Svetlana Markova

Very important pharmacogene summary: ABCB1 (MDR1, P-glycoprotein).

ABCB1 descriptionABCB1 (MDR1) is one of many ubiquitous adenosine triphosphate (ATP)-binding cassette (ABC) genes present in all kingdoms of life that is responsible for cellular homeostasis [1–3]. ABC genes encode transporter and channel proteins possessing multiple membrane-spanning domains that f

Attenuation of Cisplatin-Induced Renal Injury by Inhibition of Soluble Epoxide Hydrolase Involves Nuclear Factor κB Signaling

Acute kidney injury is associated with a significant inflammatory response that has been the target of renoprotection strategies. Epoxyeicosatrienoic acids (EETs) are anti-inflammatory cytochrome P450-derived eicosanoids that are abundantly produced in the kidney and metabolized by soluble epoxide hydrolase (sEH; Ephx2) to less active dihydroxyeicosatrienoic acids. Genetic disruption of Ephx2 and chemical inhibition of sEH were used to test whether the anti-inflammatory effects of EETs, and other lipid epoxide substrates of sEH, afford protection against cisplatin-induced nephrotoxicity. EET hydrolysis was significantly reduced in Ephx2(−/−) mice and was associated with an attenuation of cisplatin-induced increases in serum urea nitrogen and creatinine levels. Histological evidence of renal tubular damage and neutrophil infiltration was also reduced in the Ephx2(−/−) mice. Likewise, cisplatin had no effect on renal function, neutrophil infiltration, or tubular structure and integrity in mice treated with the potent sEH inhibitor 1-adamantan-1-yl-3-(1-methylsulfonyl-piperidin-4-yl-urea) (AR9273). Consistent with the ability of EETs to interfere with nuclear factor-κB (NF-κB) signaling, the observed renoprotection was associated with attenuation of renal NF-κB activity and corresponding decreases in the expression of tumor necrosis factor (TNF) α, TNF receptor (TNFR) 1, TNFR2, and intercellular adhesive molecule-1 before the detection of tubular injury. These data suggest that EETs or other fatty acid epoxides can attenuate cisplatin-induced kidney injury and sEH inhibition is a novel renoprotective strategy.

Association of CYP2C9*2 With Bosentan‐Induced Liver Injury

Bosentan (Tracleer) is an endothelin receptor antagonist prescribed for the treatment of pulmonary arterial hypertension (PAH). Its use is limited by drug‐induced liver injury (DILI). To identify genetic markers of DILI, association analyses were performed on 56 Caucasian PAH patients receiving bosentan. Twelve functional polymorphisms in five genes (ABCB11, ABCC2, CYP2C9, SLCO1B1, and SLCO1B3) implicated in bosentan pharmacokinetics were tested for associations with alanine aminotransferase (ALT), aspartate aminotransferase (AST), and DILI. After adjusting for body mass index, CYP2C9*2 was the only polymorphism associated with ALT, AST, and DILI (β = 2.16, P = 0.024; β = 1.92, P = 0.016; odds ratio 95% CI = 2.29−∞, P = 0.003, respectively). Bosentan metabolism by CYP2C9*2 in vitro was significantly reduced compared with CYP2C9*1 and was comparable to that by CYP2C9*3. These results suggest that CYP2C9*2 is a potential genetic marker for prediction of bosentan‐induced liver injury and warrants investigation for the optimization of bosentan treatment.

The relationship of polymorphisms in ABCC2 and SLCO1B3 with docetaxel pharmacokinetics and neutropenia: CALGB 60805 (Alliance)

Docetaxel-related neutropenia was associated with polymorphisms in the drug transporters ABCC2 and SLCO1B3 in Japanese cancer patients. We hypothesized that this association is because of reduced docetaxel clearance, associated with polymorphisms in those genes. We studied 64 US cancer patients who received a single cycle of 75 mg/m of docetaxel monotherapy. We found that the ABCC2 polymorphism at rs-12762549 trended to show a relationship with reduced docetaxel clearance (P=0.048), but not with neutropenia. There was no significant association of the SLCO1B3 polymorphisms with docetaxel clearance or neutropenia. We conclude that the relationship between docetaxel-associated neutropenia and polymorphisms in drug transporters identified in Japanese patients was not confirmed in this cohort of US cancer patients.

