Claude Aubert

Capillary gas chromatographic-mass spectrometric method for the identification and quantification of some benzodiazepines and their unconjugated metabolites in plasma

A gas chromatographic-mass spectrometric method for the identification and/or quantification of diazepam, clobazam, flunitrazepam, triazolam, midazolam, oxazepam and lorazepam and some of their desmethylated and hydroxylated metabolites in plasma is described. Benzodiazepines were extracted from plasma with butyl acetate at pH 9; the hydroxylated compounds were then silylated with N,O-bis (trimethylsilyltrifluoroacetamide). Analysis was performed using a compact mass-selective detector operating in the electron-impact mode. Depending on the concentration, identification was performed either by direct comparison of the observed mass spectra with reference spectra or by the relative intensities of the most intense and characteristic ions in the selected-ion monitoring (SIM) mode. Quantification was performed in the SIM mode using the most intense ion. The intra-assay precision and accuracy were better than 5-6%; linearity was satisfactory up to 1-2 micrograms/ml. The detection limit was 1-5 ng/ml for most of the benzodiazepines. This method can be easily used in clinical situations when a safe and rapid response is essential for patient treatment.

Interspecies variability and drug interactions of clozapine metabolism by microsomes

Abstract— Cytochrome P450 expression in liver is influenced by several factors, including species, sex and strain. We compared metabolism formation of clozapine in different species (rat, mouse, guinea‐pig, dog, monkey and man) so as to choose between species to further validate interaction studies. Liver microsomes of male and female Sprague‐Dawley rats, hairless rats, OFI mice, Balb C mice and Dunkin‐Hartley albino guinea‐pigs, male beagle dogs, male cynomolgus monkeys and man were used to investigate in vitro metabolism of clozapine. This process was dependent on the presence of NADPH and on the presence of microsome protein. In addition, we observed the formation of desmethyl‐ and N‐oxide metabolites, with the rate of formation of each of these compounds varying with species, sex and strain of microsomes incubated. The desmethyl‐ and N‐oxide metabolites formed were statistically greater in male than in female rats, mice in the two strains studied, as well as for the guinea‐pigs. Levels of desmethyl clozapine formed were high for the rats and no significant difference in clozapine biotransformation was observed between Sprague‐Dawley and hairless rats. For man, the formation of metabolites of clozapine was comparable with guinea‐pig, dog and monkey. In addition, we screened the effect of 52 molecules, representative of 11 different therapeutic classes, on the metabolism of clozapine by rat liver microsomes. We found that most of the calcium channel blockers (diltiazem, felodipine, isradipine, lacidipine, nicardipine and nitrendipine), antifungals (ketoconazole, miconazole) and two anticancer drugs (paclitaxel, teniposide) caused more than 50% inhibition of clozapine metabolism in vitro. The extent of inhibition was increased in a concentration‐dependant manner. Complementary clinical and pharmacokinetic studies should be performed to confirm these results.

Determination of Chloroquine and Monodesethylchloroquine in Hair

Using thin-layer and gas chromatography and mass spectrometry, chloroquine and its major metabolite (monodesethylchloroquine) were identified in hair samples of numerous patients who received this antimalarial drug for several months. In two patients the amounts of chloroquine were, respectively, 310 and 145 mg/kg hair and those of the monodesethylchloroquine 23 and 11 mg/kg. The respective proportions (93 and 7%) are the same in the two subjects. The chloroquine percentage was near those in the spleen or stomach wall after poisoning. Other metabolites in hair are being identified. Hair analysis may provide a good toxicologic and forensic science complement to the blood, urine, and tissues. It may be useful for the control of chloroquine therapy.

Determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma by high-performance liquid chromatography and fluorescence detection.

A high-performance liquid chromatographic method for the simultaneous determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma is described. After extraction with an organic solvent, the compounds and their internal standard (7-amino-methylclonazepam or Ro 5-3384) are derivatized with fluorescamine and chromatographed on a reversed-phase muBondapak C18 column using pH 8 buffer solution-acetonitrile (3:1) as mobile phase. The detection is performed by a fluorometer at excitation and emission wavelengths of 390 and 470 nm, respectively. The sensitivity limit is about 1 ng/ml of plasma for both 7-amino-flunitrazepam and 7-amino-desmethylflunitrazepam. The method has been applied to the determination of plasma levels of these substances during pharmacokinetic studies of flunitrazepam, desmethylflunitrazepam and 7-amino-flunitrazepam.

