Abstract
A rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of organic anion transporting polypeptide 1B1 (OATP1B1) and cytochrome P450 (P450) probe substrates and their phase I metabolites in human plasma was developed. The OATP1B1 (pitavastatin) and five P450 probe substrates, caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A) and their metabolites were extracted from human plasma (50 µL) using methanol. Analytes were separated on a C18 column followed by selected reaction monitoring detection using MS/MS. All analytes were separated simultaneously within a 9 min run time. The developed method was fully validated over the expected clinical concentration range for all analytes tested. The intra- and inter-day precisions for all analytes were lower than 11.3% and 8.82%, respectively, and accuracy was 88.5–117.3% and 96.1–109.2%, respectively. The lower limit of quantitation was 0.05 ng/mL for dextromethorphan, dextrorphan, midazolam, and 1′-hydroxymidazolam; 0.5 ng/mL for losartan, EXP-3174, omeprazole, 5′-hydroxyomeprazole, and pitavastatin; and 5 ng/mL for caffeine and paraxanthine. The method was successfully used in a pharmacokinetic study in healthy subjects after oral doses of five P450 and OATP1B1 probes. This analytical method provides a simple, sensitive, and accurate tool for the determination of OATP1B1 and five major P450 activities in vivo drug interaction studies.
Highlights
Cytochrome P450 (P450) enzymes are responsible for the oxidative metabolism of xenobiotics and endogenous substrates, and are major sources of variability in drug metabolism and pharmacokinetics [1,2].Currently, 57 different isoforms have been characterized in humans [3]
Drug interactions mediated by permeability-glycoprotein (P-gp) and organic anion transporting polypeptides (OATPs) have been reported for their association with clinically important drug interactions [8]
For the first time, we developed an LC-MS/MS method to simultaneously analyze five P450s specific probe drugs and their metabolites as well as an organic anion transporting polypeptide 1B1 (OATP1B1) probe drug using LC-MS/MS after protein precipitation in human plasma
Summary
Cytochrome P450 (P450) enzymes are responsible for the oxidative metabolism of xenobiotics and endogenous substrates, and are major sources of variability in drug metabolism and pharmacokinetics [1,2].Currently, 57 different isoforms have been characterized in humans [3]. Five P450 isoforms, CYP1A2, 2C9, 2C19, 2D6, and 3A are involved in the metabolism of more than 90% of marketed drugs [4]. CYP3A4 and CYP3A5 are major isoforms implicated in the biotransformation of macrolide antibiotics, antihistamines, benzodiazepines, calcium channel blockers, and statins [5]. CYP1A2, 2C9, 2C19, and 2D6 are implicated in the biotransformation of many drugs (CYP1A2 for caffeine, phenacetin, and tizanidine; CYP2C9 for angiotensin blockers, nonsteroidal anti-inflammatory drugs, and sulfonylureas; CYP2C19 for proton pump inhibitors and antiepileptics; and CYP2D6 for beta blockers, antidepressants, and antipsychotics) [6,7]. In addition to drug metabolizing enzymes, drug transporters can cause various pharmacological consequences. Recent data have suggested that OATP1B1 is involved in the pharmacokinetics of some protease inhibitors (saquinavir and ritonavir [9]) and statin drugs (pravastatin and pitavastatin [10])
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