Marie-Claude Menet

Sprague–Dawley rats display metabolism-mediated sex differences in the acute toxicity of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)

The use of the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has been associated with unexplained deaths. Male humans and rodents are more sensitive to acute toxicity than are females, including a potentially lethal hyperthermia. MDMA is highly metabolized to five main metabolites, by the enzymes CYP1A2 and CYP2D. The major metabolite in rats, 3,4-methylenedioxyamphetamine (MDA), also causes hyperthermia. We postulated that the reported sex difference in rats is due to a sexual dimorphism(s). We therefore determined (1) the LD50 of MDMA and MDA, (2) their hyperthermic effects, (3) the activities of liver CYP1A2 and CYP2D, (4) the liver microsomal metabolism of MDMA and MDA, (5) and the plasma concentrations of MDMA and its metabolites 3 h after giving male and female Sprague-Dawley (SD) rats MDMA (5 mg.kg(-1) sc). The LD50 of MDMA was 2.4-times lower in males than in females. MDMA induced greater hyperthermia (0.9 degrees C) in males. The plasma MDA concentration was 1.3-fold higher in males, as were CYP1A2 activity (twice) and N-demethylation to MDA (3.3-fold), but the plasma MDMA concentration (1.4-fold) and CYP2D activity (1.3-fold) were higher in females. These results suggest that male SD rats are more sensitive to MDMA acute toxicity than are females, probably because their CYP1A2 is more active, leading to higher N-demethylation and plasma MDA concentration. This metabolic pathway could be responsible for the lethality of MDMA, as the LD50 of MDA is the same in both sexes. These data strongly suggest that the toxicity of amphetamine-related drugs largely depends on metabolic differences.

Sprague–Dawley rats display sex-linked differences in the pharmacokinetics of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolite 3,4-methylenedioxyamphetamine (MDA)

The use of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has increased in recent years; it can lead to life-threatening hyperthermia and serotonin syndrome. Human and rodent males appear to be more sensitive to acute toxicity than are females. MDMA is metabolized to five main metabolites by the enzymes CYP1A2, CYP2D and COMT. Little is presently known about sex-dependent differences in the pharmacokinetics of MDMA and its metabolites. We therefore analyzed MDMA disposition in male and female rats by measuring the plasma and urine concentrations of MDMA and its metabolites using a validated LC-MS method. MDA AUC(last) and C(max) were 1.6- to 1.7-fold higher in males than in females given MDMA (5 mg/kg sc), while HMMA C(max) and AUC(last) were 3.2- and 3.5-fold higher, respectively. MDMA renal clearance was 1.26-fold higher in males, and that of MDA was 2.2-fold higher. MDMA AUC(last) and t(1/2) were 50% higher in females given MDMA (1 mg/kg iv). MDA C(max) and AUC(last) were 75-82% higher in males, with a 2.8-fold higher metabolic index. Finally, the AUC(last) of MDA was 0.73-fold lower in males given 1 mg/kg iv MDA. The volumes of distribution of MDMA and MDA at steady-state were similar in the two sexes. These data strongly suggest that differences in the N-demethylation of MDMA to MDA are major influences on the MDMA and MDA pharmacokinetics in male and female rats. Hence, males are exposed to significantly more toxic MDA, which could explain previously reported sexual dysmorphism in the acute effects and toxicity of MDMA in rats.

The role of stearic acid in ascorbic acid protection from degradation: a heterogeneous system for homogeneous thermodynamic data

A heterogeneous system between vitamin C and stearic acid was characterized by thermal and crystallographic analyses. The results showed that such a system prevents vitamin C from thermal decomposition. The evidence was provided by implementing a new protocol associating chromatic assays and thermal analyses in order to quantify the percentage of non-degraded vitamin C. The results collected with the vitamin C–stearic acid mixtures allowed deducing coherent interpretation of the results obtained with pure vitamin C at different heating scan rates. Vitamin C mainly degrades upon melting but also in the solid state for temperature close to the melting point when the heating rates are very low. Under these conditions, the temperature determined at the onset of the DSC graphs cannot be associated with the melting temperature but with a fusion-degradation phenomenon. At higher scan rates, the onset as well as the endothermic value of the signal increase to reach plateau values. These values have been identified as the temperature and enthalpy values of melting of vitamin C according to the results obtained from the heterogeneous system.