Development of stealth liposome formulation of 2'-deoxyinosine as 5-fluorouracil modulator: in vitro and in vivo study.

PurposeThe aims of this study were to develop a stealth, pegylated liposomal formulation of 2′-deoxyinosine (d-Ino), a 5-fluorouracil (5-FU) modulator, to evaluate its efficacy in vitro and in tumor-bearing mice, and to study its pharmacokinetics in rats.MethodAfter designing a pegylated liposome encapsulating d-Ino (L-d-Ino), we evaluated its efficacy as 5-FU modulator in vitro. Antiproliferative assays, thymidylate synthase (TS) inhibition, and apoptosis studies were carried out to check whether an optimization of 5-FU action was achieved on the 5-FU-resistant SW620 cell line. Animal pharmacokinetic and ex vivo studies were next performed to confirm that L-d-Ino displayed a slower plasma elimination pattern than free d-Ino. Finally, effects on tumor growth of L-d-Ino + 5-FU combination was evaluated in xenografted mice.ResultsWe developed a stable, sterile, and homogenous 100-nm population of pegylated liposomes encapsulating 30% of d-Ino. Liposomal d-Ino exhibited a strong potential as 5-FU modulator in vitro by enhancing TS inhibition and subsequent apoptosis induction, while displaying a better pharmacokinetic profile in animals, with a near seven times clearance reduction as compared with the free form. When used in tumor-bearing mice in combination with 5-FU, our results showed next that the association led to 70% of tumor reduction with a doubling median survival time as compared with untreated animals, whereas 5-FU alone was ineffective.ConclusionOur data show that liposomal d-Ino, through an optimized pharmacokinetic profile, displays apotenteffect as fluoropyrimidines modulator, both in vitro and in xenografted mice. Besides, we showed here that itispossible to reverse a resistant phenotype to 5-FU, a major drug extensively described in clinical oncology.

Monitoring of the intracellular activation of 5-fluorouracil to deoxyribonucleotides in HT29 human colon cell line: application to modulation of metabolism and cytotoxicity study.

Abstract— An HPLC method was developed for in vitro detection and monitoring of intracellular metabolites of [3H]‐5‐fluorouracil (FUra). Results showed a preferential activation of FUra to ribonucleoside and ribonucleotide derivatives (FURd, FUMP, FUDP and FUTP) in the human colorectal HT29 cell line. We screened various agents so as to determine if they could act as modulators of metabolism and/or toxicity of FUra by reversing the activation pathway of FUra from ribo‐ to deoxyribonucleotides, thus enhancing FdUMP formation. Different drugs (efflux inhibitors, catabolism inhibitors and enzymatic cofactors) were tested for enhancement of cytotoxicity when associated with FUra. The most promising agents were further studied by assessment of their ability to modulate intracellular activation of FUra to enhance thymidylate synthase (TS) inhibition by FUra and to increase the subsequent induction of apoptosis. 2′‐Deoxyinosine (d‐Ino), a deoxyribose 1‐phosphate donor increasing thymidine phosphorylase activity, stood out as the best modulating agent we screened. Results showed an up to 30‐fold increase of cytotoxicity along with a stronger inhibition of TS when FUra was associated with d‐Ino, while FUra alone exhibited a lesser effect on TS activity. Besides, HPLC analysis revealed a compléte reversal of the activation pathway of FUra, thus leading to an intracellular accumulation of deoxyribonucleotides. Assessment of cell cycle distribution showed a marked increase (+480%) of apoptosis in cells exposed to FUra/d‐Ino compared to FUra alone. The HPLC method we developed is a convenient tool for assessing to what extent modulators will actually act on the intracellular activation of FUra. This study confirms the potentiality of d‐Ino to modulate FUra metabolism in vitro. It proved to be an agent able to orientate the mechanism of action of FUra towards the inhibition of TS in cells where the normal activation pathway of the drug does not result in the intracellular accumulation of the active metabolite FdUMP.

