Distribution and functional characterization of pituitary adenylate cyclase-activating polypeptide receptors in the brain of non-human primates.

The distribution and density of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites have been investigated in the brain of the primates Jacchus callithrix (marmoset) and Macaca fascicularis (macaque) using [(125)I]-PACAP27 as a radioligand. PACAP binding sites were widely expressed in the brain of these two species with particularly high densities in the septum, hypothalamus and habenula. A moderate density of recognition sites was seen in all subdivisions of the cerebral cortex with a heterogenous distribution, the highest concentrations occurring in layers I and VI while the underlying white matter was almost devoid of binding sites. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed intense expression of the mRNAs encoding the short and hop-1 variants of pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) in the cortex of both marmoset and macaque, whereas vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 1 (VPAC1-R) and vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 2 (VPAC2-R) mRNAs were expressed at a much lower level. In situ hybridization histochemistry showed intense expression of PAC1-R and weak expression of VPAC1-R mRNAs in layer IV of the cerebral cortex. Incubation of cortical tissue slices with PACAP induced a dose-dependent stimulation of cyclic AMP formation, indicating that PACAP binding sites correspond to functional receptors. Moreover, treatment of primate cortical slices with 100 nM PACAP significantly reduced the activity of caspase-3, a key enzyme of the apoptotic cascade. The present results indicate that PACAP should exert the same neuroprotective effect in the brain of primates as in rodents and suggest that PAC1-R agonists may have a therapeutic value to prevent neuronal cell death after stroke or in specific neurodegenerative diseases.

The neuropeptide pituitary adenylate cyclase-activating polypeptide exerts anti-apoptotic and differentiating effects during neurogenesis: focus on cerebellar granule neurones and embryonic stem cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from ovine hypothalamus on the basis of its hypophysiotrophic activity. It has subsequently been shown that PACAP and its receptors are widely distributed in the central nervous system of adult mammals, indicating that PACAP may act as a neurotransmitter and/or neuromodulator. It has also been found that PACAP and its receptors are expressed in germinative neuroepithelia, suggesting that PACAP could be involved in neurogenesis. There is now compelling evidence that PACAP exerts neurotrophic activities in the developing cerebellum and in embryonic stem (ES) cells. In particular, the presence of PACAP receptors has been demonstrated in the granule layer of the immature cerebellar cortex, and PACAP has been shown to promote survival, inhibit migration and activate neurite outgrowth of granule cell precursors. In cerebellar neuroblasts, PACAP is a potent inhibitor of the mitochondrial apoptotic pathway through activation of the MAPkinase extracellular regulated kinase. ES cells and embryoid bodies (EB) also express PACAP receptors and PACAP facilitates neuronal orientation and induces the appearance of an electrophysiological activity. Taken together, the anti-apoptotic and pro-differentiating effects of PACAP characterised in cerebellar neuroblasts as well as ES and EB cells indicate that PACAP acts not only as a neurohormone and a neurotransmitter, but also as a growth factor.

[Toxicology of reproduction: predictive value of experimental models. Round Table No 1 at Giens XIII]. / Toxicologie de la reproduction: valeurs prédictives des modèles expérimentaux. Table Ronde No 1 de Giens XIII.

The carrying out of clinical trials with a view to the marketing of drugs for human use is directly related to results of some animal studies. This workshop was devoted to evaluation of the quality and interest of these experimental models in reproductive toxicology. The predictive ability of preclinical trials to make extrapolations from animals to man decreases from foetotoxic to tetratogenic risks respectively and from the effects on fertility in both sexes to postnatal risks. As a result of this workshop, we propose the following improvements: (1) standardization and generalization of fertility test evaluations, especially the spermogram, in order to improve animal and human correlations; (2) development of knowledge and standardization of the follow up of the oestral cycle; (3) improvement of standardization, harmonization and diffusion of postnatal tests that prove relevant in animals; (4) increase in initiatives aimed at better mutual understanding of all drug partners; (5) creation of registers for new drugs, as soon as possible during clinical trials, to study their effects on the whole reproductive process; (6) recommendations for the creation of guidelines for International Conference on Harmonisation (ICH) to enable classification of observed effects in experimental models. This could lead to specific (potentially for each phase of the reproductive cycle) guidelines, precautions for use and/or contraindications which are listed in the summary of product characteristics.