Ultra high performance liquid chromatography-quadrupole-time of flight analysis for the identification and the determination of resveratrol and its metabolites in mouse plasma.

Resveratrol is a polyphenol that has numerous interesting biological properties, but, per os, it is quickly metabolized. Some of its metabolites are more concentrated than resveratrol, may have greater biological activities, and may act as a kind of store for resveratrol. Thus, to understand the biological impact of resveratrol on a physiological system, it is crucial to simultaneously analyze resveratrol and its metabolites in plasma. This study presents an analytical method based on UHPLC-Q-TOF mass spectrometry for the quantification of resveratrol and of its most common hydrophilic metabolites. The use of (13)C- and D-labeled standards specific to each molecule led to a linear calibration curve on a larger concentration range than described previously. The use of high resolution mass spectrometry in the full scan mode enabled simultaneous identification and quantification of some hydrophilic metabolites not previously described in mice. In addition, UHPLC separation, allowing run times lower than 10 min, can be used in studies that requiring analysis of many samples.

Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes

Flavone-8-acetic acid (FAA) is a potent anticancer agent in mouse but has not shown activity in humans. Because FAA metabolism could play a role in this interspecies difference, our aim was to identify the metabolites formed in vitro using mouse microsomes compared with those in human microsomes. Mouse microsomes produced six metabolites as detected by reversed-phase high-performance liquid chromatography-mass spectrometry (MS). Three metabolites were identified as the 3′-, 4′-, or 6-hydroxy-FAA, by comparison with retention times and UV and MS spectra of standards. Two metabolites presented a molecular weight of 296 (FAA = 280) indicating the presence of one oxygen but did not correspond to any monohydroxylated FAA derivative. These two metabolites were identified as epoxides because they were sensitive to epoxide hydrolase. The position of the oxygen was determined by the formation of the corresponding phenols under soft acidic conditions: one epoxide yielded the 3′- and 4′-hydroxy-FAA, thus corresponding to the 3′,4′-epoxy-FAA, whereas the other epoxide yielded 5- and 6-hydroxy-FAA, thus identifying the 5,6-epoxy-FAA. The last metabolite was assigned to the 3′,4′-dihydrodiol-FAA because of its molecular weight (314) and sulfuric acid dehydration that indicated that the 3′- and 4′-positions were involved. Compared with mouse microsomes, human microsomes (2 pools and 15 individual microsomes) were unable to metabolize FAA to a significant extent. In conclusion, we have identified six new FAA metabolites formed by mouse microsomes, whereas human microsomes could not metabolize this flavonoid to a significant extent. The biological importance of the new metabolites identified herein remains to be evaluated.

Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model

Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp−/−) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murine Bcrp promoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.

Resveratrol metabolism in a non-human primate, the grey mouse lemur (Microcebus murinus), using ultra-high-performance liquid chromatography-quadrupole time of flight.

The grey mouse lemur (Microcebus murinus) is a non-human primate used to study the ageing process. Resveratrol is a polyphenol that may increase lifespan by delaying age-associated pathologies. However, no information about resveratrol absorption and metabolism is available for this primate. Resveratrol and its metabolites were qualitatively and quantitatively analyzed in male mouse-lemur plasma (after 200 mg.kg−1 of oral resveratrol) by ultra-high performance liquid chromatography (UHPLC), coupled to a quadrupole-time-of-flight (Q-TOF) mass spectrometer used in full-scan mode. Data analyses showed, in MSE mode, an ion common to resveratrol and all its metabolites: m/z 227.072, and an ion common to dihydro-resveratrol metabolites: m/z 229.08. A semi-targeted study enabled us to identify six hydrophilic resveratrol metabolites (one diglucurono-conjugated, two monoglucurono-conjugated, one monosulfo-conjugated and two both sulfo- and glucurono-conjugated derivatives) and three hydrophilic metabolites of dihydro-resveratrol (one monoglucurono-conjugated, one monosulfo-conjugated, and one both sulfo- and glucurono-conjugated derivatives). The presence of such metabolites has been already detected in the mouse, rat, pig, and humans. Free resveratrol was measurable for several hours in mouse-lemur plasma, and its two main metabolites were trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate. Free dihydro-resveratrol was not measurable whatever the time of plasma collection, while its hydrophilic metabolites were present at 24 h after intake. These data will help us interpret the effect of resveratrol in mouse lemurs and provide further information on the inter-species characteristics of resveratrol metabolism.

