Differentiation of in vitro transcriptional repression and activation profiles of selective glucocorticoid modulators.

The SAR at C-5 of the 10-methoxy-2,2,4-trimethylbenzopyrano[3,4-f]quinoline core leading to identification of (-) anti 1-methylcyclohexen-3-yl as the optimum substituent that imparts minimal GR mediated in vitro transcriptional activation while maintaining full transcriptional repression is described. The in vitro profile of these candidates in human cell assays relevant to the therapeutic window of glucocorticoid modulators is outlined.

Nonsteroidal selective glucocorticoid modulators: the effect of C-10 substitution on receptor selectivity and functional potency of 5-allyl-2,5-dihydro-2,2,4-trimethyl-1H-[1]benzopyrano[3,4-f]quinolines.

The preparation and characterization of a series of C-10 substituted 5-allyl-2,5-dihydro-2,2,4-trimethyl-1H-[1]benzopyrano[3,4-f]quinolines as a novel class of selective ligands for the glucocorticoid receptor is described. Substitution at the C-10 position of the tetracyclic core with linear, two-atom appendages (OCH(3), OCF(2)H, NHMe, SMe, CH=CH(2), Ctbd1;CH, CH(2)OH) provided molecules of high affinity (K(i) = 2-8 nM) for the human glucocorticoid receptor (hGR) with limited cross-reactivity with other steroid receptors (PR, MR, AR, ER). Optimal analogues showed slightly less potent but highly efficacious E-selectin repression with reduced levels of GRE activation efficacy in reporter gene assays relative to prednisolone. Preliminary SAR of analogues containing substitution at the C-9 and C-10 positions identified the 9-OH, 10-OMe analogue 50 and the 9-OH, 10-Cl analogue 58 as compounds that demonstrated potent, GR-mediated inhibition in a conconavalin A stimulated T-cell proliferation assay in both rodent and human whole blood monocytes. When evaluated for their in vivo effects in carrageenan-induced paw edema in rats, 50, 58, and 10-OCF(2)H analogue 35 showed dose-dependent anti-inflammatory effects (50, ED(50) = 16 mg/kg; 58, ED(50) = 15 mg/kg; 35, ED(50) = 21 mg/kg vs ED(50) = 15 mg/kg for 18 and ED(50) = 4 mg/kg for prednisolone).

A novel antiinflammatory maintains glucocorticoid efficacy with reduced side effects.

Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.

17beta-Estradiol inhibits cytokine induction of the human E-selectin promoter.

Estradiol has been shown to decrease levels of the cell adhesion molecule E-selectin in cultured cells and in women on hormone replacement therapy. We set out to determine if the mechanism of estradiol action on E-selectin is at the level of its promoter. It was found that estradiol repressed the cytokine-stimulated induction of luciferase activity driven by the human E-selectin promoter in a reporter plasmid (hE-sel-LUC) in co-transfected human hepatoma cells (Hep G2) and human umbilical cord endothelial cells (ECV-304). Repression by estradiol was dependent on the presence of transfected estrogen receptor (ER) alpha or beta expression vectors. The ER antagonist ICI-182,780 blocked the repression by estradiol, confirming the receptor-dependence of the effect. The intact DNA-binding domain of ERalpha was required for estradiol repression of the cytokine-induced stimulation of the promoter in each cell line as demonstrated by the inability of an ER construct with two point mutations in the DNA-binding domain to inhibit reporter activity. Mutation of the NFK-B site at -94 to -85 within the E-selectin promoter led to less stimulation of hE-sel-LUC by interleukin one beta (IL-1beta). Estradiol did not inhibit this IL-1beta stimulated luciferase activity, indicating that the NFK-B site is necessary for ER-mediated inhibition of this promoter. Mutation of the AP-1 site at -500 to -494 within the E-selectin promoter had no effect on the ability of IL-1beta to stimulate its transcription, and estradiol repressed this activation in an ER-dependent manner with identical efficacy and potency in comparison with the wild-type promoter. Therefore, the E-selectin promoter is down-regulated by estradiol working through either ERalpha or ERbeta and requires the NFK-B site at -94 to -85 within the promoter.