FUNCTIONAL CHARACTERIZATION OF ABCC2 PROMOTER POLYMORPHISMS AND ALLELE SPECIFIC EXPRESSION

Multidrug resistance protein 2 (MRP2, ABCC2) is an efflux membrane transporter highly expressed in liver, kidney and intestine with important physiological and pharmacological roles. The goal of this study was to investigate the functional significance of promoter region polymorphisms in ABCC2 and potential allele-specific expression. Twelve polymorphisms in the 1.6 kb region upstream of the translation start site were identified by resequencing 247 DNA samples from ethnically diverse individuals. Luciferase reporter gene assays showed that ABCC2 −24C>T both alone and as part of a common haplotype (−24C>T/−1019A>G/−1549G>A) increased promoter function 35% compared with the reference sequence (P<0.0001). No other common variants or haplotypes affected ABCC2 promoter activity. Allele-specific expression was also investigated as a mechanism to explain reported associations of the synonymous ABCC2 3972C>T variant with pharmacokinetic phenotypes. In Caucasian liver samples (n=41) heterozygous for the 3972C>T polymorphism, the 3972C allele was preferentially transcribed relative to the 3972T allele (P<0.0001). This allelic imbalance was particularly apparent in samples with haplotypes containing two or three promoter/untranslated region variants (−1549G>A, −1019A>G and −24C>T). The observed allelic imbalance was not associated with hepatic or renal ABCC2 mRNA expression. Additional mechanisms will need to be explored to account for the interindividual variation in ABCC2 expression and MRP2 function.

Pharmacogene regulatory elements: from discovery to applications

Regulatory elements play an important role in the variability of individual responses to drug treatment. This has been established through studies on three classes of elements that regulate RNA and protein abundance: promoters, enhancers and microRNAs. Each of these elements, and genetic variants within them, are being characterized at an exponential pace by next-generation sequencing (NGS) technologies. In this review, we outline examples of how each class of element affects drug response via regulation of drug targets, transporters and enzymes. We also discuss the impact of NGS technologies such as chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq), and the ramifications of new techniques such as high-throughput chromosome capture (Hi-C), chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and massively parallel reporter assays (MPRA). NGS approaches are generating data faster than they can be analyzed, and new methods will be required to prioritize laboratory results before they are ready for the clinic. However, there is no doubt that these approaches will bring about a systems-level understanding of the interplay between genetic variants and drug response. An understanding of the importance of regulatory variants in pharmacogenomics will facilitate the identification of responders versus non-responders, the prevention of adverse effects and the optimization of therapies for individual patients.

Discovery of regulatory elements in human ATP-binding cassette transporters through expression quantitative trait mapping

ATP-binding cassette (ABC) membrane transporters determine the disposition of many drugs, metabolites and endogenous compounds. Coding region variation in ABC transporters is the cause of many genetic disorders, but much less is known about the genetic basis and functional outcome of ABC transporter expression level variation. We used genotype and mRNA transcript level data from human lymphoblastoid cell lines to assess population and gender differences in ABC transporter expression, and to guide the discovery of genomic regions involved in transcriptional regulation. Nineteen of 49 ABC genes were differentially expressed between individuals of African, Asian and European descent, suggesting an important influence of race on expression level of ABC transporters. Twenty-four significant associations were found between transporter transcript levels and proximally located genetic variants. Several of the associations were experimentally validated in reporter assays. Through influencing ABC expression levels, these single-nucleotide polymorphisms may affect disease susceptibility and response to drugs.

