Yvonne S. Lin

EVIDENCE OF SIGNIFICANT CONTRIBUTION FROM CYP3A5 TO HEPATIC DRUG METABOLISM

CYP3A4 and CYP3A5 exhibit significant overlap in substrate specificity but can differ in product regioselectivity and formation activity. To further explore this issue, we compared the kinetics of product formation for eight different substrates, using heterologously expressed CYP3A4 and CYP3A5 and phenotyped human liver microsomes. Both enzymes displayed allosteric behavior toward six of the substrates. When it occurred, the “maximal” intrinsic clearance was used for quantitative comparisons. Based on this parameter, CYP3A5 was more active than CYP3A4 in catalyzing total midazolam hydroxylation (3-fold) and lidocaine demethylation (1.4-fold). CYP3A5 exhibited comparable metabolic activity as CYP3A4 (90-110%) toward dextromethorphan N-demethylation and carbamazepine epoxidation. CYP3A5-catalyzed erythromycin N-demethylation, total flunitrazepam hydroxylation, testosterone 6β-hydroxylation, and terfenadine alcohol formation occurred with an intrinsic clearance that was less than 65% that of CYP3A4. Using two sets of human liver microsomes with equivalent CYP3A4-specific content but markedly different CYP3A5 content (group 1, predominantly CYP3A4; group 2, CYP3A4 + CYP3A5), we assessed the contribution of CYP3A5 to product formation rates determined at low substrate concentrations (≤Km). Mean product formation rates for group 2 microsomes were 1.4- to 2.2-fold higher than those of group 1 (p < 0.05 for 5 of 8 substrates). After adjusting for CYP3A4 activity (itraconazole hydroxylation), mean product formation rates for group 2 microsomes were still significantly higher than those of group 1 (p < 0.05 for 3 substrates). We suggest that, under conditions when CYP3A5 content represents a significant fraction of the total hepatic CYP3A pool, the contribution of CYP3A5 to the clearance of some drugs may be an important source of interindividual variability.

In-vivo phenotyping for CYP3A by a single-point determination of midazolam plasma concentration

We investigated whether a single plasma midazolam concentration could serve as an accurate predictor of total midazolam clearance, an established in-vivo probe measure of cytochrome P450 3A (CYP3A) activity. In a retrospective analysis of data from 224 healthy volunteers, non-compartmental pharmacokinetic parameters were estimated from plasma concentration-time curves following intravenous (IV) and/or oral administration. Based on statistical moment theory, the concentration at the mean residence time (MRT) should be the best predictor of the total area under the curve (AUC). Following IV or oral midazolam administration, the average MRT was found to be approximately 3.5 h, suggesting that the optimal single sampling time to predict AUC was between 3 and 4 h. Since a 4-h data point was common to all studies incorporated into this analysis, we selected this time point for further investigation. The concentrations of midazolam measured 4 h after an IV or oral dose explained 80 and 91% of the constitutive interindividual variability in midazolam AUC, respectively. The 4-h midazolam measurement was also an excellent predictor of drug-drug interactions involving CYP3A induction and inhibition. Compared with baseline values, the direction and magnitude of change in midazolam AUC and the 4-h concentration were completely concordant for all study subjects. We conclude that a single 4-h midazolam concentration following IV or oral administration represents an accurate marker of CYP3A phenotype under constitutive and modified states. Moreover, the single-point approach offers an efficient means to phenotype and identify individuals with important genetic polymorphisms that affect CYP3A activity.

MDR1 genotype is associated with hepatic cytochrome P450 3A4 basal and induction phenotype

Variant cytochrome P450 (CYP) 3A4 alleles cannot explain human variation in CYP3A4 expression. This study investigated whether common single‐nucleotide polymorphisms (SNPs) in multidrug resistance 1 (MDR1), encoding P‐glycoprotein, or the pregnane X receptor (PXR) were associated with basal or inducible CYP3A4 expression.

