Temperature effects on the trophic stages of perennial rye grass anaerobic digestion.

Continuous Stirred Tank Reactors (CSTRs), operated in batch mode, were used to evaluate the feasibility of psychrophilic (low temperature) digestion of perennial rye grass in a long term experiment (150 days) for the first time. The reactors were operated in parallel at 3 different temperatures, 10, 15 and 37 degrees C. Hydrolysis, acidification and methanogenesis were assessed by VS degradation, by soluble chemical oxygen demand (SCOD) and volatile fatty acids (VFA) production, and by methane production, respectively. Hydrolysis was the rate-limiting step at all temperatures and the rates and extent of hydrolysis were considerably lower at 15 and 10 degrees C, than at 37 degrees C. The total VS degradation was 53%, 34% and 19% at 37, 15 and 10 degrees C, respectively. Acidification was not affected by temperature and VFA production and consumption was balanced in all cases, except at 10 degrees C. Methane yields were 0.215 m3 CH4 kg(-1) VS(-1) added, 0.160 m3 CH4 kg(-1) VS(-1) added and 0.125 m3 CH4 kg(-1) VS(-1) added at 37, 15 and 10 degrees C, respectively. Methanogenesis was not strongly affected at 15 C but it became rate-limiting at 10 degrees C. Overall, the solid degradation and methane production performance under psychrophilic conditions was encouraging and greater than previously reported. Considering the non-acclimated, mesophilic nature of the inoculum, there are grounds to believe that low-temperature anaerobic digestion of grass could be feasible if coupled to efficient hydrolysis of the biomass.

Sensitive determination of tenofovir in human plasma samples using reversed-phase liquid chromatography.

A new high-performance liquid chromatography assay was developed for the determination of tenofovir, a nucleotide analogue, in plasma. A solid-liquid extraction procedure was coupled with a reversed-phase HPLC system. The system requires a mobile phase containing Na(2)HPO(4) buffer, tetrabutylammonium hydrogen sulfate and acetonitrile for different elution through a C(18) column with UV detection. The method proved to be accurate, precise and linear between 10 and 4000 ng/ml. The method was applied to determine trough levels of tenofovir in 11 HIV-infected patients with virologic failure under multiple antiretroviral therapy. This method was also successfully applied to a pharmacokinetic study in an HIV infected patient with renal failure.

Intestinal absorption and metabolism of chlorpheniramine enantiomers in rat.

Chlorpheniramine (CPAM) is a chiral antihistaminic drug commercialized as a racemic mixture. The intestinal absorption and metabolism of CPAM have been investigated in rat using in vivo (oral and IV administration), in situ (intestinal loop model), and in vitro (everted sac model) experiments. Oral and IV administrations of 20 mg/kg of the racemic mixture show that the pharmacokinetics of CPAM are stereoselective, with higher AUCs for the (+)-S-enantiomer compared to its antipode. The monodesmethyl metabolite (DCPM) was quantifiable in blood and its pharmacokinetics are stereoselective after oral but not after IV administration. Experiments using intestinal loops and everted sacs showed that the absorption is not stereoselective and that in vivo stereoselective formation of DCPM is presumably due to stereoselective hepatic metabolism. Moreover, the in vitro and in situ absorption of CPAM are not modified by modulators of P-glycoprotein and cytochromes P450 (cyclosporin A, ketoconazole).

Stereospecific versus nonstereospecific assessments for the bioequivalence of two formulations of racemic chlorpheniramine.

Chlorpheniramine (chlorphenamine, CPAM) is a racemic antihistaminic H1 drug containing two enantiomers. The aim of this study was to assess the bioequivalence of two formulations (reference and Vietnamese-tested formulation) of racemic chlorpheniramine combined with phenylpropanolamine in an open-labeled, randomized, crossover two-period study, after administration of 8 mg of racemic chlorpheniramine in 12 healthy Vietnamese subjects. First, dissolution of both formulations was tested in vitro according to USP requirements. Then the 12 subjects received both formulations after an overnight fast and a 7-day wash-out period. Plasma samples were collected up to 168 h. Plasma concentrations of total chlorpheniramine and its individual enantiomers were determined with a validated chiral HPLC method and pharmacokinetic parameters were estimated using model-independent analysis. For the reference formulation, Cmax and AUC values were higher for (+)S-chlorpheniramine ((+)S-CPAM) compared to (-)R-chlorpheniramine ((-)R-CPAM) (13.3 vs. 6.8 ng/ml and 409 vs. 222 ng/ml/h, respectively) while Clt/F and Vd/F were lower (9.8 vs. 17.6 l/h and 321 vs. 627 l, respectively). No difference was observed for Tmax, t(1/2), and MRT. Pharmacokinetic parameters were similar for the reference and the Vietnamese-tested formulation. Bioequivalence was assessed by Schuirmann test, as recommended by the current FDA and European Community criteria. Dissolution tests showed that both formulations were equivalent. A nonstereospecific, but not a stereospecific, approach indicated bioequivalence between the formulations.

