Description: Glucocorticoids are standard of care for many inflammatory conditions, but chronic use is associated with a broad array of side effects. This has led to a search for dissociative glucocorticoids—drugs able to retain or improve efficacy associated with transrepression [nuclear factor-B (NF-B) inhibition] but with the loss of side effects associated with transactivation (receptor-mediated transcriptional activation through glucocorticoid response element gene promoter elements). We investigated a glucocorticoid derivative with a -9,11 modification as a dissociative steroid. The -9,11 analog showed potent inhibition of tumor necrosis factor-α-induced NF-B signaling in cell reporter assays, and this transrepression activity was blocked by 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU-486), showing the requirement for the glucocorticoid receptor (GR). The -9,11 analog induced the nuclear translocation of GR but showed the loss of transactivation as assayed by GR-luciferase constructs as well as mRNA profiles of treated cells. The -9,11 analog was tested for efficacy and side effects in two mouse models of muscular dystrophy: mdx (dystrophin deficiency), and SJL (dysferlin deficiency). Daily oral delivery of the -9,11 analog showed a reduction of muscle inflammation and improvements in multiple muscle function assays yet no reductions in body weight or spleen size, suggesting the loss of key side effects. Our data demonstrate that a -9,11 analog dissociates the GR-mediated transcriptional activities from anti-inflammatory activities. Accordingly, -9,11 analogs may hold promise as a source of safer therapeutic agents for chronic inflammatory disorders.

Description: The glucocorticoid-induced leucine zipper (GILZ) is an important mediator of anti-inflammatory corticosteroid action. The pharmacokinetic/pharmacodynamic/pharmacogenomic effects of acute and chronic methylprednisolone (MPL) dosing on the tissue-specific dynamics of GILZ expression were examined in rats. A mechanism-based model was developed to investigate and integrate the role of MPL and circadian rhythms on the transcriptional enhancement of GILZ in multiple tissues. Animals received a single 50-mg/kg intramuscular bolus or a 7-day 0.3-mg/kg/h subcutaneous infusion of MPL and were euthanized at several time points. An additional group of rats were euthanized at several times and served as 24-hour light/dark (circadian) controls. Plasma MPL and corticosterone concentrations were measured by high-performance liquid chromatography. The expression of GILZ and glucocorticoid receptor (GR) mRNA was quantified in tissues using quantitative real-time reverse-transcription polymerase chain reaction. The pharmacokinetics of MPL were described using a two-compartment model. Mild-to-robust circadian oscillations in GR and GILZ mRNA expression were characterized in muscle, lung, and adipose tissues and modeled using Fourier harmonic functions. Acute MPL dosing caused significant down-regulation (40%–80%) in GR mRNA and enhancement of GILZ mRNA expression (500%–1080%) in the tissues examined. While GILZ returned to its rhythmic baseline following acute dosing, a new steady-state was observed upon enhancement by chronic dosing. The model captured the complex dynamics in all tissues for both dosing regimens. The model quantitatively integrates physiologic mechanisms, such as circadian processes and GR tolerance phenomena, which control the tissue-specific regulation of GILZ by corticosteroids. These studies characterize GILZ as a pharmacodynamic marker of corticosteroid actions in several tissues.

Description: Corticosteroids (CS) regulate the expression of numerous genes at the mRNA and protein levels. The time course of CS pharmacogenomics and proteomics were examined in livers obtained from adrenalectomized rats given a 50-mg/kg bolus dose of methylprednisolone. Microarrays and mass spectrometry-based proteomics were employed to quantify hepatic transcript and protein dynamics. One-hundred, sixty-three differentially expressed mRNA and their corresponding proteins (163 genes) were clustered into two dominant groups. The temporal profiles of most proteins were delayed compared with their mRNA, attributable to synthesis delays and slower degradation kinetics. On the basis of our fifth-generation model of CS, mathematical models were developed to simultaneously describe the emergent time patterns for an array of steroid-responsive mRNA and proteins. The majority of genes showed time-dependent increases in mRNA and protein expression before returning to baseline. A model assuming direct, steroid-mediated stimulation of mRNA synthesis was applied. Some mRNAs and their proteins displayed down-regulation following CS. A model assuming receptor-mediated inhibition of mRNA synthesis was used. More complex patterns were observed for other genes (e.g., biphasic behaviors and opposite directionality in mRNA and protein). Models assuming either stimulation or inhibition of mRNA synthesis coupled with dual secondarily induced regulatory mechanisms affecting mRNA or protein turnover were derived. These findings indicate that CS-regulated gene expression manifested at the mRNA and protein levels are controlled via mechanisms affecting key turnover processes. Our quantitative models of CS pharmacogenomics were expanded from mRNA to proteins and provide extended hypotheses for understanding the direct, secondary, and downstream mechanisms of CS actions.