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  • Given the molecular pharmacology and in

    2020-02-11

    Given the molecular pharmacology and in vitro metabolism data, we proceeded to evaluate the in vivo pharmacokinetics of . Mice (=3) were subsequently administered a subcutaneous dose (5mg/kg) with intermittent plasma collections to measure systemic levels of (). Compound achieved a maximum plasma concentration () of 504nM (±167) 2h () following subcutaneous administration and displayed an area-under-the-curve (AUC) of 7508nMh. To evaluate as an antagonist of EP1 and EP3 in vivo, we measured blockade of mEP1 and mEP3 acute vasopressor activity in mice. Left common carotid arteries and right jugular veins of anesthetized mice were cannulated. Direct arterial pressure was measured via carotid catheter. Vasoactive substances were administered via jugular catheter. 17PTPGE was used to acutely raise mean arterial pressure (MAP) via mEP1 and sulprostone was used for mEP3 (). Agonists were administered IV through the jugular catheter 2 h after subcutaneous administration of . Pretreatment of mice with 5mg/kg administered subcutaneously significantly attenuated the pressor activity of an IV bolus of 20μg/kg 17PTPGE (ΔMAP 50.3±5.5mmHg vs 27.0±3.6mmHg). Pretreatment with also significantly suppressed pressor activity of an IV bolus of 10μg/kg sulprostone (ΔMAP 53.3±2.3mmHg vs 32.0±3.5mmHg). To ensure the observed effect was selective for EP-mediated vasoconstriction, phenylephrine (10μg/kg) was shown to be unaffected by pretreatment with (data not shown). In conclusion, we have identified a novel, dual-selectivity antagonist () of the mouse EP1 and mouse EP3 receptors possessing an acylsulfonamide bioisostere for the prototypical carboxylic GYKI 52466 dihydrochloride moiety of EP ligands. was found to have indistinguishable affinity for mEP1 as for mEP3 (mEP1 p vs mEP3 p, =0.40, Student’s two-tailed test). had improved selectively over mEP2 and mTP. was less stable in mouse hepatic microsomes than , due in part to hydrolysis of to , a problem effectively circumvented by subcutaneous administration of . Finally, we confirmed is a functional antagonist of mEP1 and mEP3 in vivo by blocking mEP1/mEP3-mediated acute vasopressor activity in anesthetized mice. While the attenuation of pressor activity appears to be incomplete, these results recapitulate experiments performed in mice with genetic disruptions of EP1. Dual specificity EP1/EP3 antagonists represent a novel class of potential ESRD therapeutics we hypothesize will be more beneficial than blocking either receptor alone. Acknowledgements The authors acknowledge Kwangho Kim of the Vanderbilt Institute of Chemical Biology Synthesis Core and Christina Bartlett for their technical expertise. This work was supported by National Institutes of Health grantsR01 DK46205 (R.M.B.), R01 DK37097 (R.M.B.), P50 GM015431 (R.M.B.) and the Integrative Training in Therapeutic Discovery programT90 DA022873 (J.D.D.). R.M.B. has a Merit Award from the Department of Veterans Affairs.
    Prostanoids are important lipid mediators involved in a broad spectrum of physiological and pathophysiological events. They exert their effects through specific interactions with prostanoid receptors, which are all members of the G-protein coupled receptor superfamily. In particular, prostaglandin E (PGE), the most abundant mammalian prostaglandin, elicits its effects primarily through interaction with four distinct receptors designated EP, EP, EP and EP. Recent studies with EP receptor knockout mice suggest that this receptor is involved in PGE-induced allodynia and in acute inflammatory pain. The involvement of the EP receptor in pain is further supported by the demonstrated efficacy of the EP selective antagonist ONO-8711 in rat models of allodynia, postoperative pain, and neuropathic pain. Another EP antagonist, ZD6416, is reported to attenuate secondary esophageal hyperalgesia in man. Further studies with knockout mice and the antagonists ONO-8711 and ONO-8713 suggest a role for the EP receptor in colon, and breast carcinogenesis, blood pressure regulation, adaptive gastric cytoprotection, and diabetic nephropathy. Previous reports from this laboratory described the SAR of various prostanoid analogs and of a family of dibenzazocinone derivatives with the human EP receptor. Previous work also permitted the identification of 2,3-diarylthiophene as a prototypical EP receptor antagonist. Compound binds with good affinity to the EP receptor (=15nM) and is shifted 15-fold to a of 220nM in the presence of 2% human serum albumin (HSA). The selectivity ratio of is at least 100-fold against the EP, EP, FP, and IP receptors. Its selectivity versus the EP, DP, and TP receptors is 67-, 11-, and 10-fold, respectively. While this antagonist presents an acceptable pharmacokinetic profile in rats (=51%; =18μM at 0.5h, 10mg/kg P.O., =3h), only a modest brain–blood ratio of 0.1 is measured in the same species.