Evaluation of peripheral versus central effects of GABA(B) receptor activation using a novel, positive allosteric modulator of the GABA(B) receptor ADX71943, a pharmacological tool compound with a fully peripheral activity profile.

BACKGROUND AND PURPOSE: The GABA(B) receptor agonist, baclofen, has shown promising effects in patients suffering from pain, post-traumatic stress disorder, alcoholism, overactive bladder and gastroesophageal reflux disease. However, baclofen's short duration of action and side effects limit its wider use. Here we characterized a novel, GABA(B) receptor positive allosteric modulator (PAM) ADX71943. EXPERIMENTAL APPROACH: In vitro, ADX71943 was assessed for pharmacological activity and selectivity using recombinant and native GABA(B) receptors. In vivo ADX71943 was assessed in the acetic acid-induced writhing (AAW) test in mice and formalin tests (FTs) in mice and rats. Marble burying (MB) and elevated plus maze (EPM) tests, rotarod, spontaneous locomotor activity (sLMA) and body temperature (BT) tests in mice and rats were used to investigate centrally-mediated effects. KEY RESULTS: In vitro, in the presence of GABA, ADX71943 increased the potency and efficacy of agonists and showed selectivity at the GABA(B) receptor. ADX71943 reduced pain-associated behaviours in AAW; an effect blocked by GABA(B) receptor antagonist CGP63360. ADX71943 reduced pain in the FT in mice and rats, but was inactive in the MB and EPM despite reaching high concentrations in plasma. ADX71943 had no effect on BT, rotarod and sLMA. CONCLUSIONS AND IMPLICATIONS: ADX71943 showed consistent and target-related efficacy in tests of disorders that have a significant peripheral component (acute and chronic pain), while having no effect in those associated with centrally-mediated anxiety-like reactivity and side effects. Thus, ADX71943 is a useful pharmacological tool for delineation of peripherally- versus centrally-mediated effects of GABA(B) receptor activation.

High-throughput optimization by statistical designs: example with rat liver slices cryopreservation.

The purpose of this study was to optimize cryopreservation conditions of rat liver slices in a high-throughput format, with focus on reproducibility. A statistical design of 32 experiments was performed and intracellular lactate dehydrogenase (LDHi) activity and antipyrine (AP) metabolism were evaluated as biomarkers. At freezing, modified University of Wisconsin solution was better than Williams'E medium, and pure dimethyl sulfoxide was better than a cryoprotectant mixture. The best cryoprotectant concentrations were 10% for LDHi and 20% for AP metabolism. Fetal calf serum could be used at 50 or 80%, and incubation of slices with the cryoprotectant could last 10 or 20 min. At thawing, 42 degrees C was better than 22 degrees C. After thawing, 1h was better than 3h of preculture. Cryopreservation increased the interslice variability of the biomarkers. After cryopreservation, LDHi and AP metabolism levels were up to 84 and 80% of fresh values. However, these high levels were not reproducibly achieved. Two factors involved in the day-to-day variability of LDHi were identified: the incubation time with the cryoprotectant and the preculture time. In conclusion, the statistical design was very efficient to quickly determine optimized conditions by simultaneously measuring the role of numerous factors. The cryopreservation procedure developed appears suitable for qualitative metabolic profiling studies.

In vitro metabolism and drug interaction potential of a new highly potent anti-cytomegalovirus molecule, CMV423 (2-chloro 3-pyridine 3-yl 5,6,7,8-tetrahydroindolizine I-carboxamide).

