Matthieu L. Kaltenbach

Simultaneous determination of amoxicillin and clavulanic acid in human plasma by HPLC with UV detection.

A simple and accurate high-performance liquid chromatographic method with ultraviolet detection at 220 nm has been validated for the simultaneous determination of amoxicillin and clavulanic acid in human plasma. Plasma samples were pretreated by direct deproteinization with methanol. A good chromatographic separation between both compounds was achieved using a reversed phase C8 column and a mobile phase, consisting of acetonitrile-phosphate solution-tetramethyl ammonium chloride solution. The calibration curves were linear over the concentration range of 0.625-20 mg l(-1) for amoxicillin and 0.3125-10 mg l(-1) for clavulanic acid with determination coefficients > 0.998. The method is accurate (bias < 7%) and reproducible (intra- and inter-day R.S.D. < 15%), with a quantitation limit of 0.625 and 0.3125 mg l(-1) for amoxicillin and clavulanic acid, respectively. Analytical recoveries from human plasma ranged from 91 to 102% for both components. This fully validated method, which allows the simultaneous measurement of amoxicillin and clavulanic acid in biological samples, is rapid (total run time < 10 min) and requires only a 100 microl sample. This assay is suitable for biomedical applications and was successfully applied to a pilot pharmacokinetics study in healthy volunteers after a single-oral administration of amoxicillin/clavulanic acid combination (500/125 mg).

Trichobilharzia spp. in natural conditions in Annecy Lake, France

Annecy Lake is a well-known focus of human cercarial dermatitis in France. Identification of the parasites, however, was not performed in the past. Previous studies suspected two species, Trichobilharzia franki and Trichobilharzia regenti, based on the presence of parasites in mallards and/or morphological identification of snails emitting ocellate furcocercariae. Following a standardized molecular approach, we studied snails and furcocercariae and compared their haplotypes with those deposited in GenBank. The selected markers were the second internal transcribed spacer ITS-2 for the snails and ITS-2 and D2 domain of the ribosomal DNA for the parasites. Our results confirm the presence of T. franki and T. regenti and two probable new species that could be potential agents of cercarial dermatitis. All the snails emitting the ocellate furcocercariae belong to the same species identified as Radix peregra (=Radix ovata = Radix balthica). Parasite–host relationships between species of the genus Trichobilharzia and snails of the genus Radix do not seem to be as specific as supposed previously.

Systématique moléculaire des phlebotominae : étude pilote. paraphylie du genre phlebotomus

Phylogenetic relationships among Phlebotominae were inferred through a pilot study using parsimony analysis of the D2 domain of ribosomal DNA sequences: 455 pairs of bases were sequenced in nine species of Phlebotomine sandflies which belong to the genera Lutzomyia, Phlebotomus and Sergentomyia. Two taxa are used as outgroups: Psychoda sp. and Nemapalpus flavus which is the sister group of the Phlebotominae. The South American genus Lutzomyia appears to be monophyletic. The Mediterranean species Sergentomyia dentata is its sister group and is not clustered with the Old World genus Phlebotomus. The latter is a paraphyletic genus with an early individualization of the branch including the closely related subgenera Phlebotomus and Paraphlebotomus, and a late individualization of the subgenus Larroussius. These results have some consequences on the biogeography of the leishmaniasis in the Old World.

Avian schistosomes in French aquatic birds: a molecular approach.

The prevalence of human cercarial dermatitis (HCD) caused by bird schistosomes appears to be increasing in France, in light of the impact of tourism combined with high densities of wild aquatic hosts in freshwater areas. The present work expands our knowledge of schistosome systematics by including samples of bird schistosomes collected from their natural hosts in France. Heads (318) and viscera (81) of aquatic birds belonging to 16 species from five orders, collecting during the hunting seasons or found dead, were autopsied for nasal and visceral schistosomes. Eggs and/or adults were analysed by molecular methods using the D2 domain and the second internal transcribed spacer (ITS-2) region of rDNA to determine species. Even if nasal eggs were polymorphic according to the host, all haplotypes were similar to that of Trichobilharzia regenti. Marked diversity of visceral species was observed. Final hosts under natural conditions were reported. For the first time, Trichobilharzia franki is reported in its natural bird hosts, Anas platyrhynchos, Anas crecca, Aythya fuligula and Cygnus olor. We also identified T. szidati in A. crecca and Anas clypeata. Bilharziella polonica was found in six species of aquatic birds, including Grus grus. This finding is the first record of bird schistosomes in this aquatic bird. Three new taxa of visceral schistosomes in Anser anser are strongly suspected according to their haplotypes. Futhermore, a new haplotype of visceral schistosomes isolated in Cygnus olor and similar to Allobilharzia visceralis was identified.

Pharmacokinetics of methotrexate in the extracellular fluid of brain C6-glioma after intravenous infusion in rats.