Correlation and validation of alternative methods to the Draize eye irritation test (OPAL project).

A multicentre study of alternative methods to the Draize eye irritation test, involving six different laboratories was organized by OPAL (Oeuvre Pour l'Assistance aux Animaux de Laboratoire). Forty chemicals (including solvents, surfactants, acids, bases, and others) were selected for testing by three methods, namely Griffith's test (a low-volume eye irritation test on rabbits), the hen's egg chorioallantoic membrane (HET-CAM) assay for the evaluation of hyperaemia, haemorrhage and coagulation, and neutral red uptake by SIRC cells for the assessment of cytotoxicity. Each method was used in two or three laboratories. Intralaboratory reproducibility was good for each laboratory with values, for error, close to 10%. Interlaboratory agreement was also good, particularly for the cell culture method, a quantitative and objective technique. Griffith's test correlated well with the Draize test (r = 0.846; n = 37), while for the HET-CAM test (r = 0.670; n = 32) and the cell culture method (r = 0.579; n = 32) the correlation was satisfactory. A more complete statistical analysis is currently under way to confirm and extend these preliminary findings.

Determination of cyclosporine in plasma: specific radioimmunoassay with a monoclonal antibody and liquid chromatography compared.

A radioimmunoassay of cyclosporine (Sandimmune) involving use of a mouse monoclonal antibody was tested to monitor specifically the parent drug in plasma. The cyclosporine concentrations obtained by RIA were compared with those obtained by the HPLC method. For the RIA method, the within- and between-assay CVs are less than 6%, the limit of detection is about 10 micrograms/L for a 50-microL sample of plasma. For the HPLC method, the within- and between-assay CVs are less than 20%, the limit of detection is about 15 micrograms/L for a 1-mL sample of plasma. The concentrations by RIA correlated well with those by HPLC in samples from patients receiving bone-marrow (n = 39), heart (n = 52), or liver (n = 51) transplants. In all indications, the ratio of values by RIA to those by HPLC for these samples remained stable and close to 1 during the drug-monitoring period, i.e., for up to 202 days. Therefore, the specific RIA can be used instead of HPLC to measure the parent drug in plasma.

[Pharmacokinetics of cyclosporin A in patients with nephrotic syndrome]. / Pharmacocinétique de la ciclosporine A chez les patients atteints de syndrome néphrotique.

The pharmacokinetics of Cy A have been studied in patients with N.S.. Eight patients (7 M, 1 F) received a single 12.5 mg/kg oral dose and a single 4 mg/kg intravenous dose. Plasma was separated from red blood cells at 22 degrees C, at least 2 hr after drawing. Cy A plasma levels were determined by reverse HPLC. The comparison of our pharmacokinetic parameters for the oral route with those from reference patients showed significant differences for T1/2 Ka and Tmax which were decreased in N.S. For the I.V. route we found a decrease in total plasma clearance (CLTP). Absolute bioavailability (18%) was also diminished. Moreover total cholesterol and B apolipoprotein were increased in our nephrotic population. Cy A is highly bound to lipoprotein and we found a significant negative correlation between CLTP of Cy A and either B apolipoprotein or total cholesterol. We conclude that the increase of lipoprotein in N.S. is probably responsible of the modifications in pharmacokinetics of Cy A. Nevertheless Cy A dosage can be not modified in N.S. when oral route is used and divided by a factor two for the I.V. route.

Species differences in metabolism of ketotifen in rat, rabbit and man: demonstration of similar pathways in vivo and in cultured hepatocytes.

In vitro drug metabolism by cultured rat, rabbit and human adult hepatocytes has been studied, using ketotifen (ZADITEN) as a model substrate because it is biotransformed in vivo by various metabolic pathways in man and animals. The major in vivo pathways were demonstrated in vitro, namely oxidation in rat hepatocytes, oxidation, glucuronidation and sulfation in rabbit hepatocytes, reduction and glucuronidation in human hepatocytes. Human hepatocytes were the most stable in culture, displaying ketotifen biotransformation for at least one week. These results clearly demonstrated that cultured hepatocytes retain their in vivo specific drug metabolizing activities, including inter-species polymorphism, for a few days. Therefore, pure hepatocyte cultures represent a useful system suitable for drug metabolism studies.

