P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP, and UDP, regulating several physiologic responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. P2 receptor characterization has been hampered by the lack of selective antagonists. The aim of the current study was to investigate the mRNA and protein expression of P2X and P2Y receptors in human SMC and in endothelial cells (EC). Smooth muscle cells were obtained from human mammary artery and EC from human umbilical vein. Using real-time PCR, the authors established quantitative mRNA assays. Protein expression was studied using Western blotting with recently developed antibodies. The P2X1 receptor was highly specific for human SMC, while the P2X4 was the highest expressed receptor in EC. The P2Y2 receptor was present in both SMC and EC. UTP-mediated effects in these cells are likely to be mediated by P2Y2 and not P2Y4 receptors since the latter had considerably lower expression. The P2Y6 receptor was expressed in both SMC and EC. The P2Y1 and surprisingly the P2Y11 receptors were the most abundantly expressed P2Y receptors in the endothelium. Overall, Western blotting confirmed the mRNA findings in most aspects, and most interestingly, indicated oligomerization of the P2Y1 receptor that may be important for its function. In conclusion, P2X1, P2Y2, and P2Y6 are the most expressed P2 receptors in SMC and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1, and P2Y2 are the most expressed P2 receptors in EC, and are most likely mediating release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), and t-PA induced by extracellular nucleotides. These findings will help to direct future cardiovascular drug development against the large P2 receptor family.

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A drug discovery program in search of novel 5-lipoxygenase activating protein (FLAP) inhibitors focused on driving a reduction in lipophilicity with maintained or increased ligand lipophilic efficiency (LLE) compared to previously reported compounds led to the discovery of AZD6642 (15b). Introduction of a hydrophilic tetrahydrofuran (THF) ring at the stereogenic central carbon atom led to a significant shift in physicochemical property space. The structure-activity relationship exploration and optimization of DMPK properties leading to this compound are described in addition to pharmacokinetic analysis and an investigation of the pharmacokinetic (PK)-pharmacodynamic (PD) relationship based on ex vivo leukotriene B4 (LTB4) levels in dog. AZD6642 shows high specific potency and low lipophilicity, resulting in a selective and metabolically stable profile. On the basis of initial PK/PD relation measured, a low dose to human was predicted.

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The cytotoxic nucleoside cytarabine (Ara-C) is a cornerstone of AML induction and consolidation therapies, but drug resistance contributes to disease relapse. Among clinically relevant mechanisms of Ara-C resistance is the over-expression of cytidine deaminase (CDA). CDA degrades Ara-C through deamination into inactive metabolites and its increased expression in AML cells results in drug resistance. Therefore, nucleoside analogues that are not degraded by CDA may be new therapeutic agents for this disease. Through our efforts to develop novel nucleoside analogues that overcome mechanisms of resistance to Ara-C, we identified 5-fluorotroxacitabine (5FTRX), a chain-terminating cytidine-based L-nucleoside. First, we tested whether 5FTRX was a substrate of CDA by evaluating its cytotoxicity in HEK-293 cells over-expressing CDA. Compared to wild type cells, Ara-C was 6-fold less active in cells over-expressing CDA (IC50 wild type HEK293: 3.7µM (95% CI: 3.2-4.2uM) vs IC50 CDA over-expression: 25.6uM (95% CI 21.6-30.3uM), consistent with degradation of Ara-C by CDA. In contrast, cells over-expressing CDA were more sensitive to 5FTRX, compared to wild type cells (IC50 wild type HEK293: 5.5uM (95% CI 4.7-6.3uM) vs IC50 CDA over-expression: 0.4uM (95% CI 0.4-0.5uM), potentially due to depletion of endogenous nucleotide pools by increased CDA. Thus, 5FTRX is not a substrate of CDA. We then treated 6 AML cell lines for 72 hours with increasing concentrations of 5FTRX and then measured cell growth and viability using the MTS assay. 5FTRX reduced the growth of 5 of 6 tested AML cell lines, with mean IC50 values (n= > 3) of 92nM (TEX), 130nM (KG1a), 150nM (MV4-11), 410nM (NB4), and 250nM (OCI-AML2). In contrast, K562 cells that have mutant p53 were resistant with an IC50 >50,000nM. The K562 cells were also resistant to Ara-C. 5FTRX reduced the clonogenic growth of primary AML samples (n=2) with a >90% reduction in growth at 50nM, demonstrating that 5FTRX targets leukemia initiating cells. To test whether 5FTRX induced DNA damage in AML cells, we measured changes in phosphorylated H2AX (pH2AX) after 5FTRX treatment. In the tested cell lines (OCI-AML2, TEX, NB4 and MV4-11 cells), 5FTRX increased pH2AX at concentrations associated with loss of viability. Finally, we evaluated the efficacy and toxicity of 5FTRX in mouse models of AML. 5FTRX displayed robust and dose-dependent inhibition of MV4-11 and OCI-AML2 tumors in mouse xenograft models, with complete tumor regressions and long-lasting tumor growth delays (>20 days at 100mg/kg in MV4-11 and >30 days in OCI-AML2) after a single cycle of 5 days of once-daily drug treatment, with no changes in body weight or behavior. The efficacy of 5FTRX was superior to Ara-C dosed for daily for 10 days at its MTD, 60 mg/kg (35%TGI, no regressions). MV4-11 tumors treated with 5FTRX displayed induction of pH2AX, reduction in proliferation (BrdU incorporation) and induction of necrosis, consistent with the mode of action and dramatic tumor regressions. The anti-leukemic effects of 5FTRX were further evaluated in mice engrafted intrafemorally with primary AML cells. 5FTRX (100 mg/kg i.p, x 5 days) reduced primary AML engraftment >95% compared to controls without toxicity (Fig 1). In summary, 5FTRX was identified as a potent inhibitor of AML cell proliferation in vitro and in vivo. In contrast to Ara-C, 5FTRX was not a substrate for CDA. Further development of analogues of 5FTRX is ongoing using protide prodrugs of the 5FTRX monophosphate to further increase potency and evade additional resistance mechanisms. Taken together, our findings support the further development of 5FTRX-based therapies for the treatment of AML, including AML patients with reduced sensitivity to Ara-C through high CDA expression. #AB and TPS contributed equally to this work. #ADS and MA contributed equally to this work. Disclosures Rizoska: Medivir AB: Employment, Equity Ownership. Rydergård:Medivir AB: Employment, Equity Ownership. Kylefjord:Medivir AB: Employment, Equity Ownership. Rraklli:Medivir AB: Employment. Eneroth:Medivir AB: Employment, Equity Ownership. Pinho:Medivir AB: Employment, Equity Ownership. Norin:Medivir AB: Employment, Equity Ownership. Bylund:Medivir AB: Employment, Equity Ownership. Moses:Medivir AB: Employment, Equity Ownership. Bethell:Medivir AB: Employment, Equity Ownership. Targett-Adams:Medivir AB: Employment, Equity Ownership. Schimmer:Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy; Medivir AB: Research Funding. Albertella:Medivir AB: Employment, Equity Ownership.

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