Amarjit S. Chaudhry

Liver X receptor α and farnesoid X receptor are major transcriptional regulators of OATP1B1

Organic anion transporting polypeptide 1B1 (OATP1B1) is a liver‐enriched transporter involved in the hepatocellular uptake of many endogenous molecules and several structurally divergent drugs in clinical use. Although OATP1B1 coding region polymorphisms are known to make an impact on substrate drug disposition in humans, little is known regarding the mechanisms underlying the transcriptional regulation of this transporter. In this study, we note that messenger RNA (mRNA) expression of OATP1B1 in a large human liver bank exhibited marked interindividual variability that was not associated with coding region polymorphisms. Accordingly, we hypothesized that such variability in expression is reflective of nuclear receptor‐mediated transcriptional regulation of this transporter. We tested prototypical ligands for the nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), liver X receptor (LXR) α, and farnesoid X receptor (FXR) in a human hepatoma‐derived cell line and noted induction of OATP1B1 mRNA when the cells were treated with LXRα or FXR ligands. To confirm a direct role for LXRα and FXR to OATP1B1 expression, we performed detailed promoter analysis and cell‐based reporter gene assays resulting in the identification of two functional FXR response elements and one LXRα response element. The direct interaction between nuclear receptors with the identified response elements was assessed using chromatin immunoprecipitation assays. Using isolated primary human hepatocytes, we show that LXRα or FXR agonists, but not PXR or CAR agonists, are capable of OATP1B1 induction. Conclusion: We note that OATP1B1 transcriptional regulation is under dual nuclear receptor control through the oxysterol sensing LXRα and the bile acid sensor FXR. Accordingly, the interplay between OATP1B1 and nuclear receptors may play an important and heretofore unrecognized role during cholestasis, drug‐induced liver injury, and OATP1B1 induction–related drug interactions. (HEPATOLOGY 2010)

Ontogeny and Sorafenib Metabolism

Purpose: To investigate the role of ontogeny in sorafenib metabolism to the equipotent active metabolite sorafenib N-oxide. Experimental Design: Steady-state pharmacokinetic studies of sorafenib and metabolites were conducted in 30 children and young adults (17 males; median age, 9.5 years) receiving sorafenib 150 mg/m2 or 200 mg/m2 twice daily. Sorafenib metabolism was evaluated in vitro at 10 μmol/L using a panel of purified human cytochrome P450 (CYP) enzymes. Sorafenib metabolism and CYP3A4 expression was evaluated in 52 human liver samples from donors of ≤20 years old. The drug–drug interaction potential between sorafenib and azole antifungal agents was evaluated in vitro and in vivo. Results: No age-related differences in sorafenib apparent oral clearance were observed. Mean sorafenib N-oxide metabolite ratio was 0.27 ± 0.14. In children of ≤10 years of age, boys had approximately 2-fold higher N-oxide ratios than girls (0.40 ± 0.15 vs. 0.22 ± 0.12, P = 0.026). Of the CYPs evaluated, sorafenib was exclusively metabolized to sorafenib N-oxide by CYP3A4. A trend for increased N-oxide formation in boys was observed in liver samples, which correlated with CYP3A4 mRNA expression. Posaconazole and voriconazole potently inhibited sorafenib N-oxide formation in vitro, and reduced sorafenib N-oxide formation in 3 children given sorafenib concurrent with azoles. Conclusion: We have identified several factors affecting interpatient variability in sorafenib metabolism to the active N-oxide metabolite including age, sex, and concurrent treatment with azole antifungals. This knowledge may provide important considerations for the clinical use of sorafenib in children and possibly other kinase inhibitors undergoing CYP3A4-mediated metabolism. Clin Cancer Res; 18(20); 5788–95. ©2012 AACR.

Hepatic organic anion transporting polypeptide transporter and thyroid hormone receptor interplay determines cholesterol and glucose homeostasis.

The role of organic anion transporting polypeptides (OATPs), particularly the members of OATP1B subfamily, in hepatocellular handling of endogenous and exogenous compounds is an important and emerging area of research. Using a mouse model lacking Slco1b2, the murine ortholog of the OATP1B subfamily, we have demonstrated previously that genetic ablation causes reduced hepatic clearance capacity for substrates. In this study, we focused on the physiological function of the hepatic OATP1B transporters. First, we studied the influence of the Oatp1b2 deletion on bile acid (BA) metabolism, showing that lack of the transporter results in a significantly reduced expression of Cyp7a1, the key enzyme of BA synthesis, resulting in elevated cholesterol levels after high dietary fat challenge. Furthermore, Slco1b2−/− mice exhibited delayed clearance after oral glucose challenge resulting from reduced hepatic glucose uptake. In addition to increased hepatic glycogen content, Slco1b2−/− mice exhibited reduced glucose output after pyruvate challenge. This is in accordance with reduced hepatic expression of phosphoenolpyruvate carboxykinase (PEPCK) in knockout mice. We show that this phenotype is due to the loss of liver‐specific Oatp1b2‐mediated hepatocellular thyroid hormone entry, which then leads to reduced transcriptional activation of target genes of hepatic thyroid hormone receptor (TR), including Cyp7a1 and Pepck but also Dio1 and Glut2. Importantly, we assessed human relevance using a cohort of archived human livers in which OATP1B1 expression was noted to be highly associated with TR target genes, especially for glucose facilitating transporter 2 (GLUT2). Furthermore, GLUT2 expression was significantly decreased in livers harboring a common genetic polymorphism in SLCO1B1. Conclusion: Our findings reveal that OATP1B‐mediated hepatic thyroid hormone entry is a key determinant of cholesterol and glucose homeostasis. (HEPATOLOGY 2011;)

