Jatinder K. Lamba

Disrupted Bile Acid Homeostasis Reveals an Unexpected Interaction among Nuclear Hormone Receptors, Transporters, and Cytochrome P450

Sister of P-glycoprotein (SPGP) is the major hepatic bile salt export pump (BSEP). BSEP/SPGP expression varies dramatically among human livers. The potency and hierarchy of bile acids as ligands for the farnesyl/bile acid receptor (FXR/BAR) paralleled their ability to induce BSEP in human hepatocyte cultures. FXR:RXR heterodimers bound to IR1 elements and enhanced bile acid transcriptional activation of the mouse and human BSEP/SPGP promoters. In FXR/BAR nullizygous mice, which have dramatically reduced BSEP/SPGP levels, hepatic CYP3A11 and CYP2B10 were strongly but unexpectedly induced. Notably, the rank order of bile acids as CYP3A4 inducers and activators of pregnane X receptor/steroid and xenobiotic receptor (PXR/SXR) closely paralleled each other but was markedly different from their hierarchy and potency as inducers of BSEP in human hepatocytes. Moreover, the hepatoprotective bile acid ursodeoxycholic acid, which reverses hydrophobic bile acid hepatotoxicity, activates PXR and efficaciously induces CYP3A4 (a bile-metabolizing enzyme) in primary human hepatocytes thus providing one mechanism for its hepatoprotection. Because serum and urinary bile acids increased in FXR/BAR −/− mice, we evaluated hepatic transporters for compensatory changes that might circumvent the profound decrease in BSEP/SPGP. We found weak MRP3 up-regulation. In contrast, MRP4 was substantially increased in the FXR/BAR nullizygous mice and was further elevated by cholic acid. Thus, enhanced hepatocellular concentrations of bile acids, due to the down-regulation of BSEP/SPGP-mediated efflux in FXR nullizygous mice, result in an alternate but apparent compensatory up-regulation of CYP3A, CYP2B, and some ABC transporters that is consistent with activation of PXR/SXR by bile acids.

Natural allelic variants of breast cancer resistance protein (bcrp) and their relationship to Bcrp expression in human intestine

The aim of this study was to identify the extent of genetic variability in breast cancer resistance protein (BCRP) in humans. We first analysed the sequence of BCRP cDNA from human livers and from human intestines phenotyped for expression of intestinal BCRP. We then determined the frequency of all known coding single nucleotide polymorphisms (cSNPs) using DNA from individuals representing 11 different ethnic populations. Nine SNPs including four non-synonymous and three synonymous cSNPs and two intronic SNPs were identified. Of the missense mutations, exon 2 SNP (G34A) resulted in a V12M change; exon 5 SNP (C421A) resulted in a Q141K substitution; exon 6 SNP (A616C) resulted in an I206L amino acid substitution; and exon 15 SNP (A1768T) resulted in a N590Y change in the BCRP protein. The two most frequent polymorphisms identified in the human population studied were the G34A and C421A transitions. There was marked variation in BCRP genotypes and allele frequencies in the different populations. BCRP mRNA was phenotyped in human small bowel intestinal samples by real-time polymerase chain reaction and BCRP protein was analysed on immunoblots of tissue from the same individuals. There was a 78-fold variation in expression of BCRP mRNA and significant variation in BCRP protein expression in human intestine. Expression of intestinal BCRP mRNA and protein was not different between persons expressing the common Gln141 allele compared to the Lys141 allele. Thus, common natural allelic variants of BCRP have been identified, and did not influence interindividual variation in expression of BCRP mRNA in human intestine, but remain to be tested for their effect on BCRP function.

Tacrolimus Dosing in Pediatric Heart Transplant Patients is Related to CYP3A5 and MDR1 Gene Polymorphisms

Tacrolimus is a substrate for P‐glycoprotein (P‐gp) and cytochrome (CYP) P4503A. P‐gp is encoded by the multiple drug resistance gene MDR1 and CYP3A is the major enzyme responsible for tacrolimus metabolism. Both MDR1 and CYP3A5 genes have multiple single nucleotide polymorphisms. The objective of this study was to evaluate whether the MDR1 exon21 and exon26 polymorphisms and the CYP3A5 polymorphism are associated with tacrolimus disposition in pediatric heart transplant patients. At 3, 6 and 12 months post transplantation, a significant difference in tacrolimus blood level per dose/kg/day was found between the CYP3A5 *1/*3 (CYP3A5 expressor) vs. *3/*3 (nonexpressor) genotypes with the *1/*3 patients requiring a larger tacrolimus dose to maintain the same blood concentration. There were no significant differences in tacrolimus blood level per dose/kg/day between MDR1 exon21 G2677T and exon 26 C3435T at 3 months, but both were found to have a significant association with tacrolimus blood level per dose/kg/day at 6 and 12 months. We conclude that specific genotypes of MDR1 and CYP3A5 in pediatric heart transplant patients require larger tacrolimus doses to maintain their tacrolimus blood concentration, and that this information could be used prospectively to manage patients immunosuppressive therapy.

