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M Martinet

Publications and source records attributed to M Martinet.

At least 19 recordsLinked to original sources
PubMed2004

Liquid chromatography-accurate radioisotope counting and microplate scintillation counter technologies in drug metabolism studies.

The present study involves an analysis of the performance of liquid chromatography (LC)-accurate radioisotope counting (ARC) and microplate scintillation counter (TopCount) technologies in drug metabolism studies. For the purpose of evaluating these systems, biological samples resulting from the metabolism of a radiolabeled [14C] compound, known as compound B, are analyzed using LC-ARC and TopCount under similar high-performance LC conditions. Counting efficiency is 83% for LC-ARC, 77% for TopCount, and linearity is R2 of 0.9998 versus 0.9984, respectively. The limit of detection for LC-ARC is 12 disintegrations per minute (dpm) with 1-min/fraction counting, yet for TopCount it is 8.7 dpm with 5-min/fraction counting. Under optimal conditions for each, the total run time of LC-ARC is approximately half that of TopCount. These results indicate that there is no significant difference between these two systems in terms of efficiency, linearity, and limit of detection. However, the LC-ARC system does not involve any manual operations, yet TopCount requires manual sample transfer and data import. This study shows that impressive progress has been made in the technology of radioisotope counting in drug metabolism using LC-ARC. This system enhances the resolution of radiochromatograms and is able to measure volatile metabolites that TopCount cannot detect at all. The ability to acquire mass spectra online is also a major advancement. The overall results suggest that the combination of LC-ARC with radioactivity detection and mass spectrometry has great potential as a powerful tool for radioisotope measurement in metabolite identification studies during drug discovery and development.

Chromatography, Liquid↗
PubMed2001

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.

Adolescent↗
PubMed1998

Role of human cytochrome P450 3A4 and 3A5 in the metabolism of taxotere and its derivatives: enzyme specificity, interindividual distribution and metabolic contribution in human liver.

Taxotere, a promising anticancer agent, is metabolized almost exclusively in liver and excreted from bile in all species. To determine which cytochrome P450 is involved in taxotere biotransformation, 11 cDNA-expressed human cytochrome P450s were examined for their activity in the metabolism of taxotere and its derivatives. Of all P450s, cytochrome P450 3A4 and 3A5 were the most active for the oxidation of taxotere to the primary metabolite RPR104952 and for subsequent oxidation of RPR104952 to RPR111059 and RPR111026. RP70617, an epimer of taxotere was also metabolized by both P450 3A enzymes to form metabolite XII. The activity of 3A4/5 enzymes for these substrates was 4-50-fold greater than the other P450s examined. The Kms of 3A4 and 3A5 for taxotere were 0.91 and 9.28 microM, and Vmax for the formation of RPR104952 were 1.17 and 1.36 m(-1), respectively. The contribution of the 3A enzyme complex to the metabolism of taxotere in human livers from 21 individuals was assessed with the inhibitory monoclonal antibody and ranged from 64-93%. The primary oxidative metabolism of taxotere by human liver microsomes was well correlated with 3A4-dependent reactions for testosterone 6beta-hydroxylation (r2 = 0.84), taxol aromatic hydroxylation (r2 = 0.67) and aflatoxin B1 3alpha-hydroxylation (r2 = 0.63); whereas a poor correlation was found for reactions specifically catalysed by other P450s (all r2 < or =O.17). The extent of taxotere metabolism also closely correlated with levels of 3A4 enzyme in human livers quantified with immunoblot monoclonal antibody (r2 = 0.61). These results demonstrate that the P450 3A4 and 3A5 enzymes are major determinants in taxotere oxidation and suggest that care must be taken when administering this drug with other drugs that are also substrates for these enzymes.

Adolescent↗
PubMed1997

Human liver CYP2B6-catalyzed hydroxylation of RP 73401.

