Influence of different calcic antagonists on the Caco-2 cell monolayer integrity or "TEER, a measurement of toxicity?".

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.

Transepithelial transport of bepridil in the human intestinal cell line, Caco-2, using a "dynamic model".

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.

Isotopic effect study of propofol deuteration on the metabolism, activity, and toxicity of the anesthetic.

The use of isotopic substitution to delay the oxidative metabolism of the anesthetic propofol 1 was studied. The aromatic hydrogens of propofol 1 were replaced by deuterium to produce the mono- and trideuterated derivatives 4 and 5. In vitro metabolic studies on human hepatic microsomes showed no isotopic effect in the para hydroxylation of propofol, and 1, 4, and 5 display similar hypnotic activity and toxicity in mice.

High-performance liquid chromatographic determination of phenylephrine and tropicamide in human aqueous humor.

A high-performance liquid chromatographic method for the simultaneous determination of phenylephrine and tropicamide in human aqueous humor was developed. After centrifugation, an aliquot of the supernatant was injected onto the column and the eluent was monitored at 280 nm then 254 nm after 5 min. Separation was performed on a CN column with 0.01 M Pic B8 (octane sulfonic acid)-acetonitrile (65:35, v/v) as mobile phase. The standard curves were linear in the detection range. The precision of the method (expressed by relative standard deviation) and the accuracy (mean error in per cent) were <5% for both intra- and interassays.

Transepithelial transport of bepridil in the human intestinal cell line, Caco-2, using two media, DMEMc and HBSS.

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.

Simple and sensitive method for determination of metronidazole in human serum by high-performance liquid chromatography.

A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml(-1) using 1 ml of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile-aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 microg ml(-1). Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 microg ml(-1) and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans.

Comparative study of the biotransformation of bepridil analogs in isolated liver cells from one rat. Relationships between structure and in vitro liver toxicity.

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.

Determination of oxeladin in human plasma by gas chromatography--mass spectrometry.

A capillary gas chromatographic method with mass-selective detection was developed for the determination of oxeladin in human plasma. Plasma samples (1 mL) were alkalinized and extracted using 5mL of hexane: isoamyl alcohol (99:1). The method was demonstrated to be sensitive (limit of quantitation at 1 ng/mL), linear between 1 and 150 mg/mL, accurate and precise enough (mean error and mean coefficient of variation at the limit of quantitation were 2.3 and 13.3%, respectively) to support pharmacokinetic evaluation of the drug at doses down to 30 mg.

Biotransformation study of para-substituted phenylpiperazines in beagle dogs by gas chromatography-mass spectrometry.

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.

N-dephenylated and N-phenyl urinary metabolites of mociprazine (CERM 3517) in beagle dogs after oral administration. A mass spectrometric determination.

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.