The emerging role of electronic medical records in pharmacogenomics.

Health‐care information technology and genotyping technology are both advancing rapidly, creating new opportunities for medical and scientific discovery. The convergence of these two technologies is now facilitating genetic association studies of unprecedented size within the context of routine clinical care. As a result, the medical community will soon be presented with a number of novel opportunities to bring functional genomics to the bedside in the area of pharmacotherapy. By linking biological material to comprehensive medical records, large multi‐institutional biobanks are now poised to advance the field of pharmacogenomics through three distinct mechanisms: (i) retrospective assessment of previously known findings in a clinical practice‐based setting, (ii) discovery of new associations in huge observational cohorts, and (iii) prospective application in a setting capable of providing real‐time decision support. This review explores each of these translational mechanisms within a historical framework.

Pharmacogenetic characteristics of indinavir, zidovudine, and lamivudine therapy in HIV-infected adults: A pilot study

Objective: The aim of the study was to investigate relationships among indinavir, lamivudine-triphosphate, and zidovudine-triphosphate pharmacokinetics and pharmacodynamics with polymorphisms in CYP3A5, MDR1, MRP2, MRP4, BCRP, and UGT1A1 genes. Study Design: Retrospective pilot investigation among 33 subjects who participated in a randomized pharmacological study of indinavir, lamivudine, and zidovudine. Subjects were defined as genetic variant carriers or not. Relationships were investigated with multivariable regression. Indinavir clearance was adjusted for African American race; triphosphates for sex; and HIV-response for study arm, drug exposure, and baseline HIV-RNA. Results: Genetically determined CYP3A5 expressors had 44% faster indinavir oral clearance versus nonexpressors (P = 0.002). MRP2-24C/T variant carriers had 24% faster indinavir oral clearance (P = 0.05). Lamivudine-triphosphate concentrations were elevated 20% in MRP4 T4131G variant carriers (P = 0.004). A trend for elevated zidovudine-triphosphates was observed in MRP4 G3724A variant carriers (P = 0.06). The log10 changes in HIV-RNA from baseline to week 52 were -3.7 for MDR1 2677 TT, -3.2 for GT, and -2.2 for GG (P < 0.05). Bilirubin increases were 2-fold higher in UGT1A1 [TA]7/[TA]7 genotypes. No relationships were identified with BCRP. Discussion: Novel relationships were identified among genetic variants in drug transporters and indinavir, lamivudine-triphosphate, and zidovudine-triphosphate concentrations. CYP3A5 expression was associated with faster indinavir oral clearance. These pilot data provide a scientific basis for more rational utilization of antiretroviral drugs.

Tacrolimus Dosing in Adult Lung Transplant Patients Is Related to Cytochrome P4503A5 Gene Polymorphism

Tacrolimus is a potent immunosuppressive agent used in lung transplantation and is a substrate for both P‐glycoprotein (P‐gp, encoded by the gene MDR1) and cytochrome (CYP) P4503A. A previous study by the authors identified a correlation between the tacrolimus blood level per dose with CYP3A5 and MDR1 gene polymorphisms in pediatric heart transplant patients. The objective of this study was to confirm the influence of these polymorphisms on tacrolimus dosing in adult lung transplant patients. Adult lung transplant patients who had been followed for at least 1 year after lung transplantation were studied. Tacrolimus blood level (ng/mL) per dose (mg/day) at 1, 3, 6, 9, and 12 months after transplantation was calculated as [L/D]. DNA was extracted from blood. MDR1 3435 CC, CT, and TT; MDR1 2677 GG, GT, and TT; and CYP3A5*1 (expressor) and *3 (nonexpressor) genotypes were determined by PCR amplification, direct sequencing, and sequence evaluation. Eightythree patients were studied. At 1, 3, 6, 9, and 12 months after the transplant, a significant difference in [L/D] was found between the CYP3A5 expressor versus nonexpressor genotypes (mean ± SD of 1.49 ± 0.88 vs. 3.11 ± 4.27, p = 0.01; 1.23 ± 0.82 vs. 3.44 ± 8.97, p = 0.05; 1.32 ± 0.96 vs. 3.81 ± 6.66, p = 0.005; 0.95 ±1.19 vs. 3.74 ±5.98, p = 0.0015; and 0.45 ± 0.2 vs. 3.76 ± 6.75, p = 0.0001, respectively). MDR1 G2677T and C3435T genotypes had only minimal effects on [L/D] at 1 and 3 months after transplantation. This study confirms the relationship of CYP3A5 polymorphisms to tacrolimus dosing in organ transplant patients. CYP3A5 expressor genotypes required a larger tacrolimus dose to achieve the same blood levels than the CYP3A5 nonexpressors at all time points during the first posttransplant year. This was not uniformly true for MDR1. The authors therefore conclude that tacrolimus dosing in adult lung transplant patients is associated with CYP3A5 gene polymorphisms.

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.

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.