[Generic drugs and right of substitution. Relating to Article 29 of the 1999 *social Security Financing Act]. / Génériques et droit de substitution. A propos de l'article 29 de la loi de financement de la Sécurité sociale pour 1999.

The 1999 Social Security Financing Act grants the right of substitution to pharmacists. If the prescriptor does not object, the pharmacist can replace a medicine by its generic. After reiterating that generic medicines are essentially similar but not strictly identical to the original or to each other, we analyze the discussions which took place at the National Assembly and Senate which should have resulted in some further modifications to the recently adopted legislative text.

Binding of chlorpheniramine enantiomers to human plasma proteins.

The in vitro binding of RS-chlorpheniramine to human proteins was studied by equilibrium dialysis. The binding to total plasma proteins and to individual albumin and alpha-glycoprotein acid is stereoselective. (+)S-chlorpheniramine is more extensively bound than its antipode to total plasma proteins (38% vs. 23%), to albumin (20% vs. 15%) and to alpha-glycoprotein acid (23% vs. 5%).

Stereoselective binding of zopiclone to human plasma proteins.

The binding of racemic zopiclone (ZOP) and of its two enantiomers to plasma proteins, albumin and alpha 1-acid glycoprotein were compared. Our work shows that the binding of ZOP to human plasma proteins is stereoselective. The total plasma protein binding percentages were 79.3 +/- 5.5%, 83.8 +/- 5.2%, and 75.1 +/- 2.1%, for racemic zopiclone, (-)zopiclone and (+)zopiclone, respectively. These results were confirmed by the analysis of samples obtained from healthy volunteers after the oral administration of ZOP. The anticoagulant used for sampling was also shown to have an influence on the percentage binding and on its stereoselectivity. Considering albumin and alpha 1-acid glycoprotein separately, stereoselectivity was also observed.

Residual glutaraldehyde levels in fiberoptic endoscopes: measurement and implications for patient toxicity.

Most gastroenterology societies recommend glutaraldehyde for fiberoptic endoscope disinfection. However, glutaraldehyde toxicity has been suspected in patients examined with endoscopes disinfected with this compound. The aim of our study was to determine the residual levels of glutaraldehyde in fiberoptic endoscopes after either manual or automatic disinfection and to evaluate the extent of toxicity. Furthermore, the procedures for disinfection currently performed by the department were compared with the new French guidelines. We used both manual and automatic disinfection procedures and flushed sterile distilled water through the lumens of endoscopes before use. Residual glutaraldehyde levels were determined using liquid chromatography coupled to spectrophotometric detection. In a total of 92 measurements it was found that residual glutaraldehyde levels were higher and more variable after manual disinfection (< 0.2-159.5 mg/L) than after automatic disinfection (< 0.2-6.3 mg/L). We conclude that local procedures for disinfection need to be improved to conform to the new French guidelines. Since thresholds for the toxic dose of glutaraldehyde and international norms for levels of residual glutaraldehyde in equipment have not been defined, additional studies combining accurate measurements in fiberoptic endoscopes and clinical observations of endoscopy patients will be required to draw more definitive conclusions.

Chiral chromatographic method to determine the enantiomers of halofantrine and its main chiral desbutyl metabolite in erythrocytes.

We describe a direct liquid chromatographic method with spectrofluorimetric detection to quantify the two enantiomers of halofantrine and the two enantiomers of its main chiral N-monodesbutylated metabolite in erythrocyte pellets. The method involves a Chiralpak AD column and a rapid one-step extraction procedure with acetonitrile. The method was validated for the four enantiomers within the range 0-1000 ng/ml. The absence of stereoconversion was studied in samples stored frozen for up to eight months. The optical rotation of the halofantrine and metabolite enantiomers was determined after separation on a semi-preparative Chiralcel OD column with polarimetric detection.

Determination of the enantiomers of chlorpheniramine and its main monodesmethyl metabolite in urine using achiral-chiral liquid chromatography.

