Scaling in vitro activity of CYP3A7 suggests human fetal livers do not clear retinoic acid entering from maternal circulation.

All-trans-retinoic acid (atRA), the active metabolite of vitamin A, is a critical signaling molecule during embryonic and fetal development and is necessary for maternal health. Fetal exposure to endogenous atRA is tightly regulated during gestation in a tissue specific manner and maternal exposure to exogenous retinoids during pregnancy is teratogenic. The clearance of atRA is primarily mediated by the cytochrome P450 (CYP) 26 enzymes, which play an essential role in controlling retinoid gradients during organogenesis. We hypothesized that CYP26 enzymes in the human fetal liver also function as a protective barrier to prevent maternal atRA reaching fetal circulation. Using human fetal liver tissue, we found that the mRNA of CYP26A1 and CYP26B1 enzymes is expressed in the human fetal liver. However, based on inhibition studies, metabolite profiles and correlation of atRA metabolism with testosterone hydroxylation, clearance of atRA in the fetal livers was mediated by CYP3A7. Based on in vitro-to-in vivo scaling, atRA clearance in the fetal liver was quantitatively minimal, thus providing an insufficient maternal-fetal barrier for atRA exposure.

Toxicokinetics and Physiologically Based Pharmacokinetic Modeling of the Shellfish Toxin Domoic Acid in Nonhuman Primates.

Domoic acid (DA), a neurotoxin, is produced by marine algae and has caused toxications worldwide in animals and humans. However, the toxicokinetics of DA have not been fully evaluated, and information is missing on the disposition of DA following oral exposures at doses that are considered safe for human consumption. In this study, toxicokinetics of DA were investigated in cynomolgus monkeys following single doses of 5 µg/kg DA intravenously, 0.075 mg/kg DA orally, and 0.15 mg/kg DA orally. After intravenous dosing, DA had a systemic clearance of 124 ± 71 (ml/h)/kg, volume of distribution at steady state of 131 ± 71 ml/kg and elimination half-life of 1.2 ± 1.1 hours. However, following oral dosing, the average terminal half-life of DA was 11.3 ± 2.4 hours, indicating that DA disposition follows flip-flop kinetics with slow, rate-limiting absorption. The absorption of DA was low after oral dosing with absolute bioavailability of 6% ± 4%. The renal clearance of DA was variable [21-152 (ml/h)/kg] with 42% ± 11% of the intravenous DA dose recovered in urine. A physiologically based pharmacokinetic model was developed for DA in monkeys and humans that replicated the flip-flop kinetics observed after oral administration and allowed simulation of urinary excretion and brain and kidney distribution of DA following intravenous and oral dosing. This study is the first to characterize DA disposition at exposure levels close to the current estimated tolerable daily intake and to mechanistically model DA disposition in a model species, providing important information of the toxicokinetics of DA for human safety assessment.

Ultra-high performance liquid chromatography tandem mass-spectrometry for simple and simultaneous quantification of cannabinoids.

Cannabis is used widely in the United States, both recreationally and for medical purposes. Current methods for analysis of cannabinoids in human biological specimens rely on complex extraction process and lengthy analysis time. We established a rapid and simple assay for quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy Δ9-tetrahydrocannabinol (11-OH THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannbinol (THCCOOH) in human plasma by U-HPLC-MS/MS usingΔ9-tetrahydrocannabinol-D3 (THC-D3) as the internal standard. Chromatographic separation was achieved on an Acquity BEH C18 column using a gradient comprising of water (0.1% formic acid) and methanol (0.1% formic acid) over a 6 min run-time. Analytes from 200µL plasma were extracted using acetonitrile (containing 1% formic acid and THC-D3). Mass spectrometry was performed in positive ionization mode, and total ion chromatogram was used for quantification of analytes. The assay was validated according to guidelines set forth by Food and Drug Administration of the United States. An eight-point calibration curve was fitted with quadratic regression (r2>0.99) from 1.56 to 100ngmL-1 and a lower limit of quantification (LLOQ) of 1.56ngmL-1 was achieved. Accuracy and precision calculated from six calibration curves was between 85-115% while the mean extraction recovery was >90% for all the analytes. Several plasma phospholipids eluted after the analytes thus did not interfere with the assay. Bench-top, freeze-thaw, auto-sampler and short-term stability ranged from 92.7 to 106.8% of nominal values. Application of the method was evaluated by quantification of analytes in human plasma from six subjects.

