Vishal Lamba

The orphan nuclear receptor HNF4α determines PXR- and CAR-mediated xenobiotic induction of CYP3A4

The drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is thought to be involved in the metabolism of nearly 50% of all the drugs currently prescribed. Alteration in the activity or expression of this enzyme seems to be a key predictor of drug responsiveness and toxicity. Currently available studies indicate that the ligand-activated nuclear receptors pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) regulate CYP3A4 expression. However, in cell-based reporter assays, CYP3A4 promoter activity was most pronounced in liver-derived cells and minimal or modest in non-hepatic cells, indicating that a liver-specific factor is required for physiological transcriptional response. Here we show that the orphan nuclear receptor hepatocyte nuclear factor-4α (HNF4α; HNF4A) is critically involved in the PXR- and CAR-mediated transcriptional activation of CYP3A4. We identified a specific cis-acting element in the CYP3A4 gene enhancer that confers HNF4α binding and thereby permits PXR- and CAR-mediated gene activation. Fetal mice with conditional deletion of Hnf4α had reduced or absent expression of CYP3A. Furthermore, adult mice with conditional hepatic deletion of Hnf4α had reduced basal and inducible expression of CYP3A. These data identify HNF4α as an important regulator of coordinate nuclear-receptor–mediated response to xenobiotics.

Pharmacogenetic pathway analysis of docetaxel elimination

The purpose of this study was to evaluate the affinity of docetaxel for 14 transporter proteins and assess the functional significance of 17 variants in five genes involved in drug elimination. Among the transfected models investigated, OATP1B3 (SLCO1B3) was identified as the most efficient influx transporter for docetaxel. None of the observed genotypes (SLCO1B3, ABCB1, and ABCC2) was related with docetaxel clearance in 92 white patients (P > 0.17). However, the simultaneous presence of the CYP3A4*1B and CYP3A5*1A alleles was associated with a 64% increase in docetaxel clearance (P = 0.0015), independent of both sex and CYP3A activity (as determined using the erythromycin breath test). This haplotype was also associated with increased midazolam clearance in another population (P = 0.0198). An analysis of the CYP3A locus among CEPH‐HapMap samples revealed that CYP3A4*1B is present exclusively among a subset of CYP3A5 expressors. Therefore, future studies should first stratify the population on the basis of CYP3A5 genotype and then compare CYP3A activity between individuals with and without the CYP3A4*1B allele.

Novel Single Nucleotide Polymorphisms in the Promoter and Intron 1 of Human Pregnane X Receptor/NR1I2 and Their Association with CYP3A4 Expression

The hypothesis was tested that sequence diversity in pregnane X receptor (PXR) cis-regulatory regions is a significant determinant of variation in inducible and constitutive CYP3A4 expression. A combination of comparative genomics and computational algorithms was used to select regions of the human PXR promoter and intron 1 that were resequenced in the polymorphism discovery resource 24 DNA subset. PXR single nucleotide polymorphisms (SNP) were then genotyped in donor human livers phenotyped for CYP3A4 and multidrug resistance protein 1 mRNA and primary human hepatocytes phenotyped for basal and rifampin-inducible CYP3A4 activity. The human PXR promoter [16.9 kilobase (kb)] was significantly larger than in rodents (2.9 kb). Eighty-nine SNPs were identified in the promoter and intron 1 of PXR. The SNPs most consistently associated with CYP3A4 phenotypic measures were a 44477T>C(-1359) promoter SNP (in linkage disequilibrium with SNP 463970, a 6-base pair deletion in intron 1a, and SNP 46551, a C nucleotide insertion in intron 1b); SNP 63396C>T in intron 1 (in linkage disequilibrium with SNP 63704A>G, a 63813(CAAA)(CA) variable repeat, and SNP 65104T>C); and SNP 56348C>A, SNP 69789A>G, and SNP 66034T>C. Donor livers with the variant PXR alleles had altered hepatic expression of PXR targets compared with livers with PXR wild-type alleles. These results identified PXR promoter and intron 1 SNPs associated with PXR target gene expression (CYP3A4) in donor livers and cultured hepatocytes and that a striking number of the linked intron 1 SNPs will affect putative binding sites for hepatic nuclear factor 3β (FOXA2), a transcription factor linked with PXR expression.