RP 73401 is a potent inhibitor of cyclic nucleotide phosphodiesterase type IV. RP 73401 is metabolized by human liver microsomes almost exclusively by transhydroxylation of the cyclopentyl group to RPR 113406. Liquid chromatography/mass spectrometry/mass spectrometry analysis of plasma from patients given RP 73401 also revealed a molecular ion and fragmentation consistent with RPR 113406. Thus, the objective was to determine the oxidative enzyme(s) responsible for RP 73401 hydroxylation. Kinetic constants of RP 113406 formation ranged from 8 to 26 MM and 0.83 to 5.99 nmol/min/mg protein for K(m) and V(max), respectively (n = 3). Enzyme activity varied 23-fold among 15 human liver microsome samples and correlated with CYP2A6-catalyzed coumarin hydroxylase (r2 = 0.85, P < .01) and CYP2B6-catalyzed 7-ethoxytrifluoromethylcoumarin O-deethylase (r2 = 0.82, P < .01) activities. Chemical inhibition studies showed a 63% decrease in RP 73401 hydroxylation by 500 microM orphenadrine. Coumarin (10 microM), however, did not inhibit RP 73401 hydroxylation. Also, anti-CYP2B1 IgG produced 85% inhibition of RP 73401 hydroxylation, but only a negligible decline in coumarin hydroxylase activity. Of the 10 expressed P450 forms studied, only CYP2B6 catalyzed RP 73401 hydroxylation. Finally, expressed CYP2B6 showed a high affinity (K(m) = 22.5 microM) for RP 73401 hydroxylation, similar to the human liver microsome studies.

Aryl Hydrocarbon Hydroxylases↗
PubMed1997

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.

Biotransformation↗
PubMed1996

Hepatic biotransformation of docetaxel (Taxotere) in vitro: involvement of the CYP3A subfamily in humans.

Docetaxel metabolism mediated by cytochrome P450-dependent monooxygenases was evaluated in human liver microsomes and hepatocytes. In microsomes, the drug was converted into four major metabolites resulting from successive oxidations of the tert-butyl group on the synthetic side chain. Enzyme kinetics appeared to be biphasic with a V(max) and apparent K(m) for the high-affinity site of 9.2 pmol/min/mg and 1.1 microm, respectively. the intrinsic metabolic clearance in human liver microsomes (V(max)/K(m), 8.4 ml/min/g protein) was comparable to that in rat and dog liver microsomes, but lower in mouse liver microsomes. Although the metabolic profile was identical in all subjects, a large quantitative variation in docetaxel biotransformation rates was found in a human liver microsome library, with a ratio of 8.9 in the highest:lowest biotransformation rates. Docetaxel biotransformation was correlated significantly (0.7698; P < 0.0001) with erythromycin N-demethylase activity, but not with aniline hydroxylase or debrisoquine 4-hydroxylase. It was inhibited, both in human hepatocytes and in liver microsomes, by typical CYP3A substrates and/or inhibitors such as erythromycin, ketoconazole, nifedipine, midazolam, and troleandomycin. Docetaxel metabolism was induced in vitro in human hepatocytes by dexamethasone and rifampicin, both classical CYP3A inducers. These data suggest a major role of liver cytochrome P450 isoenzymes of the CYP3A subfamily in docetaxel biotransformation in humans. Finally, some Vinca alkaloids and doxorubicin were shown to inhibit docetaxel metabolism in human hepatocytes and liver microsomes. These findings may have clinical implications and should be taken into account in the design of combination cancer chemotherapy regimens.

Animals↗
PubMed1995

Immunoglobulin G, F(AB')2, and fab fragment uptake kinetics in isolated perfused rat liver and rat hepatic cells.