Induction of CYP26A1 by metabolites of retinoic acid: evidence that CYP26A1 is an important enzyme in the elimination of active retinoids.

All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxo-atRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARß were increased 300- to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, >60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD(+), suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (Km = 63 nM and intrinsic clearance = 90 µl/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action.

The relative contributions of CYP3A4 and CYP3A5 to the metabolism of vinorelbine.

Vinorelbine is a semisynthetic vinca alkaloid used in the treatment of advanced breast and non-small cell lung cancers. Vincristine, a related vinca alkaloid, is 9-fold more efficiently metabolized by CYP3A5 than by CYP3A4 in vitro. This study quantified the relative contribution of CYP3A4 and CYP3A5 to the metabolism of vinorelbine in vitro using cDNA-expressed human cytochrome P450s (P450s) and human liver microsomes (HLMs). CYP3A4 and CYP3A5 were identified as the P450s capable of oxidizing vinorelbine using a panel of human enzymes and selective P450 inhibitors in HLMs. For CYP3A4 coexpressed with cytochrome b5 (CYP3A4+b5) and CYP3A5+b5, the Michaelis-Menten constants for vinorelbine were 2.6 and 3.6 µM, respectively, but the Vmax of 1.4 pmol/min/pmol was common to both enzymes. In HLMs, the intrinsic clearance of vinorelbine metabolism was highly correlated with CYP3A4 activity, and there was no significant difference in intrinsic clearance between CYP3A5 high and low expressers. When radiolabeled vinorelbine substrate was used, there were clear qualitative differences in metabolite formation fingerprints between CYP3A4+b5 and CYP3A5+b5 as determined by NMR and mass spectrometry analysis. One major metabolite (M2), a didehydro-vinorelbine, was present in both recombinant and microsomal systems but was more abundant in CYP3A4+b5 incubations. We conclude that despite the equivalent efficiency of recombinant CYP3A4 and CYP3A5 in vinorelbine metabolism the polymorphic expression of CYP3A5, as shown by the kinetics with HLMs, may have a minimal effect on systemic clearance of vinorelbine.

Pharmacogenetic predictors of nausea and vomiting of pregnancy severity and response to antiemetic therapy: a pilot study.

BACKGROUND: Nausea and vomiting of pregnancy (NVP) is a common condition. The objective of this study was to evaluate the association between response to antiemetics in the treatment of NVP and genetic polymorphisms in the serotonin receptor subunit genes HTR3A and HTR3B. METHODS: Pregnant women ≥18 years of age with NVP starting antiemetic therapy with promethazine, prochlorperazine, metoclopramide, or ondansetron at ≤ 16 weeks gestational age were eligible. The study recruited 29 women with complete data and sampling who returned for their one week follow-up and were genotyped for HTR3A and HTR3B polymorphisms. Severity of NVP was captured (using Pregnancy Unique Quantification of Emesis (PUQE) and Quality of Life (QOL) tools) upon enrollment and after one week of antiemetic therapy. These measures were correlated with pharmacogenetic variability. RESULTS: Subjects with genotype associated with high serotonin affinity of the 5-HT3B receptor (rs1176744, CC) required more antiemetic medications (p < 0.001) than other subjects. Those with genotypes associated with increased expression of the 5-HT3A receptor subunit (rs1062613, CT or TT) had worse final PUQE scores (p = 0.01) than other subjects while rs3782025 variants carriers had significantly better initial (p = 0.02) and final (p = 0.02) PUQE scores than other subjects. CONCLUSIONS: HTR3B and HTR3A gene variants may contribute to variability in response to antiemetic therapy for NVP.

Hepatic Cyp2d and Cyp26a1 mRNAs and activities are increased during mouse pregnancy.