The enantiomers of chlorpheniramine and its monodesmethyl metabolite were determined separately in urine by using a coupled achiral-chiral chromatographic system. The two enantiomers of the studied compound and the internal standard were separated from the biological matrix on a cyanopropyl column and reinjected into a chiral amylose AD column where the two enantiomers were separated and quantified by UV detection. The method was validated for chlorpheniramine and for the metabolite within the range 0-1000 ng/ml. It was also applied in a pilot pharmacokinetic study to samples from a volunteer given 8 mg of racemic chlorpheniramine by mouth.

Variations of teicoplanin concentrations in neutropenic patients.

The aim of this study was to measure the trough plasma concentrations of teicoplanin in neutropenic patients with a view to optimizing the loading dose. Teicoplanin trough plasma concentrations were followed in 11 neutropenic patients after repeated administration of a 6 mg/kg i.v. bolus. The first three injections were given at 12-h intervals, and the rest every 24 h. Trough plasma concentrations at 48 h varied from 5.6 to 13.1 mg/I. The mean trough plasma concentration-time curve indicated a trend towards accumulation. In conclusion, the loading dose of teicoplanin should be tailored to individual neutropenic patients.

Fast specific separation and sensitive quantification of bactericidal and sporicidal aldehydes by high-performance liquid chromatography: example of glutaraldehyde determination.

This article describes the design and the validation of the HPLC determination of glutaraldehyde at g/l and mg/l concentrations, after derivatization by 2,4-dinitrophenylhydrazine and using the external standard method. At low concentrations, the reaction mixture needs to be heated and a weight ratio of 500 for the 2,4-dinitrophenylhydrazine reagent and the glutaraldehyde ensures a linear calibration curve. In contrast, high concentrations do not require heating of the reaction mixture and a weight ratio of 32 proved to be sufficient. The optimized HPLC method has been validated for both ranges of concentrations. Between 1.25 and 10 mg/l, the content can be determined by the external standard method, with a repeatability of 0.5%. The detection limit is 0.2 mg/l. Between 0.31 and 2.5 g/l, the content can also be determined by the external standard method, with a repeatability of 0.4%. Finally, statistical analysis has demonstrated that aqueous solutions of glutaraldehyde are stable for at least three days at 4 degrees C within the mg to g range.

[Strategy of quality of the application of the Huriet law in hospitals]. / Stratégie de la qualité dans l'application de la loi Huriet en milieu hospitalier.

Due to the "Huriet" law, the hospital pharmacist has new responsibilities in biomedical research. This article reports the activities of Pitié-Salpêtrière hospital. We also expose the organisation of drug dispensation and storage in our hospital pharmacy.

Carbamazepine and its epoxide: an open study of efficacy and side effects after carbamazepine dose increment in refractory partial epilepsy.

We evaluated the efficacy, development of adverse effects, and possible correlation between the plasma concentration of carbamazepine (CBZ) and its major metabolite, carbamazepine-10,11-epoxide (CBZ-E), in a group of epileptic patients in whom selective increases in CBZ doses were made. Eighteen patients with refractory partial epilepsy participated in an open trial. Five were on monotherapy and 13 on polytherapy. All the patients were on CBZ before the trial and had plasma levels within the therapeutic range (17-42 mumol/L). After a baseline period, CBZ doses were progressively increased either to reach a 50% reduction in seizure frequency for 2 months or until side effects appeared. Thirty-nine percent of the patients had a 50% decline in seizure frequency, but only 17% improved for > 6 months. Mild or moderate side effects were observed in 78% of the patients. Side effects were correlated with CBZ plasma levels but not with CBZ-E plasma levels. Correlation between CBZ and CBZ-E plasma levels were found in the monotherapy group, but not in the polytherapy group. Our results confirm that higher doses of CBZ can successfully be used in some patients with refractory partial epilepsy. Furthermore, the plasma level of CBZ-E does not seem to be a useful indicator of toxicity in CBZ-treated ambulatory epileptic patients.

Pharmacokinetics of zopiclone and its enantiomers in Caucasian young healthy volunteers.