The interaction of 125I-radiolabeled immunoglobulin G (IgG), F(ab')2, and Fab fragments with different modes of production (polyclonal or monoclonal), belonging to different subclasses (IgG1 and IgGT) and derived from different sources (mouse, rat, and horse) with liver, was investigated by using isolated perfused rat liver and isolated rat hepatic parenchymal cells (PCs) and non-parenchymal cells (NPCs) in suspension. Lactosaminated-bovine serum albumin (Lac-BSA) and formaldehyde-bovine serum albumin were used as markers of specific binding to PCs and NPCs, respectively. Using the isolated perfused rat liver model, data clearly indicated a very weak hepatic extraction ratio (< 0.003) for IgGs and fragments in comparison with Lac-BSA (extraction ratio = 0.398) over the 3 hr of the experiments. No breakdown or higher molecular weight compounds were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Biliary excretion of IgGs and fragments ranged from 0.07 to 0.3%, mainly as free iodine-125. In contrast, 7% of Lac-BSA was excreted unchanged in bile, and 10% of free iodine was excreted at 3 hr. In vitro binding studies showed no specific binding of any antibody and fragment proteins at 4 degrees C or 37 degrees C. In contrast, saturable uptake was observed for Lac-BSA with PCs and formaldehyde-bovine serum albumin with NPCs. Both models demonstrated that nonspecific antibody/fragment interactions occurred with rat liver. Several hypotheses can be formulated to explain why liver-antibody interactions depend on more complex antibody molecular states (aggregated structure and immune complex) rather than the monomeric structure investigated in the present study.

Animals↗
PubMed1990

Comparison of anticholinergic effects of cibenzoline, disopyramide, and atropine.

The anticholinergic effects of cibenzoline, disopyramide, and atropine were compared on experimental models. Using inhibition of specific binding of 3H-quinuclidinyl benzylate (3H-QNB) in rat heart and cerebral cortex, Ki values were 15.8 +/- 1.6, 12 +/- 3.5, and 0.013 +/- 0.001 microM, respectively, for heart membranes and 31.6 +/- 1.5, 7.8 +/- 1.3, and 0.006 +/- 0.001 microM, respectively, for cerebral cortex membranes. In isolated guinea pig ileum, disopyramide was about 15 times more anticholinergic than cibenzoline but about 900 times less so than atropine. In anesthetized dogs, the three drugs administered by intravenous bolus reduced bradycardia caused by vagal stimulation. The effect of cibenzoline at 7 mg/kg i.v. (double the antiarrhythmic dose) was approximately the same as that of disopyramide at 2.5 mg/kg (half the antiarrhythmic dose). The drugs were infused for 1 h at 0.17 mg/kg/h for atropine, 11.6 mg/kg/h for disopyramide, and 5.5 mg/kg/h for cibenzoline. The maximal inhibition of the vagal stimulation was 98, 95, and 52%, respectively, for the three drugs. In nonanesthetized dogs, inhibition of the vagal-tone-induced tachycardia reached 33 +/- 4, 134 +/- 20, and 206 +/- 19% for cibenzoline, disopyramide and atropine, respectively. These results show cibenzoline to exert less potent anticholinergic effects than disopyramide.

Animals↗
PubMed1989

[The handicap].

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Persons with Disabilities↗
PubMed1985

In vitro and in vivo antagonistic regulation by estradiol and progesterone of the rat pituitary domperidone binding sites: correlation with ovarian steroid regulation of the dopaminergic inhibition of prolactin secretion in vitro.

The influences of in vivo and in vitro estradiol (E2) and progesterone (P) treatments on the characteristics of [3H]domperidone binding to intact and ovariectomized (OVX) rat pituitary membranes were analyzed and compared to the modulation by these steroids of dopamine (DA) inhibition of PRL secretion in vitro from intact and OVX rat pituitaries. Using intact rat pituitaries, high and low affinity binding sites for domperidone were detected; the dose-dependent DA inhibition curve of PRL secretion was biphasic (range, 10(-13) - 10(-10) M DA, IC50 = 6 X 10(-12) M; range, 10(-10) - 10(-6) M DA, IC50 = 2 X 10(-8) M). Using OVX rat pituitaries, only the high affinity sites for domperidone were detected, and the dose-dependent DA inhibition curve of PRL secretion was monophasic (range, 10(-10) - 10(-6) M DA, IC50 = 10(-8) M). E2 and P did not modify the characteristics of the high affinity sites either after in vivo treatment or when directly added to the in vitro binding assay. However, using in vivo and in vitro tests, a modulation of the low affinity sites by E2 and P was demonstrated. When E2 is in excess and P levels are low or undetectable, these sites are not detectable, and P is able to restore there presence. A parallelism has been established between this antagonistic E2 and P regulation and the modulation of DA inhibition of PRL secretion (range, 10(-13) - 10(-10) M DA). When intact rat pituitaries are perifused in the presence of 10(-8) M E2, the biphasic dose-dependent inhibition curve of the control is changed into the monophasic curve of the OVX rat pituitaries. Conversely, when OVX rat pituitaries are perifused in the presence of 10(-6) M P, the monophasic curve of the control is changed into the biphasic curve of the intact rat pituitaries. Thus, the DA inhibition in the range 10(-13) - 10(-10) M might result from an interaction between DA and the low affinity site for domperidone. In summary, the biological regulation of PRL by DA at the pituitary level may be mediated by two different DA sites, one being submitted to an antagonistic E2 and P regulation directly at the membrane level. The consequence of this regulation is that, whereas E2 decreases the sensitivity of the cell to DA, P is necessary for a normal DA response of the lactotroph.