Simultaneous assay of triazolam and its main hydroxy metabolite in plasma and urine by capillary gas chromatography

A gas--liquid chromatographic method for the simultaneous determination of triazolam and its major hydroxy metabolite (1-hydroxymethyltriazolam) in human plasma and urine is described. After addition of two internal standards to the biological fluid, extraction at pH 9, acid washing, back-extraction, and derivatization, the analysis was performed on a wall-coated superior capacity open-tubular (WSCOT) CP-Sil 5 capillary column with electron-capture detection. The detection limit was 0.1-0.2 ng/ml; reproducibility was about 6-7% for plasma concentrations below 1 ng/ml. No interference from other possible minor hydroxy metabolites of triazolam was found. Gas chromatography coupled with mass spectrometry validated the chromatographic results. The method was successfully applied to plasma specimens collected from healthy human volunteers following a single intravenous administration of 1 mg of triazolam or 1-hydroxymethyltriazolam.

Cytotoxic Effects of Haplamine and its Major Metabolites on Human Cancer Cell Lines

Haplamine, extracted from Haplophyllum perforatum, is widely used in Central Asia for treating various diseases, including testicular cancer. The purpose of the present study was to investigate in vitro the cytotoxic properties of haplamine and its major metabolites (trans/cis-3,4-dihydroxyhaplamine) on human pancreatic cancer, colorectal cancer and hepatic cancer cell lines. The efficacy of haplamine was compared with those of the respective reference drugs for treating digestive cancers (e. g., 5-FU, gemcitabine). Finally, the implication of apoptosis in haplamine-induced cell death was investigated. The IC50 values of of haplamine were 52.5 +/- 2.6, 24.3 +/- 0.7; 41.5 +/- 2.5, 72 +/- 2, 32 +/- 2.2 and 59.7 +/- 2.1 microM in human pancreatic cancer (Capan1 and Capan2), colorectal cancer (LS174T, HT29, and SW620) and hepatic cancer (HepG2) cells, respectively. The IC50 values of trans/cis-3,4-dihydroxyhaplamine were both > 200 microM, thus suggesting that the previously reported cytotoxic efficacy of haplamine was supported by the parent drug only. Besides, our data showed that haplamine leads to cell death through the induction of early/late apoptosis in the target cells. Interestingly, we found that haplamine showed significant antiproliferative efficacy on resistant SW620 colorectal cells, whereas the reference drug 5-FU was ineffective (32 vs. 73 microM, p < 0.01 t- test), thus suggesting that haplamine could be of interest for treating digestive cancers resistant to standard fluoropyrimidines. Similarly, haplamine proved to be significantly more potent in pancreatic cells than gemcitabine, the reference cytotoxic drug for treating pancreatic carcinomas. Overall, these results confirm the anticancer properties of haplamine suggested by its traditional use, and indicate that it could be further considered in various other solid tumours frequently encountered in adults, including those resistant to standard chemotherapy.

Plasma determination of 3-methylclonazepam by capillary gas chromatography.

A capillary gas-liquid chromatographic method for the determination of 3-methylclonazepam in plasma was developed. This method involved a single extraction by butyl acetate followed by analysis of the organic extract on a CP-Sil 5 glass capillary column with detection by electron capture. The detection limit was about 0.1 ng/ml, and the inter- and intra-assay precision did not exceed 8% for the concentration range 0.1-6.0 ng/ml. Specificity towards some of the possible metabolites in human plasma was demonstrated. This method was used for the measurement of the pharmacokinetic parameters of 3-methylclonazepam in healthy volunteers after a single intravenous administration of 1 mg, and oral administrations of 1 and 4 mg.

Determination of Clobazam, n-desmethylclobazam and their hydroxy metabolites in plasma and urine by high-performance liquid chromatography

Owing to the pharmacological and clinical importance of the determination of plasma and urine levels of the hydroxy metabolites of clobazam and N-desmethylclobazam in healthy volunteers and in epileptic patients, a high-performance liquid chromatographic (HPLC) method was developed that permits the determination of all these compounds in the same plasma or urine sample. The method involved ether extraction at pH 13 with diazepam as internal standard for the measurement of clobazam and N-desmethylcobazam, followed by ether extraction at pH 9 with nitrazepam as internal standard for the measurement of the hydroxy derivatives. The limit of detection was about 10-20 ng/ml for each of these compounds. Applications to patients were limited by chromatographic interferences between the hydroxy metabolites and some medications currently associated with clobazam in the treatment of epilepsy. The only interference in clobazam and N-desmethylclobazam analysis was from N-desmethyldiazepam. Despite these inconveniences, this HPLC procedure appears to be the only available method for the simultaneous quantification of clobazam and its three main metabolites.