Prediction of Intravenous Busulfan Clearance by Endogenous Plasma Biomarkers Using Global Pharmacometabolomics.

INTRODUCTION: High-dose busulfan (busulfan) is an integral part of the majority of hematopoietic cell transplantation conditioning regimens. Intravenous (IV) busulfan doses are personalized using pharmacokinetics (PK)-based dosing where the patient's IV busulfan clearance is calculated after the first dose and is used to personalize subsequent doses to a target plasma exposure. PK-guided dosing has improved patient outcomes and is clinically accepted but highly resource intensive. OBJECTIVE: We sought to discover endogenous plasma biomarkers predictive of IV busulfan clearance using a global pharmacometabolomics-based approach. METHODS: Using LC-QTOF, we analyzed 59 (discovery) and 88 (validation) plasma samples obtained before IV busulfan administration. RESULTS: In the discovery dataset, we evaluated the association of the relative abundance of 1885 ions with IV busulfan clearance and found 21 ions that were associated with IV busulfan clearance tertiles (r2 ≥ 0.3). Identified compounds were deoxycholic acid and/or chenodeoxycholic acid, and linoleic acid. We used these 21 ions to develop a parsimonious seven-ion linear predictive model that accurately predicted IV busulfan clearance in 93% (discovery) and 78% (validation) of samples. CONCLUSION: IV busulfan clearance was significantly correlated with the relative abundance of 21 ions, seven of which were included in a predictive model that accurately predicted IV busulfan clearance in the majority of the validation samples. These results reinforce the potential of pharmacometabolomics as a critical tool in personalized medicine, with the potential to improve the personalized dosing of drugs with a narrow therapeutic index such as busulfan.

An inducible cytochrome P450 3A4-dependent vitamin D catabolic pathway.

Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4ß,25-dihydroxyvitamin D(3) [4ß,25(OH)(2)D(3)] dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4ß,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4ß,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4ß,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.

Simultaneous measurement of plasma vitamin D(3) metabolites, including 4ß,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry.

Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that permits the quantification of major circulating vitamin D(3) metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid-liquid extraction, and Diels-Alder derivatization procedure prior to LC-MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D(3) peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] concentrations. This interfering metabolite has been identified as 4ß,25-dihydroxyvitamin D(3) [4ß,25(OH)(2)D(3)] and was found at concentrations comparable to 1α,25(OH)(2)D(3). Quantification of 1α,25(OH)(2)D(3) in plasma required complete chromatographic separation of 1α,25(OH)(2)D(3) from 4ß,25(OH)(2)D(3). An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD(3), 24R,25(OH)(2)D(3), 1α,25(OH)(2)D(3), and 4ß,25(OH)(2)D(3) in healthy individuals. The LC-MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH)(2)D(3) as well as monitoring the newly identified circulating metabolite, 4ß,25(OH)(2)D(3).

Changes in maternal liver Cyp2c and Cyp2d expression and activity during rat pregnancy.

During human pregnancy, CYP2C9, CYP2C19, and CYP2D6 activities are altered. The aim of the current study was to determine if this phenomenon can be replicated in the rat, and to evaluate the mechanisms that contribute to the changes in Cyp2c and Cyp2d activity during pregnancy. The intrinsic clearance of dextromethorphan O-demethylation, a measure of Cyp2d2 activity, was decreased 80% at both days 9 and 19 of gestation when compared to non-pregnant controls. The decreased intrinsic clearance was a result of both decreased V(max) and increased K(m)-values at both days of gestation. Quantitative RT-PCR revealed that transcripts of Cyp2d2 and Cyp2d4 were significantly decreased at day 19 of pregnancy (p<0.05) when compared to day 9 and non-pregnant controls. The decrease in Cyp2d mRNA levels correlated with a decrease in several nuclear receptor mRNA levels (RARalpha, RXRalpha, HNF1 and HNF3beta) but not with the mRNA levels of nuclear receptors usually associated with regulation of P450 enzymes (PXR, CAR and HNF4alpha). In contrast, Cyp2c12 and Cyp2c6 transcription and protein expression were not significantly altered during rat pregnancy although the intrinsic clearance of Cyp2c6 mediated diclofenac 4'-hydroxylation was increased 2-fold on day 19 of gestation when compared to non-pregnant controls. The increase in intrinsic clearance was due to a decrease in the K(m)-value for 4'-hydroxydiclofenac formation. These data show that pregnancy significantly alters the expression and activity of drug metabolizing enzymes in an enzyme and gestational stage specific manner. These changes are likely to have toxicological and therapeutic implications.