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  • Determination of relative potency EC by dose


    Determination of relative potency (EC50) by dose response assays in vitro is subject to the same sources of variability as determination of affinity, since the EC50 and Hill slope of the dose response curve are highly dependent on the binding of ligand to a competing receptor that may be present, and on metabolism of the ligand. This is illustrated in Fig. 3A and B (compare with Fig. 1B and C). All the determinants of potency are not fully understood, since a response involves several steps downstream of progestogen binding to receptor. However, factors such as relative concentrations of cofactors [45] and SRs [4] have been shown to shift (EC50) values. For example, dose response curves for transrepression have been shown experimentally to left-shift with increasing concentrations of target receptor, with accompanying increases in Hill slope, most likely due to effects downstream of ligand binding to the receptor (Fig. 3C).Few studies have investigated relative potency and efficacy of different progestogens via particular SRs by dose response analysis in ex vivo models. For transactivation, it has been shown that Prog, MPA, dienogest, and norethisterone are all agonists for the expressed PR on a synthetic GRE promoter in COS-1 cells, but that their potencies and efficacies differ [37]. We have shown that unlike NET-A, MPA and Prog have GR agonist activity, while MPA and NET-A have AR agonist activity, and Prog has anti-AR activity via expressed receptors on synthetic GRE promoters in COS-1 muscarinic [22], [23], [39]. Using a similar approach, dose response studies with expressed receptors on another GRE promoter reported that Prog and DRSP displayed no anti-AR activity, weak MR agonist and considerable anti-MR activity in CV-1 cells [2]. Several studies have shown that transactivation efficacy for progestogens appear to be highly promoter - and cell-specific. For example, MPA was found to only minimally transactivate (partial agonist) a synthetic GRE-reporter gene via overexpressed human GR in the Jurkat T-lymphocyte cell line [46], while displaying potent transactivation activity (full agonist) on another synthetic GRE-reporter gene via endogenous mouse GR in L929sA cells [47]. For transrepression, cell line studies have shown that MPA exhibits potent GR-mediated transrepression on synthetic and endogenous AP-1 or NFκB-containing promoters [47], [48]. In contrast, NET-A is a weak partial agonist for transrepression [22]. Using expressed receptors in COS-1 cells, we have shown that Prog and NET-A, but not MPA, have partial MR agonist activity for transrepression on an AP-1 promoter-reporter, while MPA and NET-A, unlike Prog, display similar AR agonist activity for transrepression on a NFκB promoter-reporter construct [23]. Although very little work has been done comparing relative potencies and efficacies of progestogens for transactivation or transrepression on endogenous genes, we have shown that saturating concentrations of MPA, NET-A and Prog exhibit promoter- and cell-specific regulation of endogenous cytokine genes in human cervical and vaginal epithelial cell lines [49]. Other studies have reported different ‘efficacies’ of some progestogens for expression of the low density lipoprotein receptor protein in a placental cell line, using a single and possibly non-saturating concentration of progestogens [50], while others reported differential effects of progestogens on expression of thrombospondin-1 mRNA in Ishikawa cells, using three concentrations of progestogens [51]. Some of these studies illustrate the difficulties in obtaining accurate potency, efficacy and biocharacter data from cell culture studies without the use of full dose response curves. We have shown by dose response analysis that MPA, as compared to other progestogens, exhibits very different potencies, efficacies and biocharacter for expression of endogenous genes in cells where the GR is the predominantly expressed SR, as assessed by receptor knock down strategies or the use of receptor-selective antagonists (unpublished data). More of such investigations are required, including investigating tissue/cell- and promoter-specific biological activities and the involvement of particular SRs and their isoforms.