Robert Farinotti

Clinical pharmacokinetics and metabolism of chloroquine : Focus on recent advancements

SummaryThis paper presents the current state of knowledge on chloroquine disposition, with special emphasis on stereoselectivity and microsomal metabolism. In addition, the impact of the patient’s physiopathological status and ethnic origin on chloroquine pharmacokinetics is discussed.In humans, chloroquine concentrations decline multiexponentially. The drug is extensively distributed, with a volume of distribution of 200 to 800 L/kg when calculated from plasma concentrations and 200 L/kg when estimated from whole blood data (concentrations being 5 to 10 times higher).Chloroquine is 60% bound to plasma proteins and equally cleared by the kidney and liver. Following administration chloroquine is rapidly dealkylated via cytochrome P450 enzymes (CYP) into the pharmacologically active desethylchloroquine and bisdesethylchloroquine. Desethylchloroquine and bisdesethylchloroquine concentrations reach 40 and 10% of chloroquine concentrations, respectively; both chloroquine and desethylchloroquine concentrations decline slowly, with elimination half-lives of 20 to 60 days. Both parent drug and metabolite can be detected in urine months after a single dose.n In vitro and in vivo, chloroquine and desethylchloroquine competitively inhibit CYP2D1/6-mediated reactions. Limited in vitro studies and preliminary data from clinical experiments and observations point to CYP3A and CYP2D6 as the 2 major isoforms affected by or involved in chloroquine metabolism.n In vitro efficacy studies did not detect any difference in potency between chloroquine enantiomers but, in vivo in rats, S(+)-chloroquine had a lower dose that elicited 50% of the maximal effect (ED950) than that of R(−)-chloroquine. Stereoselectivity in chloroquine body disposition could be responsible for this discrepancy. Chloroquine binding to plasma proteins is stereoselective, favouring S(+)-chloroquine (67% vs 35% for the R-enantiomer). Hence, unbound plasma concentrations are higher for R(−)-chloroquine. Following separate administration of the individual enantiomers, R(−)-chloroquine reached higher and more sustained blood concentrations. The shorter half-life of S(+)-chloroquine appears secondary to its faster clearance. Blood concentrations of the S(+)-forms of desethylchloroquine always exceeded those of the R(−)-forms, pointing to a preferential metabolism of S(+)-chloroquine.

Improvement of cefpodoxime proxetil oral absorption in rats by an oil-in-water submicron emulsion.

Absolute bioavailability of cefpodoxime proxetil is both limited by its low solubility in aqueous solution and its intraluminal hydrolysis. The oil-in-water submicron emulsion was proven to be effective in protecting the prodrug from the enzymatic attack in rabbit intestinal washings. The aim of the study was to perform a pharmacokinetic study in conscious rats to confirm o/w submicron superiority in comparison to other oral formulations (hydro-alcoholic solution, suspension and coarse emulsion). The pharmacokinetic study was performed in conscious rats implanted with permanent aortic catheters. A parenteral solution of cefpodoxime was injected via this catheter, and oral formulations were administered orally. The cefpodoxime plasma level was performed by a HPLC validated method. The pharmacokinetic parameters, t1/2, Cmax, tmax, AUC and absolute bioavailability (F) were determined with a non-compartmental analysis. The results show a significant increase of F for submicron emulsion (97.4%) between the other oral formulations. No significant difference of F was found between the other oral formulations, even with the coarse o/w emulsion. The o/w submicron emulsion made the enhancement of the absolute bioavailability of cefpodoxime proxetil possible. This benefit could be explained by the low droplet size of the submicron emulsion which improve the absorption process of the prodrug.

Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT.