[Pharmacokinetics and metabolism of cyclosporin; drug interactions]. / Pharmacocinétique et métabolisme de la cyclosporine; interactions médicamenteuses.

The pharmacokinetic properties of cyclosporine and mainly its absorption and hepatic elimination can vary in each patient in relation to the subject's individual characteristics (inter-subject variability), as well as in an individual patient during his treatment because of clinical episodes or drug interactions (intra-subject variabilities). Therefore, it appears compulsory to follow regularly cyclosporine blood levels or even to characterise each subject following a dose-test during the initiation of treatment, in order to adapt an individual posology aiming to minimise as far as possible the risk of nephrotoxicity.

Mechanism of uptake of dihydroergotamine by isolated rat hepatocytes: effect of troleandomycin.

The mechanism of uptake of dihydroergotamine (DHE) was studied in isolated rat hepatocytes and the effect of troleandomycin on DHE uptake was examined. The uptake was linear for 75 sec and reached an equilibrium at 5 min with an intracellular/extracellular concentration ratio of approximately 65. The initial velocity of uptake was linearly related to the concentration of DHE in the extracellular medium with a diffusion constant of 127 pmol X min-1 X mg of protein-1 X microM-1. Metabolic inhibitors (KCN, carbonylcyanide-M-chlorophenylhydrazone and antimycin A) had no effect on DHE uptake. Replacement of sodium by choline chloride in the extracellular medium decreased slightly but significantly (P less than .02) the uptake of DHE. The addition of troleandomycin (300 microM) in the incubation medium decreased the initial velocity of uptake of DHE (control, velocity of uptake = 88 pmol X min-1 X mg of protein-1 X microM-1; troleandomycin, velocity of uptake = 55 pmol X min-1 X mg of protein-1 X microM-1; P less than .05). These results suggest that DHE enters into the hepatocytes by passive diffusion. The high intracellular/extracellular concentration ratio suggests that intracellular binding occurs and results in an accumulation of DHE in the cells.

Metabolism of ketotifen by human liver microsomes. In vitro characterization of a tertiary amine glucuronidation.

Biotransformation of ketotifen was investigated in vitro using human liver microsomes. Three of the four metabolic pathways observed in vivo in man were exhibited under the conditions of incubation, namely demethylation, N-oxidation, and N-glucuronidation, the absent route being the ketoreduction, which probably has a cytosolic localization. The kinetic parameters of the N-glucuronidation (KM for ketotifen and UDPGA and Vmax) were determined with native and detergent-treated microsomes. Treatment by Triton X-100 increased by about 3-fold the conjugation reaction. No sex difference was observed and N-glucuronidation did not seem to be inhibited either by bilirubin or by 4-nitrophenol. Thus, human liver microsomes are a useful and suitable in vitro model for studying metabolic routes, specific for man, as in the case of ketotifen. Obviously, the results obtained can only reflect partially the multiplicity of in vivo events and interpretation has to be complemented by investigations with other models.

Effects of N-alkylation and n-acylation on tobramycin activity.

The activities of tobramycin derivatives acetylated and ethylated on the 6'-N,2'-N and 3-N positions were examined. The MICs of these derivatives against tobramycin sensitive strains indicated that 2'-N-ethylated and 6'-N-ethylated derivatives have a fairly good activity, and confirmed that the 3-N position is the most important one for antibiotic activity since 3-N derivatives were less active. The MICs of these derivatives against tobramycin resistant strains, and their inactivation by tobramycin modifying enzymes were examined. These results showed that 2'-N or 6'-N ethylation protects the drug against inactivation by AAC(2') or AAC(6'), respectively, and 2'-N-ethyltobramycin and 6'-N-ethyltobramycin were active against strains containing these modifying enzymes. On the other hand, 3-N ethylation protects the drug against inactivation by AAC(3) but 3-N-ethyl tobramycin does not inhibit strains containing this enzyme.