Novel Polymorphisms Associated with Tacrolimus Trough Concentrations: Results from a Multicenter Kidney Transplant Consortium

Background. The CYP4503A5*1 genotype is associated with lower tacrolimus concentrations. Although its effect is important, it incompletely explains the variability in tacrolimus concentrations and has a relatively low minor allele frequency in whites relative to African Americans (AA). Methods. We studied clinical and recipient genetic correlates of dose-normalized tacrolimus troughs (n=12,277) in the first 6 months posttransplant using a customized single-nucleotide polymorphism chip with 2722 variants in a large, ethnically diverse (144 AA and 551 non-AA) adult kidney transplant population through a seven-center consortium. Results. During the 6-month study, AAs had consistently lower median (interquartile range) troughs than non-AAs, 6.2 (4.4–8.4) ng/mL vs. 8.3 (6.4–10.4) ng/mL (P<0.0001), despite 60% higher daily doses, 8 (5–10) mg vs. 5 (4–7) mg (P<0.0001). The median tacrolimus trough concentration in week 1 posttransplant was particularly low in AAs (2.1 [1.2–3.5] ng/mL) compared with non-AAs (5.0 [3.1–8.2] ng/mL) (P<0.0001), despite similar initial doses. In single-variant analysis, CYP3A5*3 (rs776746) was the top variant (P=2.4×10−33) associated with troughs. After adjustment for CYP3A5*3, clinical factors and race, 35 additional variants were identified (P<0.01, not significant at false discovery rate 20%). In the final multivariant, regression models beginning with these variants and clinical factors, seven variants were identified in the non-AA and seven variants in the AA group towards the first trough concentrations. Rs776746 (CYP3A5), rs2239393 (COMT) and diabetes were the only factors common in both populations. Conclusion. We identified variants beyond CYP3A5*3, which may further explain pharmacokinetic variability of tacrolimus and demonstrated that important variants differ by race.

Genetic Variants of PXR (NR1I2) and CAR (NR1I3) and Their Implications in Drug Metabolism and Pharmacogenetics

The defense mechanisms responsible for protecting the body from endogenous toxins are also involved in the metabolism of drugs and are composed of phase I and phase II drug metabolizing enzymes, as well as drug transporters. Numerous drugs and chemicals have been shown to modulate the expression of the genes involved in these three drug-detoxifying processes. Induction of these genes contributes to both auto-induction of drug clearance and to drug-drug interactions in combination therapies. The orphan nuclear receptors PXR (pregnane X receptor) and CAR (Constitutive androstane receptor) are xenosensors that mediate drug-induced changes by increasing transcription of genes that are involved in drug clearance and disposition. Co-administration of drugs, one of which is a nuclear receptor agonist or antagonist, can either lead to altered clearance of the second drug and severe toxicity, or a loss of therapeutic efficacy or an imbalance in physiological substrate concentrations, providing a novel molecular mechanism for drug-drug interactions. Thus, genetic variability in these nuclear receptors will contribute to human variation in the magnitude of clinically significant drug-drug interactions. This review describes common PXR and CAR genetic variants that have been identified to date in the human population and the functional consequence of these variant alleles. In addition, alternatively spliced variants of PXR and CAR that may also contribute to individual variability as well as tissue specific expression of these receptors are also described. Identification of PXR and CAR genetic variants and alternatively spliced mRNAs may ultimately allow predictions of interindividual differences in target gene induction and drug-drug interactions.

Variation in oral clearance of saquinavir is predicted by CYP3A5*1 genotype but not by enterocyte content of cytochrome P450 3A5

Saquinavir, a widely prescribed human immunodeficiency virus 1 protease inhibitor, has a low and variable oral bioavailability that has been attributed to extensive first‐pass extraction mediated by hepatic or intestinal cytochrome P450 (CYP) 3A4 and intestinal P‐glycoprotein (P‐gp). The polymorphic CYP3A5 has also been shown to influence the saquinavir metabolite/parent urinary ratio, suggesting a role for CYP3A5.

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.