Population Pharmacokinetics of 3,4-Methylenedioxymethamphetamine and Main Metabolites in Rats

The pharmacokinetics of the recreational drug 3,4-methylenedioxymethamphetamine (MDMA) and its mains metabolites have never been modeled together. We therefore designed a model with which to analyze the pharmacokinetics of MDMA, 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) and to test the effect of covariates like gender and body weight on the pharmacokinetics. Rats (18 males and 18 females) were given 1 mg/kg MDMA iv, and the concentrations of MDMA, MDA, and HMMA were measured by high-performance liquid chromatography-mass spectrometry. Another 30 rats (15 males) were given 1 mg/kg MDA, and MDA and HMA were measured. A population pharmacokinetic model was developed to describe the changes in MDMA, HMMA, MDA, and HMA concentrations over time and to estimate interanimal variability. The influence of gender was tested using a likelihood ratio test. Estimated exposures of males and females to MDMA and its metabolites were compared using the Wilcoxon nonparametric test. An integrated six-compartment model adequately described the data. MDMA (two compartments) was transformed irreversible to HMMA (one compartment) and MDA (two compartments), which then produced HMA (one compartment). All rate constants were first order. Females given MDMA had significantly smaller MDMA distribution volumes than males, and they converted less MDMA to MDA than did males. Our MDMA, MDA, HMA, and HMMA model is suitable for examining the relationship between drug concentrations and its pharmacological/toxicological effects. Male rats were exposed to significantly more MDA and HMA than were females, which could explain why males are more sensitive to MDMA toxic effects than females.

Determination of 3,4-methylenedioxymethamphetamine and its five main metabolites in rat urine by solid-phase extraction and high performance liquid chromatography with on line mass spectrometry

The consumption of psychostimulant amphetamine-like drugs has increased significantly in recent years. Some MDMA metabolites are probably involved in the neurotoxicity and neurodegeneration caused by prolonged use rather than MDMA itself. We recently developed a method to analyze MDMA and its five main metabolites in rat plasma [7]. We have now fully validated this method to the quantification of these drugs in rat urine. We extracted MDMA and its metabolites with Oasis WCX cartridges, separated them on a Nucleodur C18 analytical column and quantified them by ion-trap mass spectrometry. Linearity was excellent: 12.5-1250ng/mL urine for HMA, HMMA, MDA and MDMA, 25-2500ng/mL for HHMA, and 150-7500ng/mL for HHA (r(2)>0.993 for all analytes). The lower limits of quantification were 12.5ng/mL urine for MDMA, MDA, HMA and HMMA, 25ng/mL for HHMA and 150ng/mL for HHA. Reproducibility was good (intra-assay precision=1.7-6.1%; inter-assay precision=0.6-5.7%), as was accuracy (intra-assay deviation=0.1-4.8%; inter-assay deviation=0.7-7.9%). Average recoveries were around 85.0%, except for HHMA (66.2%) and HHA (53.0%) (CV<8.3%). We also checked the stability of stock solutions and the internal standards after freeze-thawing and in the autosampler. Lastly, we measured the MDMA, MDA, HHMA, HHA, HMMA and HMA in urine samples taken over 24h from rats given subcutaneous MDMA.

Incidence of the melting-degradation process of vitamin C on the determination of the phase diagram with acetaminophen enhanced by high performance liquid chromatography tools

Compounds that degrade upon melting, such as vitamin C, are not convenient for temperature-composition phase diagram survey. To bypass such a disadvantage, faster scan rates may be used to heat the samples. However, in that case, the thermodynamic solid–liquid equilibrium cannot be reached as the present study demonstrates regarding the vitamin C–acetaminophen phase diagram determination. In this work, high performance liquid chromatography has therefore been used as a complementary tool with differential scanning calorimetry to determine the appropriate liquidus point for various mole fractions of vitamin C–acetaminophen. Because a classical thermodynamic approach is not sufficient for such a study, the analytical experiments that we have conducted provide useful data, allowing us to determine the non-degraded vitamin C quantity in the vitamin C–acetaminophen mixtures treated at a given temperature and annealing time.