"In-vivo" phenotyping of CYP3A using midazolam as probe drug : development of novel approaches based on highly sensitive LC-MS/MS methods

Cytochrome P450 (CYP) 3A is the most important subfamily of drug-metabolizing enzymes in humans, responsible for metabolizing more than 50% of the currently marketed drugs. Many studies on CYP3A-mediated drug metabolism in humans have demonstrated that CYP3A activity exhibits marked interindividual variability, rendering the dosing and use of many CYP3A substrates difficult, especially those with narrow therapeutic ranges. Furthermore, many clinical drug-drug interactions have been ascribed to induction and/or inhibition of CYP3A. Direct assessment of the functional CYP3A activity ("Phenotyping") might therefore be helpful to predict enzyme induction or inhibition, to improve the therapeutic outcome, by minimizing side effects and maximizing therapeutic efficiency, and might be also useful for guiding therapeutic dosing.
Several methods for CYP3A phenotyping have been published, showing that midazolam (MDZ) is the most suitable probe drug available at present. However, these methods were using either therapeutic MDZ doses, which may cause adverse reactions, or, if employing lower doses, were not able to quantify both hydroxymetabolites. To avoid these problems, we developed a sensitive LC-MS/MS method to quantify MDZ and two of its metabolites, 1´-hydroxymidazolam (1´-OHMDZ) and 4-hydroxymidazolam (4-OHMDZ), in human plasma (PM) using liquid-liquid extraction and a MDZ isotope as internal standard. Validation data showed excellent results for all parameters studied. The analytical method has been successfully applied to a pharmacokinetic (PK) study, showing that MDZ and, to our knowledge for the first time, both hydroxymetabolites can be determined precisely in in-vivo samples following the administration of a sub-therapeutic oral MDZ dose. The method appears therefore to be useful for low-dose CYP3A phenotyping in PM, resulting in minimized adverse reactions.
So far, CYP3A phenotyping with MDZ is based on the determination of the MDZ clearance, however necessitating serial intravenous (iv) blood samples. Using oral fluid (OF) as a non-invasive matrix would be advantageous for assessing the CYP3A activity. Since MDZ is extensively bound to PM proteins and concentrations of free drug in OF are expected to be very low, a very sensitive LC-MS/MS method was developed and validated allowing for the first time the simultaneous determination of MDZ and two of its metabolites in human OF. In a single subject PK study, the assay has been shown to be suitable for analyzing OF samples after the administration of a therapeutic MDZ dose. MDZ OF concentrations were roughly two orders of magnitude lower as compared to PM. However, the kinetics of PM and OF were comparable with each other, resulting in a satisfactory correlation for MDZ (R²=0.972). Therefore, the method seems to be useful for assessing the CYP3A activity in OF.
To confirm the results obtained, a randomized, two-way cross-over study was conducted. Our main goal was to compare MDZ kinetics in PM and OF and to find out whether OF is suitable for CYP3A phenotyping. 8 healthy subjects were treated with 2mg MDZ iv or 7.5mg orally (po) under basal conditions and after CYP3A induction with rifampicin (RIF). Under basal conditions and iv administration, MDZ and 1´-OHMDZ (PM, OF), 4-OHMDZ- and 1´-OHMDZ-glucuronide (PM) were detectable. Correlation between the MDZ concentrations obtained in PM and OF, both under basal conditions and after induction was significant. After treatment with RIF, the MDZ AUC had decreased significantly. After po administration and basal conditions, MDZ, 1´-OHMDZ and 4-OHMDZ were detectable in PM and OF. Similar to iv administration, there was a significant linear correlation between MDZ PM and OF concentration, indicating that kinetics of MDZ can be reliably assessed also in OF. After treatment with RIF, the AUC of MDZ and 1´-OHMDZ had decreased significantly. The results also showed that after treatment with RIF, 1´-OHMDZ undergoes extensive glucuronidation, resulting in a decrease of 1´-OHMDZ AUC, instead of the expected increase, which might be due to an induction of UDP-glucuronosyltransferases by RIF. In conclusion, we provided evidence that MDZ and 1´-OHMDZ can be determined reliably in OF, that concentrations in OF correlate well with those in PM and that MDZ kinetics in OF may be a useful tool to differentiate between constitutive and induced CYP3A activity. Therefore, OF appears to be a suitable matrix for non-invasive CYP3A phenotyping using MDZ as a probe drug.
In a case report, we presented a patient with refractory focal nonconvulsive status epilepticus, treated with very high doses (up to 4mg/min) of iv MDZ and other antiepileptics. From the literature it was known that the half-life (t1/2) of MDZ can increase at high dosage. Therefore, the kinetics of MDZ, 1´-OHMDZ, and 4-OHMDZ were assessed at steady state and after stopping the treatment. The total body clearance of MDZ and intrinsic hepatic clearance at steady state were both 5-10 times higher than after normal therapeutic doses, demonstrating hepatic CYP3A induction. Despite the high body clearance, we observed a substantially prolonged terminal t1/2 of MDZ, mainly due to an extraordinarily large volume of distribution caused by saturation of protein binding. The free fraction of MDZ at steady state was much higher as compared to normal therapeutic doses.