Interindividual Variability in Cytochrome P450–Mediated Drug Metabolism

The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug–drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals.

Regulation of Coagulation Factor XI Expression by MicroRNAs in the Human Liver

High levels of factor XI (FXI) increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs) in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK) demonstrated a direct interaction between miR-181a-5p and 3′untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.

Genetic Variation in Aldo-Keto Reductase 1D1 (AKR1D1) Affects the Expression and Activity of Multiple Cytochrome P450s

Human liver gene regulatory (Bayesian) network analysis was previously used to identify a cytochrome P450 (P450) gene subnetwork with Aldo-keto reductase 1D1 (AKR1D1) as a key regulatory driver of this subnetwork. This study assessed the biologic importance of AKR1D1 [a key enzyme in the synthesis of bile acids, ligand activators of farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), known transcriptional regulators of P450s] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. We resequenced AKR1D1 from 98 donor livers and identified a 3′-untranslated region (UTR) (rs1872930) single nucleotide polymorphism (SNP) significantly associated with higher AKR1D1 mRNA expression. AKR1D1 3′-UTR-luciferase reporter studies showed that the variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. Consistent with AKR1D1’s putative role as a driver of the P450 subnetwork, the AKR1D1 3′-UTR SNP was significantly associated with increased hepatic mRNA expression of multiple P450s (CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6) and CYP3A4, CYP2C8, CYP2C19, and CYP2B6 activities. After adjusting for multiple testing, the association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA expression and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy and adverse drug events. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of the P450 network.

Why Do Most Human Liver Cytosol Preparations Lack Xanthine Oxidase Activity

When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.

The CYP2C19 Intron 2 Branch Point SNP is the Ancestral Polymorphism Contributing to the Poor Metabolizer Phenotype in Livers with CYP2C19*35 and CYP2C19*2 Alleles

CYP2C19 rs12769205 alters an intron 2 branch point adenine leading to an alternative mRNA in human liver with complete inclusion of intron 2 (exon 2B). rs12769205 changes the mRNA reading frame, introduces 87 amino acids, and leads to a premature stop codon. The 1000 Genomes project (http://browser.1000genomes.org/index.html) indicated rs12769205 is in linkage disequilibrium with rs4244285 on CYP2C19*2, but found alone on CYP2C19*35 in Blacks. Minigenes containing rs12769205 transfected into HepG2 cells demonstrated this single nucleotide polymorphism (SNP) alone leads to exon 2B and decreases CYP2C19 canonical mRNA. A residual amount of CYP2C19 protein was detectable by quantitative proteomics with tandem mass spectrometry in CYP2C19*2/*2 and *1/*35 liver microsomes with an exon 2 probe. However, an exon 4 probe, downstream from rs12769205, but upstream of rs4244285, failed to detect CYP2C19 protein in livers homozygous for rs12769205, demonstrating rs12769205 alone can lead to complete loss of CYP2C19 protein. CYP2C19 genotypes and mephenytoin phenotype were compared in 104 Ethiopians. Poor metabolism of mephenytoin was seen in persons homozygous for both rs12769205 and rs4244285 (CYP2C19*2/*2), but with little effect on mephenytoin disposition of CYP2C19*1/*2, CYP2C19*1/*3, or CYP2C19*1/*35 heterozygous alleles. Extended haplotype homozygosity tests of the HapMap Yorubans (YRI) showed both haplotypes carrying rs12769205 (CYP2C19*35 and CYP2C19*2) are under significant natural selection, with CYP2C19*35 having a higher relative extended haplotype homozygosity score. The phylogenetic tree of the YRI CYP2C19 haplotypes revealed rs12769205 arose first on CYP2C19*35 and that rs4244285 was added later, creating CYP2C19*2. In conclusion, rs12769205 is the ancestral polymorphism leading to aberrant splicing of CYP2C19*35 and CYP2C19*2 alleles in liver.

Predictors of Variation in CYP2A6 mRNA, Protein, and Enzyme Activity in a Human Liver Bank: Influence of Genetic and Nongenetic Factors.

Cytochrome P450 2A6 (CYP2A6) metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47–0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine.

Hepatic Abundance and Activity of Androgen- and Drug-Metabolizing Enzyme UGT2B17 Are Associated with Genotype, Age, and Sex

The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes (n = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (∼10-fold) with ∼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures.