Pharmacogenetics of Human Carbonyl Reductases

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DESCRIPTION (provided by applicant): Human carbonyl reductases (CBRs) catalyze the reduction of several drugs widely used in clinical practice including the anticancer anthracyclines doxorubicin and daunorubicin. The pharmacodynamics of these 2 drugs is unpredictable. We hypothesize that interindividual variability in CBR activity contributes to the unpredictable pharmacodynamic profiles for doxorubicin and daunorubicin. Therefore, our main goal is to characterize the molecular basis of variable CBR activity as a prerequisite for the design of more effective anticancer therapies. Thus far, we have (1) characterized functional allelic variants of carbonyl reductase 1 (CBR1) and carbonyl reductase 3 (CBR3); (2) documented the variability in CBR1 and CBR3 expression in hepatic tissue; and (3) identified a genetic risk factor (CBR3 V244M) for anthracycline-related cardiotoxicity in pediatric cancer survivors. Gene regulation studies indicate that specific DNA sequences in the promoter regions of CBR1 and CBR3 influence the level of protein expression and activity in response to various stimuli. New data indicate that CBR3 mRNA expression increases considerably (8.5-fold) in the presence of the prototypical antioxidant tert-butylhydroquinone and that the CBR3 promoter contains 2 conserved antioxidant response elements (AREs). We have designed experiments that will allow us to characterize the functional role of AREs in the induction of CBR3 expression in response to antioxidant exposure (Specific Aim 1). These experiments will also allow us to determine how 2 common CBR3 promoter polymorphisms (CBR3 -725T>C, and CBR3 -326T>A) modulate gene promoter activity in response to antioxidants. A common CBR1 polymorphism (1096G>A) dictates the synthesis of cardiotoxic doxorubicinol in human hepatic tissue. We have planned experiments to determine whether the effect of CBR1 1096G>A is mediated through the binding of specific microRNAs to the polymorphic 3'-untranslated region (Specific Aim 2). A growing amount of experimental evidence, together with our pharmacogenetic findings, suggests that CBR1 and CBR3 have a crucial role in the complex pharmacodynamics of anthracyclines in the heart. The expression of CBR1 and CBR3 in the human heart has not been characterized. We plan to document the relative contributions of CBR1 and CBR3 to the metabolism of doxorubicin and daunorubicin in 200 samples of human myocardial tissue (Specific Aim 3). In this comprehensive approach we will use quantitative real-time PCR analysis, nano-liquid chromatography coupled to triple quadruple mass spectroscopy, and enzyme activity assays with CBR substrates (e.g., doxorubicin) and inhibitors (e.g., the cardioprotective flavonoid monohydroxyethyl rutoside, or mono-HER). We will also conduct genotype-phenotype correlation studies to determine whether functional CBR1 and CBR3 polymorphisms affect the formation of cardiotoxic anthracycline metabolites in the heart. The body of knowledge gathered from the proposed research will contribute to the development of anticancer therapy that can be individualized by identifying the genetic determinants of variable CBR activity. PUBLIC HEALTH RELEVANCE: Human carbonyl reductases (CBR1 and CBR3) catalyze the reduction of several drugs including the anticancer anthracyclines doxorubicin and daunorubicin. The anthracycline alcohol metabolites synthesized by CBR activity are cardiotoxic. Three research aims will investigate (1) the regulation of polymorphic CBR3, (2) the functional impact of a common polymorphism in human CBR1, and (3) the pharmacogenetics of CBR1 and CBR3 with doxorubicin and daunorubicin in human myocardium, the target tissue for anthracycline-related cardiotoxicity.