AbstractPurpose. Establishment of the pharmacokinetic profile of methotrexate (MTX) in the extracellular fluid (ECF) of a brain C6-glioma in rats.nMethods. Serial collection of plasma samples and ECF dialysates after i.v. infusion of MTX (50 or 100 mg/kg) for 4 h. HPLC assay.nResults. Histological studies revealed the presence of inflammation, edema, necrosis, and hemorrhage in most animals. In vivo recovery (reverse dialysis) was 10.8 ± 5.3%. MTX concentrations in tumor ECF represented about 1−2% of the plasma concentrations. Rapid equilibration between MTX levels in brain tumor ECF and plasma. ECF concentrations almost reached steady-state by the end of the infusion (4 h), then decayed in parallel with those in plasma. Doubling of the dose did not modify MTX pharmacokinetic parameters (t1/2α, t1/2β, MRT, fb, Vd, and CLT), except for a 1.7-fold increase of AUCPlasma and a 3.8-fold increase in AUCECF which resulted in a 2.3-fold increase in penetration (AUCECF/AUCPlasma). In spite of an important interindividual variability, a relationship between MTX concentrations in plasma and tumor ECF could be established from mean pharmacokinetic parameters.nConclusions. High plasma concentrations promote the penetration of MTX into brain tissue. However, free MTX concentrations in tumor ECF remain difficult to predict consistently.

Influence of schedule of administration on methotrexate penetration in brain tumours

The influence of the administration schedule (intravenous (i.v.) bolus versus i.v. infusion) on the pharmacokinetics of methotrexate (MTX) in plasma and extracellular fluid (ECF) of a brain C6-glioma was investigated in rats. MTX concentrations were determined by high performance liquid chromatography (HPLC)-ultraviolet radiation (UV). MTX (50 mg/kg) was administered by i.v. bolus or i.v. infusion (4 h). Concentration-time profiles were fitted to a two-compartment open model. Maximum MTX concentrations ranged between 178 and 294 microgram/ml (i.v. bolus), and between 11 and 24 microgram/ml (i.v. infusion) in plasma. MTX rapidly entered the tumour tissue although its concentrations in the ECF were much lower than those observed in plasma for both modes of administration. In spite of an important interindividual variability, AUC(ECF) was approximately 5-fold higher and mean MTX penetration in tumour ECF (AUC(ECF)/AUC(Plasma)) was approximately 3-fold higher after i.v. bolus than after i.v. infusion administration. These results indicate that i.v. bolus administration schedules promote MTX delivery in brain tumour tissue.

Determination of Free Extracellular Levels of Methotrexate by Microdialysis in Muscle and Solid Tumor of the Rabbit

AbstractPurpose. To determine of the pharmacokinetic profile of methotrexate (MTX) in blood and extracellular fluid (ECF) of VX2 tumor and muscle in rabbits.nMethods. Microdialysis probes were inserted into VX2 tumor and in muscle tissue. Following intravenous administration of MTX (30 mg/ kg), serial collection of arterial blood samples and dialysates of muscle and tumor ECF for 4 h was carried out. Quantitation of MTX and determination of free plasma concentrations was performed by fluorescence polarization immunoassay and ultrafiltration, respectively. Correlations were established between the unbound plasma and ECF MTX concentrations.nResults. Total and free plasma concentrations exhibited a parallel three exponential decay in both healthy and tumorigenic animals. Total clearance (8.9 vs 6.5 ml−1.min−1.kg−1) and volume of distribution (4.0 vs 2.9 1.kg−1), however, tended to decrease in the tumor-bearing group. The ECF/plasma AUC ratio equaled 14.2 ± 8.8% in muscle and 23.9 ± 15.9% in tumor. The concentration-time profile of muscle ECF MTX was parallel and highly correlated (r = 0.97) to that determined in plasma. In contrast, free MTX plasma levels were not correlated with tumor ECF concentrations (r = 0.564).nConclusions. In addition to the well-known pharmacological variability in the concentration-effect relationship, the important inter-individual variability in tumor exposure to MTX may partly explain that studies in patients with solid tumors have often failed to demonstrate firm correlations between MTX blood pharmacokinetics and the chemotherapeutic response.