AIMS: To identify the enzymes involved in the metabolism of CMV423, a new anticytomegalovirus molecule, to evaluate its in vitro clearance and to investigate its potential involvement in drug/drug interactions that might occur in the clinic. METHODS: The enzymes involved in and the kinetics of CMV423 biotransformation were determined using pools of human liver subcellular fractions and heterologously expressed human cytochromes P450 (CYP) and FMO. The effect of CMV423 on CYP probe activities as well as on indinavir and AZT metabolism was determined, and 26 drugs were tested for their potential to inhibit or activate CMV423 metabolism. RESULTS: CMV423 was oxidized by CYP and not by FMO or cytosolic enzymes. The Km values for 8-hydroxylation to rac-RPR 127025, an active metabolite, and subsequent ketone formation by human liver microsomes were 44 +/- 13 microM and 47 +/- 11 microM, respectively, with corresponding Vmax/Km ratios of 14 and 4 microl min(-1) nmol(-1) P450. Inhibition with selective CYP inhibitors indicated that CYP1A2 was the main isoform involved, with some participation from CYP3A. Expressed human CYP1A1, 1A2, 2C9, 3A4 and 2C8 catalysed rac-RPR 127025 formation with Km values of < 10 microM, 50 +/- 21 microM, 55 +/- 19 microM, circa 282 +/- 61 microM and circa 1450 microM, respectively. CYP1B1, 2A6, 2B6, 2C19, 2D6, 2E1 or 3A5 did not catalyse the reaction to any detectable extent. CYP1A1 and 3A4 also catalysed ketone formation from rac-RPR 127025. In human liver microsomes, CMV423 at 1 and 10 microM inhibited CYP1A2 activity up to 31% and 63%, respectively, CYP3A4 activity up to 40% (10 microM) and CYP2C9 activity by 35% (1 and 10 microM). No effect was observed on CYP2A6, 2D6 and 2E1 activities. CMV423 had no effect on indinavir and AZT metabolism. Amongst 26 drugs tested, none inhibited CMV423 metabolism in vitro at therapeutic concentrations. CONCLUSIONS: CMV423 is mainly metabolized by CYP1A2 and 3A4. Its metabolism should not be saturable at the targeted therapeutic concentrations range (Cmax < 1 microM). CMV423 will probably affect CYP1A2 and 1A1 activities in vivo to some extent, but no other drug-drug interactions are expected.

Cryopreserved rat liver slices: a critical evaluation of cell viability, histological integrity, and drug-metabolizing enzymes.

The effects of a cryopreservation procedure on the biochemical, morphological and functional integrity of rat liver slices just after thawing and after 24 h culture were evaluated. Freshly prepared slices were incubated in modified University of Wisconsin solution containing 50% fetal calf serum and 10% dimethyl sulfoxide for 20 min on ice prior to a rapid cooling in liquid nitrogen. After 10-40 days, slices were thawed rapidly at 42 degrees C. Total protein content and (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) (MTT) reduction were well preserved at thawing, whereas ATP content was markedly decreased relative to freshly prepared slices (-83%). The major microscopic findings in sections of just-thawed liver slices consisted of hepatocellular dissociation and minimal apoptosis. The qualitative profile of antipyrine (AP) metabolism was well preserved in cryopreserved slices, but the amounts of phase I and phase II AP metabolites produced over a 3-h incubation period were markedly reduced relative to fresh slices (-58 to -71%). When cryopreserved slices were cultured for 24 h after thawing, the viability was markedly reduced, as reflected by the almost complete absence of MTT reduction and the loss of ATP content. Histological examinations showed extensive cellular necrosis. The amount of AP metabolites produced by cryopreserved slices was similar after a 3- or a 24-h culture period, indicating that AP metabolism capacities were lost at 24 h culture. In conclusion, our results suggest that cryopreserved rat liver slices may be a useful model for short-term in vitro determination of drug metabolism pathways. Further work is required to extend their use for toxicological studies.

Usefulness of statistic experimental designs in enzymology: example with recombinant hCYP3A4 and 1A2.

