Purpose: Introduction: Corticosteroids (CS) are widely used immunosuppressive agents that produce therapeutic and adverse effects through coupled genomic mechanisms (Schacke H et al., 2002). Clinical dosing of CS remains largely empirical, and preclinical knowledge on sex differences in their pharmacokinetic/pharmacodynamic (PK/PD) mechanisms is sparse. While sex-dependent PK has been reported for some drugs, mechanistic knowledge on sex differences in PD is lacking. Hormonal physiology is conserved across the rodent (4 day-estrous) and human (28 day-menstrual) cycles. In vitro evidence suggests that 17β-estradiol (E2), the major female sex hormone, antagonizes genomic CS actions (Whirledge S and Cidlowski JA, 2013). Plasma E2 is elevated during the 24-h pro-estrus period and is negligible during the 24-h estrus periods of female rats. We recently established GILZ (glucocorticoid-induced leucine zipper) as a sensitive, multi-tissue PD biomarker of CS (Ayyar VS et al., 2017). It is our hypothesis that elevated E2 production during the pro-estrous stage in females will antagonize CS-enhanced GILZ expression in tissues with estrogen receptors (ER).
Objectives: Combined experimental and modeling approaches aimed to: i) explore potential sex differences in methylprednisolone (MP) PK and PD, ii) connect the PK/PD responses of MP to estrous cycle in females, and iii) establish a model that links the multi-hormonal interactions of MP, corticosterone (the major endogenous CS), and E2 to genomic MP response (i.e. GILZ dynamics).
Methods: Experimental: Male as well as estrus (negligible E2) and pro-estrus (elevated E2) female Wistar rats were dosed IM with 50 mg/kg MP at 11 weeks of age. Estrous staging was performed by daily vaginal smears for 3 weeks prior to MP. Blood and various tissues were harvested at 12 points (15 min - 24 h) after MP (3-4 rats/point). Plasma E2 was measured via competitive ELISA. Endogenous corticosterone as well as plasma and tissue PK of MP were assayed using HPLC. MP binding in plasma and liver homogenates was analyzed by ultrafiltration. MP depletion studies were performed to estimate hepatic intrinsic clearances. GILZ and glucocorticoid receptor (GR) mRNA were quantified in liver and uterus using qRT-PCR assays.
PK/PD Modeling: The quantitative model in Figure 1 was assembled by integrating four physiological processes: i) estrous cycle regulation of E2, ii) plasma and tissue MP in both sexes, iii) suppression of endogenous CST by MP, and iv) cellular level, receptor-mediated actions of MP, and its interaction with CST and E2. Plasma and hepatic PK of MP were described using a minimal PBPK model (structure not shown) (Cao Y and Jusko WJ, 2012). Model parameter values were estimated from in vitro experiments, model fitting of in vivo data, and obtained from the literature. The model was built and implemented using ADAPT5.
Results: Plasma E2 concentrations were significantly elevated during pro-estrus compared to estrus in females, and showed an inverse relationship to baseline GILZ expression in the uterus (Fig. 2A). Sex differences in PK of MP were observed with an approximate 3-fold higher plasma and liver exposure and reduced clearance in females (Fig. 2B and 2C). Plasma CST was strongly suppressed in both sexes by 4 h after MP (Fig. 2D), although females had higher baseline concentrations. The GILZ enhancement by MP was higher by 1.7-fold in livers of estrus-staged females compared to males, attributable to higher liver drug exposure in females (not shown). Of interest, the area-under-effect curve (AUEC0→24h) of uterine GILZ expression after MP dosing in estrus females was 2-fold higher than in uterus from pro-estrous females, despite no differences in MP PK or GR mRNA, supporting our hypothesis of CS antagonism by E2 (Fig. 3). The developed model, based on current mechanistic knowledge, successfully captured MPL PK and GILZ dynamics across sex and tissue type. Model sensitivity analyses revealed ER density and occupancy as critical determinants for antagonizing MP-mediated GILZ expression in tissues.
Conclusion: Differences in PK and receptor-mediated PD of MP were identified based on sex, estrous stage, and tissue type. Antagonistic regulation of drug actions by E2 explains sex differences in CS pharmacodynamics. The developed model offers a mechanistic platform to integrate and evaluate the determinants of sex- and tissue-specificity in CS actions. This systems model may also form the basis for explaining the interactions of E2 with other drugs and xenobiotics acting via nuclear receptors.
Debra DuBois– Research Associate Professor, University at Buffalo
Richard Almon– Professor, University at Buffalo
William Jusko– SUNY Distinguished Professor, Department of Pharmaceutical Sciences, University at Buffalo, Buffalo, New York