There is considerable evidence that drug disposition is altered during human pregnancy and based on probe drug studies, CYP2D6 activity increases during human pregnancy. The aim of this study was to determine whether the changes of CYP2D6 activity observed during human pregnancy could be replicated in the mouse, and explore possible mechanisms of increased CYP2D6 activity during pregnancy. Cyp2d11, Cyp2d22, Cyp2d26 and Cyp2d40 mRNA was increased (P < 0.05) on gestational days (GD) 15 and 19 compared with the non-pregnant controls. There was no change (P > 0.05) in Cyp2d9 and Cyp2d10 mRNA. In agreement with the increased Cyp2d mRNA, Cyp2d-mediated dextrorphan formation from dextromethorphan was increased 2.7-fold (P < 0.05) on GD19 (56.8±39.4 pmol/min/mg protein) when compared with the non-pregnant controls (20.8±11.2 pmol/min/mg protein). An increase in Cyp26a1 mRNA (10-fold) and retinoic acid receptor (Rar)ß mRNA (2.8-fold) was also observed during pregnancy. The increase in Cyp26a1 and Rarß mRNA during pregnancy indicates increased retinoic acid signaling in the liver during pregnancy. A putative retinoic acid response element was identified within the Cyp2d40 promoter and the mRNA of Cyp2d40 correlated (P < 0.05) with Cyp26a1 and Rarß. These results show that Cyp2d mRNA is increased during mouse pregnancy the and mouse may provide a suitable model to investigate the mechanisms underlying the increased clearance of CYP2D6 probes observed during human pregnancy. Our findings also suggest that retinoic acid signaling in the liver is increased during pregnancy, which may have broader implications to energy homeostasis in the liver during pregnancy.

Stereoselective formation and metabolism of 4-hydroxy-retinoic Acid enantiomers by cytochrome p450 enzymes.

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the ß-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.

Comparison of the function and expression of CYP26A1 and CYP26B1, the two retinoic acid hydroxylases.

All-trans-retinoic acid (atRA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of atRA via oxidative metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are atRA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from atRA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from atRA. Quantitatively, CYP26B1 had a lower K(m) (19nM) and V(max) (0.8 pmol/min/pmol) than CYP26A1 (K(m)=50 nM and V(max)=10 pmol/min/pmol) for formation of 4-OH-RA. The major atRA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2-10-fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of atRA in tissues that function as a barrier for atRA exposure.

Increased risk of vincristine neurotoxicity associated with low CYP3A5 expression genotype in children with acute lymphoblastic leukemia.

BACKGROUND: This study evaluates the relationship between cytochrome P450 (CYP) 3A5 genotype and vincristine-induced peripheral neuropathy (VIPN) in children with precursor B cell acute lymphoblastic leukemia (preB ALL). We have shown in vitro that vincristine is metabolized significantly more efficiently by CYP3A5 than by CYP3A4. We also found that vincristine neurotoxicity is less common in African-Americans (70% express CYP3A5) than in Caucasians. We test the hypothesis that CYP3A5 expressers experience less vincristine neuropathy than do CYP3A5 non-expressers. PROCEDURE: This study of pharmacogenetics of vincristine neuropathy in children with preB ALL was completed at Indiana University Simon Cancer Center. Whole blood for DNA extraction and genotyping was collected as well as plasma from a single time-point for analysis of vincristine and primary metabolite (M1) concentrations. Vincristine neuropathy was captured via chart review and graded per the National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.0. RESULTS: Eighty-nine percent of CYP3A5 expressers experienced neurotoxicity versus 100% of non-expressers (P = 0.03). The proportion of treatment months with neurotoxicity was significantly different between the expressers and non-expressers (16% vs. 27%, P = 0.0007). Limited pharmacokinetic data suggest different rates of vincristine metabolism between CYP3A5 genotype groups with higher primary metabolite (M1) plasma concentrations (P = 0.0004) and lower metabolic ratios ([vincristine]/[M1]) (P = 0.036) in the CYP3A5 expressers compared to the CYP3A5 non-expressers. M1 concentration was also inversely related to severity of neuropathy (P = 0.0316). CONCLUSIONS: In children with preB ALL, CYP3A5 expressers experience less VIPN, produce more M1, and have lower metabolic ratios compared to CYP3A5 non-expressers.