INTRODUCTION: CYP3A subfamily of enzymes is involved in the metabolism of more than 50% of all currently used drugs in humans. Clinically most important CYP3A enzymes are CYP3A4 and CYP3A5. CYP3A5 is polymorphically expressed at approximately 20% of individuals and it shows overlapping substrate specificity with CYP3A4, but the extent of known CYP3A4 substrates metabolized by CYP3A5 at individuals polymorphically expressing it, is under investigation. In vitro studies using recombinant systems are extensively applied to predict pharmacokinetic properties of drugs in vivo, where enzyme kinetic parameters from the in vitro experiments are extrapolated to in vivo values.
AIM: Establishment of a stable THLE cell line specifically expressing human CYP3A5 enzymes was one of the aims of the present thesis. Another aim was to investigate and compare metabolism of midazolam, which is a standard in vitro probe for characterization of the CYP3A activity, in different in vitro systems expressing human CYP3A5 enzymes.
METHODS: Stable transfection of THLE cells with a DNA coding for human CYP3A5 was performed in order to obtain human cell line specifically expressing CYP3A5 enzymes. The metabolism studies of midazolam by CYP3A5 were performed in two in vitro systems: microsomes isolated from baculovirus infected insect cells specifically expressing human CYP3A5 enzymes (Supersomes®), the difference between the two preparations applied being coexpression of cytochrome b5 in only one of them.
RESULTS: The transfection of THLE cells with human DNA coding for CYP3A5 enzymes was achieved, but the cells did not express functional CYP3A5 proteins, which resulted in no metabolite formation after incubation with midazolam. The formation of the main metabolite of midazolam, 1′-hydroxy-midazolam (1′-OH-MDZ), followed Michaelis-Menten like kinetics in Supersomes® with coexpressed cytochrome b5, while the formation of the same metabolite in the Supersomes® without cytochrome b5 demonstrated substrate inhibition. Surprisingly, the formation rate of 1′-OH-MDZ appeared to be higher in the Supersomes® without cytochrome b5. Formation of the minor metabolite of midazolam, 4-hydroxy-midazolam (4-OH-MDZ), had sigmoid shape independently of the presence of cytochrome b5, but the formation rate was lower in the Supersomes® with coexpressed cytochrome b5.
CONCLUSION: Cytochrome b5 appears to have an impact on midazolam metabolism catalyzed by CYP3A5 in vitro. For further evaluation, more experiments are necessary, and the conditions need to be optimalized in order to obtain functional CYP3A5 proteins from the THLE cells, so that the comparisons of the CYP3A5 catalyzed metabolism of midazolam between insect and human microsomes can be performed.