or better. The precision (relative standard deviation) was 13% at the 5 ng/ml level and 5% at the 1000 ng/ml level. The assay is suitable to monitor routinely bepridil plasma levels during clinical studies.

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The urinary metabolites of isopropyl-4-(trifluoromethyl-3-phenyl)-2-morpholine (oxaflozane) were compared in man, the dog and the rat using thin-layer chromatography and gas chromatography. In the dog, the tritiated molecule on the morpholine ring was administered to a certain number of animals. The principal metabolites were isolated, purified and identified by IR, RMN spectrometry and mass spectrometry. The principal metabolite in man and the rat was obtained by osaflozane N-dealkylation. In the dog, 2 metabolites formed by cleavage of the morpholine ring were also observed.

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N-[(2-Phenyl-2-isoamyloxy)-ethyl-pyrrolidine]-hydrochloride (amixetrine) was administered to dogs at increasing doses, by i.v. and oral routes. Plasma determinations of active principle were carried out by gas liquid chromatography. The sensitivity limits of the method was 0.1 micrograms/ml of plasma. The evolution of the absorption coefficient and variations in half-life as a function of dose, were studied. The results indicate a non-linear, two-compartment open model. A first-pass effect and enzymatic saturation are envisaged.

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The urinary metabolism of 1-(2-methoxy-2-phenyl)-ethyl-4-(2-hydroxy-3-methoxy-3-phenyl)-propyl-piperazine dihydrochloride (zipeprol) was studied in man, the dog and the rat. Zipeprol metabolites seemed comparable in man and rat. The principal metabolites isolated from urine were identified by TLC, GLC, IR and mass spectrometry. Zipeprol is partially eliminated untransformed from the body, is mainly metabolised by N-dealkylation, oxidation, hydroxylation and methylation.

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The urinary metabolites of N-(2-phenyl-2-isoamyloxy) ethyl-pyrrolidine-hydrochloride (amixetrine) studied in man and in the dog demonstrated a comparable mode of transformation of the drugs for both species. The principal metabolites of amixetrine isolated from urine and purified with the aid of chromatographic techniques were identified by IR, NMR and mass spectrometry. Untransformed amixetrine and (1,2-phenyl-1-hydroxy)ethylpyrrolidine were found in small quantities. In man, as in the dog, the principal metabolite coming from an omega-1-oxidation of the isoamyl chain corresponded to 2-phenyl-2-butoxy-(3-methyl-3-ol)ethyl-pyrrolidine.

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