Genetic Predictors of Interindividual Variability in Hepatic CYP3A4 Expression

Variability in hepatic CYP3A4 cannot be explained by common CYP3A4 coding variants. We previously identified polymorphisms in pregnane X receptor (PXR) and ATP-binding cassette subfamily B member 1 (ABCB1) associated with CYP3A4 mRNA levels in small cohorts of human livers. However, the relative contributions of these genetic variations or of polymorphisms in other CYP3A4 regulators to variable CYP3A4 expression were not known. We phenotyped livers from white donors (n = 128) by quantitative real-time polymerase chain reaction for expression of CYP3A4, CYP3A5, and CYP3A7 and nine transcriptional regulators, coactivators, and corepressors. We resequenced hepatic nuclear factor-3-β (HNF3β, FoxA2), HNF4α, HNF3γ (FoxA3), nuclear receptor corepressor 2 (NCoR2), and regions of the CYP3A4 promoter and genotyped informative single-nucleotide polymorphisms in PXR and ABCB1 in the same livers. CYP3A4 mRNA was positively correlated with PXR and FoxA2 and negatively correlated with NCoR2 mRNA. A common silent polymorphism and a polymorphic trinucleotide (CCT) repeat in FoxA2 were associated with CYP3A4 expression. The transcriptional activity of the FoxA2 polymorphic CCT repeat alleles (wild-type, n = 14 and variant, n = 13, 15, and 19) when assayed by luciferase reporter transactivation assays was greatest for the wild-type repeat, with deviations from this number having decreased transcriptional activity. This corresponded with higher expression of FoxA2 mRNA and its targets PXR and CYP3A4 in human livers with (CCT) n = 14 genotypes. Multiple linear regression analysis was used to quantify the contributions of selected genetic polymorphisms to variable CYP3A4 expression. This approach identified sex and polymorphisms in FoxA2, HNF4α, FoxA3, PXR, ABCB1, and the CYP3A4 promoter that together explained as much as 24.6% of the variation in hepatic CYP3A4 expression.

Genetic and Clinical Determinants of Early, Acute Calcineurin Inhibitor-Related Nephrotoxicity: Results from a Kidney Transplant Consortium

Background. Calcineurin inhibitor (CNI)-related acute nephrotoxicity is a common complication of transplantation. Clinical factors and elevated CNI levels are associated with nephrotoxicity; however, they do not fully explain the risk. Genetic factors may also predispose individuals to nephrotoxicity. Methods. We enrolled 945 kidney recipients into a multicenter, prospective study. DNA was genotyped for 2724 single-nucleotide polymorphisms (SNPs) using a customized chip. Cox models, unadjusted and adjusted for clinical factors, examined the association between SNPs and time to early CNI-related acute nephrotoxicity in the first 6 months posttransplant. Results. Cyclosporine was associated with a 1.49 hazard (95% confidence interval, 1.04–2.14) of acute nephrotoxicity relative to tacrolimus. Acute nephrotoxicity occurred in 22.6% of cyclosporine and 19.8% of tacrolimus recipients. The median (interquartile range) daily dose and trough concentration at time of nephrotoxicity were 400 mg (400–500 mg) and 228 ng/mL (190–272 ng/mL) in the cyclosporine group, and 6 mg (4–8 mg) and 12.6 ng/mL (10.2–15.9 ng/mL) in the tacrolimus group, respectively. In single-SNP adjusted analysis, nine SNPs in the XPC, CYP2C9, PAX4, MTRR, and GAN genes were associated with cyclosporine nephrotoxicity. In a multi-SNP analysis, SNPs from the same genes remained significant after adjusting for the clinical factors, showing that the SNPs are jointly and independently predictive of cyclosporine nephrotoxicity. No SNPs were associated with tacrolimus nephrotoxicity. Conclusion. We identified SNPs that were potentially associated with early, acute cyclosporine-related nephrotoxicity. Identifying risk SNPs before transplantation provides an opportunity for personalization of immunosuppression by identifying those who may benefit from CNI-avoidance or minimization, or assist in selecting CNI type. These SNPs require independent validation.

Genetic determinants of mycophenolate-related anemia and leukopenia after transplantation

Background. Mycophenolate-related anemia and leukopenia are well-known toxicities after transplantation. Toxicity leads to dose reduction, addition of colony-stimulating factors or erythropoietin, or discontinuation of immunosuppressive therapy. The causes of and risk factors associated with toxicity are unclear. Methods. We studied the association between mycophenolate-related anemia and leukopenia and 2724 single nucleotide polymorphisms (SNP) in 978 patients undergoing living or deceased donor kidney transplant. Patients were followed up to time of first anemia (hemoglobin<10 gm/dL or hematocrit<30%) or first leukopenia (white blood cell [WBC] count <3000 cells/mm3), which required clinical intervention in the first 6 months after transplant. Results. Anemia occurred in 87 (9.5%) subjects and leukopenia in 224 (22.9%). In single SNP analyses, none of the SNPs were associated with time to leukopenia at a false discovery rate (FDR) of 20%. However, SNPs from the IL12A, HUS, CYP2C8 genes were associated with time to anemia, allowing for an FDR of 20%. To assess the independence of these SNPs as predictors of anemia, we conducted a multi-SNP analysis including one SNP from each of the three genes. All three SNPs were associated with time to anemia, after adjusting for recipient age, weight, posttransplant dialysis and antiviral drug use, and stratifying by clinical center. Conclusion. Although these SNPs require validation in an independent population, our results suggest that genetics may play a role in risk of mycophenolate-related hematologic toxicity. This may ultimately provide for better management of maintenance immunosuppression and gives insights into potential mechanism(s) by which toxicity occurs.