First, the effects of 10 incubation factors were screened altogether on nifedipine dehydrogenase (NIF) and methoxyresorufin O-deethylase (MROD) activities catalyzed by recombinant human CYP3A4 and 1A2, respectively. Using a statistic experimental design, only 36 assays were needed to be exhaustive. Eight factors influenced CYP3A4-mediated NIF activity: buffer type, pH, temperature, Mg/EDTA, cytochrome b5, NADPH-P450 reductase, NADH, and solvent. Two factors had no significant effect: total ionic concentration and catalase/reduced glutathione. Six factors influenced CYP1A2-mediated MROD rates: buffer type, pH, temperature, Mg/EDTA, NADH, and glycerol. Four factors had no significant effect: total ionic concentration, cytochrome b5, reductase, and NAD+. Secondly, the combined effects of ionic strength and Mg concentration on NIF/CYP3A4 were studied and easily modeled using another statistic experimental design. The effect of Mg was strong at a constant ionic strength of 100 mM and negligible at a constant ionic strength of 500 mM. Thirdly, the effects of influencing factors and their interactions on MROD/CYP1A2 were modeled after 40 assays using a third statistic experimental design. Later experiments confirmed the predictivity of the models and the efficiency of optimized conditions. This approach can be applied to other biochemistry areas.

Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro.

Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells. In vitro incubation of [14C]riluzole (15 microM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (K(m) = 30 microM) or an unidentified glucuroconjugate (K(m) = 118 microM). Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor alpha-naphthoflavone (IC50 = 0.42 microM). Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative. CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole. Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme. In vitro, human hepatic microsomal hydroxylation of riluzole (15 microM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 approximately 200-500 microM) and cimetidine (IC50 = 940 microM). Riluzole (1 and 10 microM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase. Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites. Direct glucuronidation is a relatively minor metabolic route. In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism.

[Sperm and HIV]. / Sperme et VIH.

Screening and prevention are two major weapons in the fight against AIDS. Procreation of couples where the spouse is seropositive poses a serious ethical social and epidemiological problem. We have consequently established a list of the current biological data concerning HIV in the sperm at the level of non germinal cells, seminal plasma and spermatozoa and of the clinical data on the transmission of the virus during sexual intercourse and insemination. Finally, we have presented the results of the research on predictable factors of the infectiousness of sperm. After this analysis, we consider the possibilities open to the medical corps: dissuasion, ovulation monitoring, insemination with the husband sperm, IVF and insemination with hope to be able to participate in the debate which must lead to a clear concerted and coherent position on the part of the medical corps, at least on a national level.

Detection of HIV-1 infections by PCR: evaluation in a seropositive subject population.

To study the polymerase chain reaction (PCR) performance in detecting human immunodeficiency virus (HIV) infections, we tested 53 HIV-1 seropositive patients and 29 HIV-1 seronegative subjects for four different HIV-1 DNA regions. Fifty-one seropositive patients were found positive by PCR with at least one primer pair, but two were repeatedly negative for all primers. Weekly blood samples from 12 seropositive subjects all detected positive for at least one primer pair, but for three patients an irregular primer detection pattern was found. One additional HIV-1 seropositive sample, found negative for HIV DNA, was also negative for the beta-globin PCR control. The 29 seronegative specimens were HIV-1 DNA negative, as was a HIV-2 seropositive patient. This study demonstrates that PCR is almost as good as serological tests for detecting HIV infections, with a specificity of 100% and a sensitivity of 96% and that resampling the patients may improve detection performance.

Detection of hepatitis B virus DNA in serum by a nonisotopic hybridization technique.

We developed a nonisotopic technique, Hepagene, for measuring hepatitis B virus (HBV) DNA in human serum by using a sulfonated probe that is detected by a sandwich immunoenzymatic reaction. The detection limit, determined by serum dilution tests, was 2.5 ng/L. The precision of the Hepagene test was demonstrated by the accurate reproducibility observed for low (3 ng/L) and medium (38 ng/L) concentrations of HBV DNA assayed in 24 different series. Specificity was established by assaying HBV DNA in sera from 98 patients by the Hepagene technique or by a solution hybridization assay with an 125I-labeled probe. Results by both techniques agreed for 94 sera (96%), with 68 being concordant for HBV DNA negativity and 26 for positivity. HBV DNA titers assayed by both methods also agreed. Hepagene represents the first nonisotopic HBV DNA assay involving a sulfonated probe and with performance characteristics equivalent to those of classical radioactive hybridization techniques.