The brain distribution of the enantiomers of the antimalarial drug mefloquine is stereoselective according to the species. This stereoselectivity may be related to species-specific differences in the properties of some membrane-bound transport proteins, such as P-glycoprotein (P-gp). The interactions of racemic mefloquine and its individual enantiomers with the P-glycoprotein efflux transport system have been analysed in immortalised rat brain capillary endothelial GPNT cells. Parallel studies were carried out for comparison in human colon carcinoma Caco-2 cells. The cellular accumulation of the P-glycoprotein substrate, [(3)H]vinblastine, was significantly increased both in GPNT cells and in Caco-2 cells when treated with racemic mefloquine and the individual enantiomers. In GPNT cells, the (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing cellular accumulation of [(3)H]vinblastine, while in Caco-2 cells, both enantiomers were equally effective. These results suggest that racemic mefloquine and its enantiomers are effective inhibitors of P-gp. Furthermore, a stereoselective P-glycoprotein inhibition is observed in rat cells but not in human cells. The efflux of [(14)C]mefloquine from GPNT cells was decreased when the cells were incubated with the P-gp modulators, verapamil, cyclosporin A or chlorpromazine, suggesting that MQ could be a P-gp substrate.

Clinical Pharmacokinetics of Zopiclone

SummaryZopiclone is a cyclopyrrolone hypnotic agent. It possesses a chiral centre and is commercially available as a racemic mixture. Methods involving high performance liquid chromatography (HPLC), gas chromatography, capillary electrophoresis (CE) and high performance thin layer chromatography have been developed for the quantitation of zopiclone and its 2 main metabolites in biological samples. For the chiral determination of the enantiomers of zopiclone and its metabolites, HPLC and CE methods are available.After oral administration, zopiclone is rapidly absorbed, with a bioavailability of approximately 80%. The plasma protein binding of zopiclone has been reported to be between 45 and 80%. Zopiclone is rapidly and widely distributed to body tissues including the brain, and is excreted in urine, saliva and breast milk.Zopiclone is partly metabolised in the liver to form an inactive N-demethylated derivative and an active N-oxide metabolite. In addition, approximately 50% of the administered dose is decarboxylated and excreted via the lungs. Less than 7% of the administered dose is renally excreted as unchanged zopiclone. In urine, the N-demethyl and N-oxide metabolites account for 30% of the initial dose. The terminal elimination half-life (t1/2z) of zopiclone ranges from 3.5 to 6.5 hours.The pharmacokinetics of zopiclone in humans are stereoselective. After oral administration of the racemic mixture, Cmax (time to maximum plasma concentration), AUG (area under the plasma time-concentration curve) and t1/2z values are higher for the dextrorotatory enantiomer owing to the slower total clearance and smaller volume of distribution (corrected by the bioavailability), compared with the levorotatory enantiomer. In urine, the concentrations of the dextrorotatory enantiomers of the N-demethyl and N-oxide metabolites are higher than those of the respective antipodes.The pharmacokinetics of zopiclone are altered by aging and are influenced by renal and hepatic functions. Drug interactions have been observed with erythromycin, trimipramine and carbamazepine.

Influence of tumor necrosis factor-α on the expression and function of P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT

Drug cerebral pharmacokinetics may be altered in the case of inflammatory diseases. This may be due to a modification of drug transport through the blood-brain barrier, in particular through drug interaction with the membrane efflux transporter, P-glycoprotein. The objective of this study was to investigate the influence of the inflammatory cytokine, tumor necrosis factor (TNF)-alpha, on the functionality and expression of P-glycoprotein, and on mdr1a and mdr1b mRNA expression in immortalised rat brain endothelial cells, GPNT. Cells were treated with TNF-alpha for 4 days. Levels of mdr1a and mdr1b mRNAs were quantitated using real-time RT-PCR analysis and expression of P-glycoprotein was analyzed by Western blot. The functionality of P-glycoprotein was studied by following the accumulation of [3H]vinblastine in the cells without and with a pre-treatment with a P-glycoprotein inhibitor, GF120918. TNF-alpha increased the levels of mdr1a and mdr1b mRNAs while no effect was observed on protein expression. TNF-alpha increased [3H]vinblastine accumulation indicating a time and concentration-dependent decrease of P-glycoprotein activity. This effect was eliminated when the cells were pre-treated with GF120918. Our observation of a decrease in P-glycoprotein activity could suggest that in the case of inflammatory diseases, brain delivery of P-glycoprotein-dependent drugs can be enhanced.

