Characterization of inhibition by vardenafil analogues of ATP-dependent transport of cGMP by the ABCC5 transporter.

Background: Clinical studies have reported overexpression of PDE5 and elevation of intracellular cyclic GMP in various types of cancer cells. ABCC5 transports intracellular cGMP out of the cells with high affinity, while PDE5 inhibitors prevent high affinity cGMP efflux by inhibiting ABCC5. Increasing intracellular cGMP levels through inhibition of PDE5 and PDE5 export activity is hypothesized to promote apoptosis and growth restriction in tumor cells the tumor cells. Vardenafil is a potent inhibitor of both PDE5 and ABCC5-mediated cGMP cellular efflux (Ki=3.4μl). Nineteen novel vardenafil analogs that have been predicted as potent inhibitors by VLS were chosen for tests of their ability to inhibit ATP- dependent transport of cGMP by measuring the accumulation of cyclic GMP in inside-out vesicles. Aim: In this study, we investigated the ability of nineteen new compounds to inhibit ABCC5-mediated cGMP transport. We also determined the Ki values of the six most potent compounds. Methods: Preparation of inside out vesicles and transport assay (12-well manifolds and 96-format assembly) Results: Ki values for six of nineteen compounds that showed more than 50% inhibition of cGMP transport in the screening test were determined. Two of them were more potent than the positive control, sildenafil

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ABSTRACT During the process of drug discovery, the pharmaceutical industry is faced with numerous challenges. One challenge is the successful prediction of the major routes of human clearance of new medications. For compounds cleared by metabolism, accurate predictions help provide an early risk assessment of their potential to exhibit significant interpatient differences in pharmacokinetics via routes of metabolism catalyzed by functionally polymorphic enzymes and/or clinically significant metabolic drug-drug interactions. This review details the most recent and emerging in vitro strategies used by drug metabolism and pharmacokinetic scientists to better determine rates and routes of metabolic clearance and how to translate these parameters to estimate the amount these routes contribute to overall clearance, commonly referred to as fraction metabolized. The enzymes covered in this review include cytochrome P450s together with other enzymatic pathways whose involvement in metabolic clearance has become increasingly important as efforts to mitigate cytochrome P450 clearance are successful. Advances in the prediction of the fraction metabolized include newly developed methods to differentiate CYP3A4 from the polymorphic enzyme CYP3A5, scaling tools for UDP-glucuronosyltranferase, and estimation of fraction metabolized for substrates of aldehyde oxidase. Introduction In an era where combination drug therapy to treat several conditions simultaneously is common, drug companies emphasize the need for optimal absorption, distribution, metabolism, and excretion (ADME) properties, with the purpose of optimization of efficacy and minimization of the risk of adverse events. This includes a proper assignment and an extensive understanding of the routes of metabolism to aid in the prediction of human pharmacokinetics, and to help avoid a potential "object drug" scenario when coadministration is likely. The attributes of the coadministered drug and/or the patient has the potential to increase the probability of a drug-drug interaction (DDI), i.e., enzyme inhibitor, inducer, polymorphic genotype. Hence, the greater the percentage attributed to a single metabolic route, the greater the potential for a DDI and possible "black box warning" being issued as part of the drug package insert. Furthermore, the pharmacokinetic implications of a single polymorphic enzyme being responsible for a majority of the metabolism of a drug may have either an efficacy (extensive metabolizers) or toxicological (poor metabolizers) impact on exposure. To address these concerns, early drug discovery teams strive for balanced metabolism across multiple enzymes and clearance mechanisms (hepatic, renal, biliary). These discovery efforts center on in vitro reaction phenotyping to support enhanced chemical design. There is a general agreement among the major regulatory agencies that pharmaceutical companies should provide a characterization of the metabolic profile of a new chemical entity (NCE) and understand the enzymology of the major clearance mechanisms (reaction phenotyping) using various in vitro study tools (http://www.fda.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/ucm292362.pdf). Reaction phenotyping is the semiquantitative in vitro estimation of the relative contributions of specific drug-metabolizing enzymes to the metabolism of a test compound. The relative enzyme contributions quantified The authors have no conflicts of interest to declare and have not received compensation for the preparation of this manuscript. d