[Radioimmunoassay of 5-androstene-3 beta, 17 beta-diol in plasma and breast cyst fluid]. / Dosage radio-immunologique du 5-androstène-3 beta, 17 beta-diol dans le plasma et le liquide de kyste mammaire.

A simple and reliable radioimmunoassay for the determination of 5-androstene-3 beta,17 beta-diol (5-Adiol) in peripheral plasma and in breast cyst fluid, after chromatography on Celite microcolum has been described and evaluated. The antiserum used was raised in the rabbit injected with dehydroepiandrosterone-15 alpha-carboxymethyl-bovine serum albumin. In men below 40 years of age the levels ranged from 0.85 to 2.80 ng/ml (mean +/- SEM: 1.52 +/- 0.11; n = 24) and from 0.50 to 2.20 ng/ml (mean: 0.93 +/- 0.09; n = 20) in men aged between 41 and 62 years. The mean level was significantly different (p less than 0.001) between the two groups. A significant correlation (r = -0.56 p less than 0.01) was demonstrated between age and all male levels. In women the mean plasma level was in the follicular phase: 0.81 +/- 0.07 ng/ml (range: 0.40 - 1.50; n = 17; age: 19-41 years) and in the luteal phase: 0.83 +/- 0.05 ng/ml (range: 0.40 - 1.30; n = 29; age: 18-43 years). No cyclical change and no correlation with age could be evidenced. A significant difference (p less than 0.001) was shown between females and the young male group. In breast cyst fluid the levels of unconjugated 5-Adiol ranged from 0.05 to 13.70 ng/ml (mean: 2.21 +/- 0.73; n = 23) whereas the sulfate concentrations ranged from 75 to 7,500 ng/ml (mean: 1,973 +/- 543; n = 18), thus demonstrating very wide inter-individual variations.

Radioimmunoassay of 5-androstene-3 beta,17 beta-diol in plasma and in breast cyst fluid.

A simple and reliable radioimmunoassay for the determination of 5-androstene-3 beta, 17 beta-diol in peripheral plasma and in breast cyst fluid, after a chromatography on Celite microcolumn has been described and evaluated. The antiserum used was raised in rabbits injected with dehydroepiandrosterone-15 alpha-(O-carboxymethyl)-bovine serum albumin. In men below 40 years of age the levels ranged from 0.85 to 2.80 ng/ml (mean +/- SEM: 1.52 +/- 0.11; n = 24) and from 0.50 to 2.20 ng/ml (mean +/- SEM: 0.93 +/- 0.09; n = 20) in men aged between 41 and 62 years. The mean level was significantly different (P less than 0.001) between the 2 groups. A significant correlation (r = -0.56; P less than 0.01) was demonstrated between age and all male levels. In females the mean plasma level was in the follicular phase: 0.81 +/- 0.07 ng/ml (range: 0.40-1.50; n = 17; age: 19-41 years) and in the luteal phase: 0.83 +/- 0.05 ng/ml (range: 0.40-1.30; n = 29; age: 18-43 years). No cyclical change and no correlation with age could be evidenced. A significant difference (P less than 0.001) was shown between females and the young male group. In breast cyst fluid the levels ranged from 0.05 to 13.70 ng/ml (mean +/- SEM: 2.36 +/- 0.86; n = 20) whereas the sulfate concentrations ranged from 75 to 7500 ng/ml (mean +/- SEM: 1891 +/- 565; n = 15), thus demonstrating very wide inter-individual variations.