Influence of C6 and CNS1 brain tumors on methotrexate pharmacokinetics in plasma and brain tissue

AbstractPurpose: Comparison of the influence of two different brain tumors (C6 and CNS1 glioma) on methotrexate (MTX) disposition in plasma, brain, and tumor tissue extracellular fluid (ECF).nMethods: Serial collection of plasma samples and brain ECF dialysates after i.v. bolus administration of MTX (50u2009mgu2009kg-1) for 4u2009h. Quantitation of MTX concentrations by HPLC-UV.nResults: Histological studies revealed a 3-fold higher number of blood vessels in CNS1 than in C6 tumor tissue. In vivo recoveries (reverse dialysis) were significantly different in tumor tissue (C6: 8.0 ± 3.8%; CNS1: 4.9 ± 2.5%), and in the contralateral hemisphere (C6: 6.0 ± 4.0%; CNS1: 3.9 ± 2.5%) between the two tumors. Area under the concentration–time curve (AUC) in plasma was 30% higher in CNS1 than in C6 due to a lower systemic clearance. Maximum MTX levels in brain tumor ECF were significantly higher in CNS1 than in C6, and decreased faster in CNS1 than in C6 tumor-bearing rats. Penetration in tumor ECF (AUCECF/AUCPlasma ratio) was similar in CNS1 and C6. MTX concentrations in contralateral hemisphere were significantly lower than in tumor tissue and dependent on tumor model.nConclusion: C6 and CNS1 brain tumors have a distinct yet highly variable impact on MTX penetration in brain and brain tumor ECF.

Distribution of Gacyclidine Enantiomers in Spinal Cord Extracellular Fluid

AbstractPurpose. Determination of the pharmacokinetics of gacyclidineenantiomers, a non-competitive NMDA antagonist, in plasma and spinal cordextracellular fluid (ECF) of rats.nMethods. Implantation of microdialysis probes in spinal cord (T9).Serial collection of plasma samples and ECF dialysates over 5 hoursafter IV bolus administration of (±)-gacyclidine (2.5 mg/kg). Plasmaprotein binding determined in vivo by equilibrium dialysis. ChiralGC/MS assay.nResults. Plasma concentrations of (+)-gacyclidine were ∼25% higherthan those of (−)-gacyclidine over the duration of the experiment inall animals. Plasma concentrations decayed in parallel in a biphasicmanner (t1/2α ∼9 min; t1/2β ∼90 min) with no significant differencebetween enantiomers. Clearance and volume of distribution of(−)-gacyclidine were approximately 20% higher than those of its opticalantipode (CL: 248 vs 197 ml.kg−1.min−1;Vdβ: 31.6 vs 23.5 l/kg).Protein binding (∼90%) was not stereoselective. Both gacyclidineenantiomers were quantifiable in spinal cord ECF 10 min after drugadministration and remained stable over the duration of the experimentin spite of changing blood concentrations. Penetration of(−)-gacyclidine was significantly higher (∼40%) than that of (+)-gacyclidine inall animals. Yet, exposure of spinal cord ECF was similar for bothenantiomers, and not correlated with plasma AUCs.nConclusions. The disposition of gacyclidine enantiomers isstereoselective. Both enantiomers exhibit a high affinity for spinal cord tissueand their distribution may involve a stereoselective and active transportsystem. This hypothesis could also explain the discrepancy betweendrug concentrations in plasma and spinal cord ECF.

Distribution of gacyclidine enantiomers after experimental spinal cord injury in rats: possible involvement of an active transport system.

The pharmacokinetics of gacyclidine enantiomers, a noncompetitive N-methyl-D-aspartate (NMDA) antagonist, were studied in plasma and spinal cord extracellular fluid (ECF) after experimental spinal cord injury in rats. Spinal cord trauma was produced by introducing an inflatable balloon in the dorsal subdural space. Upon implantation of microdialysis probes in spinal cord (T9) and intravenous (iv) bolus administration of (+/-)-gacyclidine (2.5 mg/kg), concentrations in plasma and ECF were monitored over 5 h and analyzed by a stereospecific gas chromatography-mass spectrometry (GC-MS) assay. In plasma, concentrations of (+)-gacyclidine were approximately 25% higher than those of (-)-gacyclidine over the duration of the experiment and decayed in parallel (t(1/2 alpha) approximately 7 min; t(1/2 beta) approximately 90 min) with no significant difference between the two enantiomers. Clearance (CL) and volume of distribution (Vd) of (-)-gacyclidine were approximately 20% higher than those of its optical antipode (CL: 285 versus 236 mL. kg(-1). min(-1); Vd(beta): 39.3 versus 31.2 l/kg). Protein binding (approximately 91%) was not stereoselective. In spinal cord ECF, both enantiomers were quantifiable within 10 min after drug administration, and their concentration remained stable over the duration of the experiment in spite of changing blood concentrations. Repeated iv bolus injections of gacyclidine did not modify these profiles. Areas under the curves (AUCs) of concentration in ECF versus time were similar for both enantiomers and not correlated with AUCs in plasma. Penetration of (-)-gacyclidine was, however, significantly higher (approximately 30%) than that of (+)-gacyclidine. In summary, the disposition of gacyclidine enantiomers is stereoselective. Both enantiomers exhibit a high affinity for spinal cord tissue, and the drug exchange between plasma and spinal cord ECF involves an active transport system. These findings contribute to the explanation of the discrepancy between drug concentrations in plasma and spinal cord ECF.