Association between abcb1 (multidrug resistance transporter) genotype and post-liver transplantation renal dysfunction in patients receiving calcineurin inhibitors

OBJECTIVE Renal dysfunction is a common and costly adverse outcome of long-term treatment with calcineurin inhibitors (CNIs). We conducted a retrospective, case-control study to test whether the risk of renal dysfunction in liver transplantation patients receiving CNIs is associated with the 2677G>T transversion in exon-21 of the gene (ABCB1) encoding P-glycoprotein. A total of 120 non-Hispanic white patients were evaluated. RESULTS The overall incidence of renal dysfunction by year 3 post-transplantation was 40%. The frequency of renal dysfunction was reduced among patients with an ABCB1 2677TT genotype, as compared to those with a 2677GG genotype. Subjects with a heterozygote genotype behaved phenotypically like the 2677GG group. Comparing those subjects with a 2677TT genotype to the combined group of subjects with a 2677GG, TG, AT, or AG genotype resulted in an odds ratio of 0.26 (0.09-0.77). When subjects were stratified by gender, the frequency of renal dysfunction was reduced among men with an ABCB1 2677TT genotype, relative to men with different genotypes. A similar odds ratio was obtained for women, but it did not achieve significance. When 18 subjects with an elevated SCr concentration just prior to surgery were excluded from the year 3 analysis, the association between the 2677TT genotype and chronic renal dysfunction in the remaining cohort was strengthened comparing genotype groups. CONCLUSIONS Based on these results, we conclude that homozygosity for the ABCB1 2677T (S893) allele is associated with reduced risk of chronic renal dysfunction among liver transplantation patients receiving an immunosuppressive regimen containing CNIs.

Interindividual Variability in the Hepatic Expression of the Human Breast Cancer Resistance Protein (BCRP/ABCG2): Effect of Age, Sex, and Genotype

Breast cancer resistance protein (BCRP), an efflux transporter expressed at the bile canalicular membrane, is responsible for the biliary clearance of many drugs. Data on the interindividual variability of hepatic BCRP expression are needed for in vitro to in vivo extrapolation of the biliary clearance of a BCRP substrate drug. Therefore, we measured the expression of BCRP in human livers (n = 65) by liquid chromatography coupled with tandem mass spectrometry. A calibration curve was generated using a synthetic signature peptide (SSLLDVLAAR) as the calibrator and the corresponding synthetic stable isotope-labeled peptide as the internal standard. The analytical method was accurate and precise. BCRP expression in 50 livers, where it was measurable, was 137.9 ± 42.1 atmol/µg of membrane protein (range 69.7-246.4 atmol/µg of membrane protein). BCRP expression was not associated with age (7-70 years), sex, or mRNA expression. BCRP expression in livers with the variant C421A (rs2231142) allele (14 heterozygotes, two homozygotes; among these, eight livers were below lower limit of quantification) was significantly lower than that in the wild-type livers (p < 0.002). Integration of these data with data on the hepatic expression of other transporters will allow refinement of physiologically based pharmacokinetic models to predict the pharmacokinetics, hepatic exposure, and drug-drug interactions of drugs (and/or their metabolites).

Herb-Drug Interactions: Challenges and Opportunities for Improved Predictions

Supported by a usage history that predates written records and the perception that “natural” ensures safety, herbal products have increasingly been incorporated into Western health care. Consumers often self-administer these products concomitantly with conventional medications without informing their health care provider(s). Such herb–drug combinations can produce untoward effects when the herbal product perturbs the activity of drug metabolizing enzymes and/or transporters. Despite increasing recognition of these types of herb–drug interactions, a standard system for interaction prediction and evaluation is nonexistent. Consequently, the mechanisms underlying herb–drug interactions remain an understudied area of pharmacotherapy. Evaluation of herbal product interaction liability is challenging due to variability in herbal product composition, uncertainty of the causative constituents, and often scant knowledge of causative constituent pharmacokinetics. These limitations are confounded further by the varying perspectives concerning herbal product regulation. Systematic evaluation of herbal product drug interaction liability, as is routine for new drugs under development, necessitates identifying individual constituents from herbal products and characterizing the interaction potential of such constituents. Integration of this information into in silico models that estimate the pharmacokinetics of individual constituents should facilitate prospective identification of herb–drug interactions. These concepts are highlighted with the exemplar herbal products milk thistle and resveratrol. Implementation of this methodology should help provide definitive information to both consumers and clinicians about the risk of adding herbal products to conventional pharmacotherapeutic regimens.