The disposition of the enantiomers of zopiclone and its two chiral metabolites was investigated after oral administration of a single dose of 15 mg of a racemic mixture (twice the usual therapeutic regimen) in 12 adult Caucasian volunteers. Determination of concentrations of zopiclone enantiomers in plasma showed that zopiclone pharmacokinetics is stereoselective with AUC0-->infinity values of 691.3 and 209.5 ng.ml-1.hr (p < 0.001), Cmax values of 87.3 and 44.0 ng.ml-1 (p < 0.001), oral CLtot/F values of 195.5 and 659.8 ml.min-1 (p < 0.001), Vd/F values of 98.6 and 192.8 liters (p < 0.01) and elimination half-life of 399.2 and 225.6 min (p < 0.01) for (+)-zopiclone and (-)-zopiclone, respectively. On the contrary, absorption half-life and Tmax values were not significantly different. In 48-hr urine, 3.6% of unchanged zopiclone was excreted, whereas 14.2% and 13.8% of both metabolites, N-desmethylzopiclone and N-oxidezopiclone, respectively, were found. Quantities of (+)-zopiclone excreted in urine were always higher compared with its antipode (-)-zopiclone for the 12 volunteers (p < 0.001). For the metabolites, quantities of both enantiomers were either equal or different and when different, it was always in favor of the (+)-enantiomer.

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.

Determination of zopiclone enantiomers in plasma by liquid chromatography using a chiral cellulose carbamate column.

The enantiomers of zopiclone were determined in human plasma using a sequential achiral-chiral liquid chromatographic method. Zopiclone was separated from the biological matrix and quantified on an achiral silica column. The limit of detection was 5 ng/ml. The eluent fraction containing zopiclone was collected, evaporated, reconstituted with the mobile phase and injected onto a chiral cellulose carbamate column where the enantiomeric ratio was calculated. This validated method, applied to a pilot study, suggests that pharmacokinetics of zopiclone is stereoselective.

[In vitro effects of 24R,25-dihydroxyvitamin D3 on alkaline phosphatase and gamma-glutamyltransferase in the kidney of hypophysectomized rats]. / Effets in vitro de la 24R,25-dihydroxyvitamine D3 sur la phosphatase alcaline et la gamma-glutamyltransferase du rein de rat hypophysectomisé.

The effects of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3 on alkaline phosphatase (PAL), gamma-glutamyltransferase (GGT) and acid phosphatase (PAC) activities were investigated on renal cortex slices of hypophysectomized rats. Indeed after hypophysectomy renal 24,25-(OH)2D3 production was increased and renal PAL and GGT activities were decreased. After 5h incubation with physiological concentrations (0.1-10 nM) of 24,25-(OH)2D3 significant increases of PAL and GGT activities were produced. The maximum stimulation obtained with 1 nM was +23% for PAL and +26% for GGT as compared to controls. PAC was not modified. The time course of these effects was studied from 45 min to 8 h. In the presence of 24,25-(OH)2D3 (1 nM), delayed (3h) stimulation of PAL and GGT appeared. It reached the maximal value after 6h, +37% for PAL and +30% for GGT and persisted again at 8h. Cycloheximide added to incubation medium with steroid inhibited the stimulating effect on PAL only. Actinomycin D suppressed the induction of both enzymes, indicating that the observed actions of 24,25-(OH)2D3 depend on protein synthesis whose responsible mechanisms were different. These protein synthesis inhibitors did not modified enzymatic activities. Physiological significance of these renal effects is to be clarified.

Phase II trial of copper zinc superoxide dismutase (CuZn SOD) in the treatment of Crohn's disease.

Bovine Cu Zn SOD was used during an 8 year period as an antiinflammatory drug in 26 patients with severe Crohn's disease (CDAI 300) usually after failure of corticotherapy or when this drug was discontinued because of side effects or infection. This was a phase II trial during which doses routes of administration and concomitant therapies were progressively modified. We obtained 73% good short term responses (judged upon CDAI and anatomic healing) and 82% positive results on long term evolution (the criteria were: i CDAI lower than 100 in between relapses, ii complete healing or notable improvement of lesions, iii no surgery needed, iv return to work. The acceptability was excellent with the free enzyme. Since the above described experience, published in Free Radical Biology and Medicine (1989, 7: 145-151), we used always the same treatment schedule (SOD 8 mg/day associated with Desferroxamine--500 mg subcutaneous every 2 days). The follow-up during the 87-89 period showed that 12 are in good health without any relapse, 9 experienced one or more relapses, and showed good responses upon resumption of treatment, 5 failed to respond to treatment, all part of the initial group on which SOD treatment had already failed, and among whom 3 were lost for follow-up before 1987, and two others took up another SOD treatment which also failed. 3 new patients (2 females, 1 male) were treated since then, and all 3 had positive results (one with disappearance of ileocoecal mass). The efficacy of SOD as an antiinflammatory drug in Crohn's disease needs to be confirmed by controlled trials.