Determination of the enantiomers of zopiclone and its two chiral metabolites in urine using an automated coupled achiral—chiral chromatographic system

The enantiomers of zopiclone and its two chiral N-desmethyl and N-oxide metabolites were determined in urine using a coupled achiral-chiral liquid chromatographic method. After liquid-liquid extraction, zopiclone and its two metabolites were quantified on a cyanopropyl column. After fluorimetric detection on the achiral system, the eluent was switched through a silica precolumn in order to trap and concentrate the analytes. Each fraction was then backflushed separately onto a carbamate cellulose chiral stationary phase in order to determine the enantiomeric ratios. The coupled system was automated with an autosampler and a switching valve programmed by an integrator. The method was validated, and a first trial was performed on urine samples of a volunteer treated with 15 mg of racemic zopiclone.

Rapid and simple method to determine chloroquine and its desethylated metabolites in human microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive and selective method was developed for the simultaneous determination of chloroquine (CQ) and its desethylated metabolites monodesethylchloroquine (DCQ) and bisdesethylchloroquine (BDCQ) in human liver microsomes. Analytes were separated on a C1 column using methanol-water (70:30, v/v) and triethylamine (0.1% v/v) as the mobile phase. The fluorescence detector was set at 250 (excitation) and 380 nm (emission). Following protein precipitation with ice-cold acetonitrile, microsomal incubation supernatants were directly injected into the HPLC system. Typically, 200 microl of incubate were diluted with 200 microl of acetonitrile and 15 microl were injected. The limit of quantitation was 78 nM of CQ or metabolite. Intra-day variability averaged 2.9% for CQ, 1.5% for DCQ and 2.5% for BDCQ. Inter-day variability was 3.1% for CQ, 3.5% for DCQ and 3.7% for BDCQ. Mean accuracies were 100% for CQ and BDCQ and 102% for DCQ.

Cefpodoxime proxetil esterase activity in rabbit small intestine : a role in the partial cefpodoxime absorption

Abstract The luminal and mucosal de-esterification of the prodrug ester cefpodoxime proxetil was studied in rabbit intestine in vitro. An enzymatic hydrolysis of the ester, releasing the active third-generation cephalosporin, was observed in both luminal washing and mucosal homogenate. The mucosal activity was mainly recovered in the soluble fraction, whereas the brush-border membranes were almost devoid of activity. Eserine and diisopropyl fluorophosphate were potent inhibitors of cefpodoxime proxetil hydrolysis in both luminal washing and mucosal homogenate, suggesting the participation of choline esterases in the hydrolysis of cefpodoxime proxetil. The luminal and mucosal activities were equally sensitive to HgCl2 and acetylsalicylic acid inhibitions but slight differences were observed concerning the 50% inhibitory concentration (IC50) of two drug esters, bacampicillin and enalapril. In vitro experiments run with rabbit jejunum mounted in Sweetana-Grass diffusion chambers showed that an extensive hydrolysis of cefpodoxime proxetil occurred in the mucosal compartment and that the accumulation of cefpodoxime in the serosal compartment was very slow. These observations support the hypothesis that the partial oral bioavailability of cefpodoxime proxetil results from a hydrolysis by luminal choline esterases.

Critical issues in chiral drug analysis in biological fluids by high-performance liquid chromatography

This review article focuses on the specificities of chiral liquid chromatography, with particular emphasis on stability, stereoconversion, enantiomeric separation, recovery and drug concentration determinations. In addition, the paper presents an overview of the different steps which have to be followed for a chiral method to be validated. Sensitivity, selectivity, linearity, precision and accuracy all have to be ensured for three chemical entities, the two enantiomers and the racemate. Only accurate and precise concentrations of the parent drug and its metabolites will lead to the reliable description of their in vitro stability and in vivo body disposition.

Whole blood concentrations of mefloquine enantiomers in healthy Thai volunteers

We studied the pharmacokinetics of the enantiomers of mefloquine in whole blood in healthy Thai volunteers after administration of a single oral dose of 750 mg of the racemic mixture.Mefloquine pharmacokinetics were stereoselective. The peak concentrations and areas under the curve of the (−) enantiomer were significantly higher than those of its antipode (0.79 versus 0.46 μg · ml-1 and 402 versus 94 μg · h · ml-1). The half-lives of (−)MQ were significantly longer than those of (+)MQ (531 versus 206 h). No stereoselectivity was observed for tmax values.