Animals↗
PubMed1983

Modifications of the high and low affinity pituitary domperidone-binding sites in chronic estrogenized rats.

The effect of chronic estrogen treatment on the anterior pituitary domperidone-binding sites was studied in female rats. The rats were implanted from 1-6 months with a Silastic capsule containing 17 beta-estradiol. The Feldman analysis of [3H]domperidone binding to anterior pituitary membranes of control or estrogenized rats revealed the presence of two sites. The binding characteristics of the higher affinity site were identical for both groups (Kd of the high affinity site, 0.30-0.45 nM; maximum number of binding sites of the high affinity site, 74-95 fmol/mg protein); however, those of the lower affinity site were affected by the estrogen treatment: the Kd of the low affinity site increased from 17.4 +/- 3.2 to 41.5 +/- 9 (+/- SE) nM, and the maximum number of binding sites of the low affinity site increased from 214 +/- 22 to 343 +/- 35 fmol/mg protein. Thus, in chronic estrogenized rats, the total number of binding sites was increased by 54%. These changes, induced by chronic estrogenization, were reversible, since 2 weeks after removal of the 17 beta-estradiol pellet, the binding characteristics were no longer different from those observed in control rats. In contrast to chronic estrogen treatment, ovariectomy reduced markedly the total number of [3H]domperidone-binding sites in anterior pituitary membranes (-70%). Feldman analysis revealed that this reduction resulted from the complete disappearance of the low affinity sites in those membranes. No significant change in the binding characteristics of the high affinity site was detected in ovariectomized rats. Since estradiol induces a decrease in the anterior pituitary content of dopamine, a denervation supersensitivity-like mechanism might be responsible for the increase in pituitary domperidone-binding sites in estrogenized rats. Conversely, a hyposensitivity mechanism could be implicated in the decrease in the total number of the pituitary domperidone-binding sites in ovariectomized rats, since pituitary dopamine levels are increased in those animals. Whether the antidopaminergic properties of estrogen are also involved in this modulation after chronic estradiol treatment requires further investigation.

Animals↗
PubMed1979

Kinetics of tryptophan accumulation into synaptosomes of various regions of rat brain.

The kinetics of synaptosomal tryptophan accumulation has been determined in five regions of the rat brain. For tryptophan concentrations ranging from 2.5 -- 20 microM, we found an active uptake in all the structures studied, i.e.: Corpus striatum, midbrain, brainstem, hypothalamus and cerebral cortex + hippocampus. The Vm of tryptophan uptake was highest in the cortex, followed in descending order by corpus striatum, hypothalamus, midbrain and brainstem, while the Km was highest in the cortex, then in descending order corpus striatum, brainstem, midbrain and hypothalamus. In spite of the possible nonspecific high affinity tryptophan uptake into serotoninergic neurons, we found a correlation between the Vm of tryptophan uptake and the different results in the literature concerning uptake and release of serotonin. These observations might indicate a correlation between the Vm of tryptophan uptake and the functional activity of serotonergic neurons.

Animals↗