Using 'omics' to define pathogenesis and biomarkers of Parkinson's disease.

Although great effort has been put forth to uncover the complex molecular mechanisms exploited in the pathogenesis of Parkinson’s disease, a satisfactory explanation remains to be discovered. The emergence of several -omics techniques, transcriptomics, proteomics and metabolomics, have been integral in confirming previously identified pathways that are associated with dopaminergic neurodegeneration and subsequently Parkinson’s disease, including mitochondrial and proteasomal function and synaptic neurotransmission. Additionally, these unbiased techniques, particularly in the brain regions uniquely associated with the disease, have greatly enhanced our ability to identify novel pathways, such as axon-guidance, that are potentially involved in Parkinson’s pathogenesis. A comprehensive appraisal of the results obtained by different -omics has also reconfirmed the increase in oxidative stress as a common pathway likely to be critical in Parkinson’s development/progression. It is hoped that further integration of these techniques will yield a more comprehensive understanding of Parkinson’s disease etiology and the biological pathways that mediate neurodegeneration.

Human Liver Expression of CYP2C8: Gender, Age, and Genotype Effects

Research investigating CYP2C8 as a drug-metabolizing enzyme has gained momentum over the past few years. CYP2C8 is estimated to oxidatively metabolize approximately 5% of therapeutically prescribed drugs. It is polymorphically expressed, and several single nucleotide polymorphisms have been identified with varying effects on the clearance of CYP2C8 substrates. However, the human liver expression of CYP2C8 and effects of genetic variation, age, and gender on mRNA and protein levels have not been fully explored. In this report, interindividual variation in CYP2C8 mRNA and protein expression in 60 livers from white individuals was examined. The livers were genotyped for CYP2C8*3 and CYP2C8*4 polymorphisms. The effects of genotype, age, and gender on hepatic CYP2C8 expression and the correlation of CYP2C8 mRNA expression with CYP3A4 and other CYP2C members were evaluated. The mean ± S.D. protein levels in CYP2C8*1/*1 livers was 30.8 ± 17.5 pmol/mg protein, and a trend for decreased protein levels was observed for CYP2C8*1/*4 livers (15.8 ± 9.7 pmol/mg, p = 0.07). The mean expression levels of CYP2C8 was comparable in males and females (p = 0.18). The mRNA expression of CYP2C8, CYP2C9, CYP2C19, and CYP3A4, but not CYP2C18, was highly correlated (p < 0.0001). Moreover, the hepatic CYP2C8 and CYP3A4 protein levels were strongly correlated (r = 0.76, p < 0.0001). This correlation is most likely due to common regulation factors for both genes. CYP2C8 mRNA or protein expression levels were not significantly affected by CYP2C8*3 or *4 genotype, gender, or age, and variation observed clinically in CYP2C8 activity warrants further investigation.

An Inducible Cytochrome P450 3A4-Dependent Vitamin D Catabolic Pathway

Vitamin D3 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 D3 (25OHD3), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD3 was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD3 hydroxylation by human liver microsomes, with the formation of 4β,25-dihydroxyvitamin D3 [4β,25(OH)2D3] dominating (Vmax/Km = 0.85 ml · min−1 · nmol enzyme−1). 4β,25(OH)2D3 was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D3, and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4β,25(OH)2D3 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 4β,25(OH)2D3, but not CYP24A1-dependent 24R,25-dihydroxyvitamin D3 formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD3 metabolism may play an important role in the regulation of vitamin D3 in vivo and in the etiology of drug-induced osteomalacia.

Simultaneous measurement of plasma vitamin D(3) metabolites, including 4β,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 4β,25-dihydroxyvitamin D(3) [4β,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 4β,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 4β,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, 4β,25(OH)(2)D(3).