Response to “CYP2C9 Polymorphism is Not a Major Determinant of Bosentan Exposure in Healthy Volunteers”

To the Editor: We appreciate the opportunity to respond to the letter by Markert et al., “CYP2C9 Polymorphism Is Not a Major Determinant of Bosentan Exposure in Healthy Volunteers.”1 In response to our study on genetic predictors of bosentaninduced liver injury in pulmonary arterial hypertension (PAH) patients, in which we demonstrated an association between the CYP2C9*2 allele, encoding the cytochrome P450 complex subunit 2C9, and liver injury,2 Markert et al. report steady-state bosentan pharmacokinetics in 36 healthy volunteers. They show that plasma bosentan levels were highly variable and not associated with CYP2C9 metabolizer phenotype. Consistent with a role for CYP2C9 in the formation of the major active metabolite hydroxy bosentan (RO48-5033), levels of RO48-5033 were 68.7% greater in extensive metabolizers than in poor metabolizers. The pharmacokinetic data obtained by Markert et al. suggest that the genetic association that we reported is not easily explained by differences in bosentan plasma concentrations between CYP2C9 genotype groups. As Markert et al. point out, CYP2C9-dependent intrahepatic bosentan concentrations may be more relevant and may not be directly related to bosentan plasma concentrations. Although hepatic transporter polymorphisms were not associated with bosentan hepatotoxicity in our study,2 the possibility exists that transporter function is altered in PAH patients as a result of disease or drug treatment. Furthermore, as discussed in our paper, there is a possibility that CYP2C9*2 is not a causative singlenucleotide polymorphism (SNP) but tags a regulatory SNP.2 The difference in study populations may also have influenced the reported results. The pharmacokinetic study by Markert et al. was carried out in healthy volunteers, whereas our genetic association study was performed in PAH patients receiving steady-state bosentan for clinical indications. As also noted by Markert et al., differences in pharmacokinetics between CYP2C9 metabolizer groups might be more apparent in PAH patients, especially because they may have a longer duration of exposure. Indeed, previously published bosentan pharmacokinetic data for PAH patients show that bosentan exposure is not proportional to dose and is approximately twofold higher than that in healthy subjects.3 In addition, because PAH affects expression of endothelin receptors,4 it may affect the tissue-to-plasma ratios of bosentan in these patients. Finally, the possibility that concurrent medications in PAH patients affect bosentan pharmacological and toxicological properties cannot be excluded. Although studies in healthy volunteers provide important information about human pharmacokinetic characteristics, it is difficult to extrapolate this information to toxicity phenotypes in relevant and complex patient populations. We welcome further investigations to elucidate mechanisms of liver injury and the potential role of pharmacogenomics in patient groups that are candidates for bosentan therapy.

Multidrug resistance-associated protein 4 is a determinant of arsenite resistance.

Although arsenic trioxide (arsenite, AsIII) has shown a remarkable efficacy in the treatment of acute promyelocytic leukemia patients, multidrug resistance is still a major concern for its clinical use. Multidrug resistance-associated protein 4 (MRP4), which belongs to the ATP-binding cassette (ABC) superfamily of transporters, is localized to the basolateral membrane of hepatocytes and the apical membrane of renal proximal tubule cells. Due to its characteristic localization, MRP4 is proposed as a candidate in the elimination of arsenic and may contribute to resistance to AsIII. To test this hypothesis, stable HEK293 cells overexpressing MRP4 or MRP2 were used to establish the role of these two transporters in AsIII resistance. The IC50 values of AsIII in MRP4 cells were approximately 6-fold higher than those in MRP2 cells, supporting an important role for MRP4 in resistance to AsIII. The capacity of MRP4 to confer resistance to AsIII was further confirmed by a dramatic decrease in the IC50 values with the addition of MK571, an MRP4 inhibitor, and cyclosporine A, a well-known broad-spectrum inhibitor of ABC transporters. Surprisingly, the sensitivity of the MRP2 cells to AsIII was similar to that of the parent cells, although insufficient formation of glutathione and/or Se conjugated arsenic compounds in the MRP2 cells might limit transport. Given that MRP4 is a major contributor to arsenic resistance in vitro, further investigation into the correlation between MRP4 expression and treatment outcome of leukemia patients treated with arsenic-based regimens is warranted.