Études métaboliques des isoenzymes du cytochrome P450 exprimées dans les ventricules de cœurs humains

CYP450 isoenzymes are greatly involved in oxidative drug metabolism and changes in their activity (drug interactions, genetic polymorphisms) can cause significant changes in plasma and tissue concentrations of the drugs. The liver predominantly governs plasma concentrations of drugs because of a high concentration of CYP450s. On average, the elimination of a drug through metabolism accounts for about 75% of clearance mechanisms compared to 20% via renal excretion and 10% via the bile excretion. The CYP450s contribute to about 75% of drug metabolism. It is thus logical to study factors influencing the expression and activity of CYP450s. Abundance and variety of expressed CYP450s differ between the liver and extrahepatic tissues. The role of these enzymes endogenous and drugs concentrations in the extrahepatic tissues is poorly studied despite their involvement and association recognized in some treatment failures, drug interactions or tissue toxicity. The ventricles of the human heart express (messenger RNA) variably some isoenzymes including CYP1A1, 2B6, 2C8, 2C9, 2D6, 2E1, 2J2, 3A5 and 4A11. The participation of these isoenzymes expressed in the cardiac ventricular wall in drug metabolism remains unknown to this day. Establishing the existence of an active participation of some CYP450s, their metabolic profile and contribution to the local clearance and determining factors responsible for variability in activity and expression (mRNA) in the cardiac ventricular tissue human is the main objective of the research presented in this thesis. The first module was the development of drug cocktails to maximize the amount of results through the use of low amount of heart tissue with selective substrates matched with highly sensitive and specific analytical methods (manuscript 1). Of the nine compounds tested 2 substrates, ebastine and chlorzoxazone, probe of CYP2E1 and CYP2J2 respectively, have demonstrated activities leading to a thorough study of their metabolic profile generated by the microsomal fraction of the ventricles of explanted human hearts (MCH). This work demonstrated preferential kinetics, described by a Michaelis-Menten model, and very high affinity towards the MCH hydroxylation pathway for ebastine. Combined with inhibition assays, a link was established, for the first time, between the hydroxylation of ebastine and the isoenzyme CYP2J2 (manuscript 2).  Hydroxylation of ebastine by MCH was also recognized as being highly variable between individuals that went through a heart transplant with life-threatening cardiomyopathies that are no longer controlled by standard therapies or treatments. The aim of this last part was to study factors (extrinsic and intrinsic, such as polymorphism of CYP2J2 * 7) known in these patients and their involvement in the variability of expression (mRNA) and enzymatic activity of CYP2J2 studied through the analysis of the enzyme kinetic profile of ebastine (Km, Vmax, CLint). It has been shown that gender, the left ventricle, use of amiodarone and clinical diagnosis of ischemia were associated with the variability of expression (mRNA) and activity (Km, Vmax, CLint) of CYP2J2 and those factors account for about 20% of the observed variability between individuals. This is the first work to explain a portion of the variability associated with the expression (mRNA) and activity of CYP2J2 in the ventricle of the human heart (manuscript 3). Overall, our results suggested that the metabolic capacity of CYP2J2 by MCH is real and important and could participate in local fluctuations of drug levels. The extrapolation of in vitro results to in vivo scale (whole body) helped to put into perspective the contribution of CYP2J2 in the local physiological context of the cardiac ventricular tissue studied. Our results support the proposal that the free plasma concentration does not necessarily reflect the effective tissue concentration as local mechanisms, in this case enzymatic metabolism by CYP2J2, could greatly provoke variation in free intracellular concentration at the site of action. The results obtained meet a need in research to understand the behavior and extent of participation of various CYP450 isoenzymes known to be expressed in the human heart on the local metabolism of drugs that are distributed locally and ultimately developing tools to predict the intracellular drug concentrations in the physiological context.

Keyphrases: Cytochrome P450, Metabolism