No abstract

The objective of the study was to establish an in vivo method for assessing cytochrome P450 3A (CYP3A) activity using therapeutically inert nanogram doses of midazolam. We administered four escalating single doses of oral midazolam (0.0001-3 mg) to 12 healthy participants, stratified according to CYP3A5 carrier status, to assess pharmacokinetics linearity. We then evaluated the interactions with the CYP3A inhibitor ketoconazole (400 mg q.d.) after nanogram and regular doses of midazolam. Area under the plasma concentration-time curve (AUC) and peak plasma concentration (C(max)) were linear over the entire range of doses. Ketoconazole reduced midazolam oral clearance by 92.8%. AUC and C(max) increased by 1,540 and 363%, respectively. CYP3A5 carrier status had no influence on midazolam oral clearance or its inhibition by ketoconazole. This is the first study showing that midazolam pharmacokinetics is linear in a 30,000-fold concentration range, and therefore that nano- and microgram doses of midazolam can reliably predict the pharmacokinetics of midazolam in therapeutic doses and can be used to assess CYP3A activity even in the presence of strong CYP3A inhibitors.

Myrcludex B acts as a hepatitis B and D virus entry inhibitor blocking the sodium taurocholate cotransporting polypeptide (SLC10A1). We investigated the effects of myrcludex B on plasma bile acid disposition, tenofovir pharmacokinetics, and perpetrator characteristics on cytochrome P450 (CYP) 3A. Twelve healthy volunteers received 300 mg tenofovir disoproxil fumarate orally and 10 mg subcutaneous myrcludex B. Myrcludex B increased total plasma bile acid exposure 19.2-fold without signs of cholestasis. The rise in conjugated bile acids was up to 124-fold (taurocholic acid). Coadministration of tenofovir with myrcludex B revealed no relevant changes in tenofovir pharmacokinetics. CYP3A activity slightly but significantly decreased by 29% during combination therapy. Myrcludex B caused an asymptomatic but distinct rise in plasma bile acid concentrations and had no relevant impact on tenofovir pharmacokinetics. Changes in CYP3A activity might be due to alterations in bile acid signaling. Long-term effects of elevated bile acids will require critical evaluation.

AIMWe aimed to establish a method to assess systemic and pre-systemic cytochrome P450 (CYP) 3A activity using ineffective microgram doses of midazolam. METHODSIn an open, one sequence, crossover study, 16 healthy participants received intravenous and oral midazolam at microgram (0.001 mg intravenous and 0.003 mg oral) and regular milligram (1 mg intravenous and 3 mg oral) doses to assess the linearity of plasma and urine pharmacokinetics. RESULTS Dose CONCLUSIONThe pharmacokinetics of an intravenous midazolam microdose is linear to the applied regular doses and can be used to assess safely systemic CYP3A activity and, in combination with oral microdoses, pre-systemic CYP3A activity. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Midazolam pharmacokinetics of oral doses are linear over a 30 000-fold range.• An oral microdose of midazolam is suitable to measure total CYP3A activity.• CYP3A inhibition with strong inhibitors can be evaluated with a microdose in healthy volunteers and patients. WHAT THIS STUDY ADDS• The pharmacokinetics of intravenous midazolam microdoses are linear to milligram doses.• The bioavailability and metabolic clearance of midazolam is similar after administration of microdoses and milligram doses.• Midazolam microdoses are a suitable tool to assess both systemic and pre-systemic CYP3A activity.

The benzodiazepine midazolam is a probe drug used to phenotype cytochrome P450 3A activity. In this situation, effective sedative concentrations are neither needed nor desired, and in fact the use of very low doses is advantageous. We therefore developed and validated an assay for the femtomolar quantification of midazolam and 1'-hydroxymidazolam in human plasma. Plasma (0.25 mL) and 96-well-based solid-phase extraction were used for sample preparation. Extraction recoveries ranged between 75 and 92% for both analytes. Extracts were chromatographed within 2 min on a Waters BEH C18 1.7 μm UPLC® column with a fast gradient consisting of formic acid, ammonia, and acetonitrile. Midazolam and 1'-hydroxymidazolam were quantified using deuterium- and (13)C-labeled internal standards and positive electrospray tandem mass spectrometry in the multiple reaction monitoring mode, which yielded lower limits of quantification of 50 fg/mL (154 fmol/L) and 250 fg/mL (733 fmol/L) and a corresponding precision of <20%. The calibrated concentration ranges were linear for midazolam (0.05-250 pg/mL) and 1'-hydroxymidazolam (0.25-125 pg/mL), with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <14% and <12%. No ion suppression was detectable, and plasma matrix effects were minimized to <15% (<25%) for midazolam (1'-hydroxymidazolam). The assay was successfully applied to assess the kinetics of midazolam in two human volunteers after the administration of single oral microgram doses (1-100 μg). This ultrasensitive assay allowed us to quantify the kinetics of midazolam and 1'-hydroxymidazolam for at least 10 h, even after the administration of only 1 μg of midazolam.

Young persons who present with agitation and cardiovascular and/or psychiatric manifestations of unclear origin and whose drug screening tests are negative may be suffering from an intoxication with a novel psychoactive substance. Physicians should know the classes of such substances and their effects. Targeted toxicological analysis can be carried out in a toxicology laboratory or a facility for forensic medicine.

Voriconazole is both a substrate and a potent inhibitor of cytochrome P450 (CYP) 3A. It has a high bioavailability and non-linear pharmacokinetics. We investigated the pharmacokinetics and metabolism of 50 mg and 400 mg doses of intravenous and oral voriconazole in 14 healthy volunteers. Concurrently, we determined systemic and presystemic CYP3A activity with microdosed midazolam. Bioavailability of voriconazole 50 mg was 39 % compared with 86 % of the 400 mg dose. Voriconazole area under the concentration-time curve extrapolated to infinity (AUC) was 416 and 16,700 h·ng/mL for the 50 and 400 mg oral doses, respectively, and 1110 and 19,760 h·ng/mL for the 50 and 400 mg intravenous doses, respectively. Midazolam metabolism was dose-dependently inhibited by voriconazole. Dose-dependent autoinhibition of CYP3A-dependent first-pass metabolism and systemic metabolism is a possible explanation for the dose-dependent bioavailability and elimination of voriconazole, either as additional mechanism to, or instead of, saturation of presystemic metabolism. Higher bioavailability and non-linear pharmacokinetics are expected to be a common property of drugs that are substrates and inhibitors of CYP3A, e.g. clarithromycin.

While ample data is available on the choice appropriate phenotyping drugs (midazolam, alfentanil, aplrazolam, buspirone, triazolam), less clinical trial data is available concerning strategies to usefully guide dosing in the clinical practice. Implementation into the clinical routine necessitates further research to identify (1) an easy-to-use and cheap test for CYP3A activity that (2) adequately predicts drug exposure to (3) allow a sound decision on dose adaptation and hence (4) improve clinical outcome and/or reduce the intensity or frequency of adverse drug effects.