Third-generation model for corticosteroid pharmacodynamics: Roles of glucocorticoid receptor mRNA and tyrosine aminotransferase mRNA in rat liver

A third-generation pharmacokinetic/pharmacodynamic model was proposed for receptor/genemediated corticosteroid effects. The roles of the messenger RNA (mRNA) for the glucocorticoid receptor (GR) in hepatic GR down-regulation and the mRNA for hepatic tyrosine aminotransferase (TAT) induction by methylprednisolone (MPL) were examined. Male adrenalectomized Wistar rats received 50 mg/kg MPL iv. Blood and liver samples were collected at various time points for a period of 18 hr. Plasma concentrations of MPL, free hepatic cytosolic GR densities, GR mRNA, TAT mRNA, and TAT activities in liver were determined. Plasma MPL profile was biexponential with a terminal t_1/2 of 0.57 hr. Free hepatic GR density rapidly disappeared from cytoplasm after the MPL dose and then slowly returned to about 60% of starting level after 16 hr. Meanwhile, GR mRNA level fell to 45% of baseline within 2 hr postdosing, and remained at that level for at least 18 hr. The GR down-regulation of GR mRNA and protein turnover rate were modeled. The TAT mRNA began to increase at about 2 hr, reached a maximum at about 5 hr, and declined to baseline by 14 hr. TAT induction followed a similar pattern, except the induction was delayed about 0.5 hr. Pharmacodynamic parameters were obtained by fitting seven differential equations in a piecewise fashion. The cascade of corticosteroid steps were modeled by a series of inductions for steroid-receptor-DNA complex, two intermediate transit compartments, TAT mRNA, and TAT activity. Results indicate that GR mRNA and TAT mRNA are major controlling factors for the receptor/gene-mediated effects of corticosteroids. This work was supported by Grant GM 24211 from the National Institute of General Medical Sciences, NIH.     

Fifth-generation model for corticosteroid pharmacodynamics: application to steady-state receptor down-regulation and enzyme induction patterns during seven-day continuous infusion of methylprednisolone in rats

A fifth-generation model for receptor/gene-mediated corticosteroid effects was proposed based on results from a 50 mg/kg IV bolus dose of methylprednisolone (MPL) in male adrenalectomized rats, and confirmed using data from other acute dosage regimens. Steady-state equations for receptor down-regulation and tyrosine aminotransferase (TAT) enzyme induction patterns were derived. Five groups of male Wistar rats (n=5/group) were subcutaneously implanted with Alzet mini-pumps primed to release saline or 0.05, 0.1, 0.2, and 0.3 mg/kg/hr of MPL for 7 days. Rats were sacrificed at the end of the infusion. Plasma MPL concentrations, blood lymphocyte counts, and hepatic cytosolic free receptor density, receptor mRNA, TAT mRNA, and TAT enzyme levels were quantitated. The pronounced steroid effects were evidenced by marked losses in body weights and changes in organ weights. All four treatments caused a dose-dependent reduction in hepatic receptor levels, which correlated with the induction of TAT mRNA and TAT enzyme levels. The 7 day receptor mRNA and free receptor density correlated well with the model predicted steady-state levels. However, the extent of enzyme induction was markedly higher than that predicted by the model suggesting that the usual receptor/gene-mediated effects observed upon single/intermittent dosing of MPL may be countered by alterations in other aspects of the system. A mean IC₅₀ of 6.1 ng/mL was estimated for the immunosuppressive effects of methylprednisolone on blood lymphocytes. The extent and duration of steroid exposure play a critical role in mediating steroid effects and advanced PK/PD models provide unique insights into controlling factors.     

Modeling receptor/gene-mediated effects of corticosteroids on hepatic tyrosine aminotransferase dynamics in rats: dual regulation by endogenous and exogenous corticosteroids

Receptor/gene-mediated effects of corticosteroids on hepatic tyrosine aminotransferase (TAT) were evaluated in normal rats. A group of normal male Wistar rats were injected with 50 mg/kg methylprednisolone (MPL) intramuscularly at the nadir of their plasma corticosterone (CST) rhythm (early light cycle) and sacrificed at various time points up to 96 h post-treatment. Blood and livers were collected to measure plasma MPL, CST, hepatic glucocorticoid receptor (GR) mRNA, cytosolic GR density, TAT mRNA, and TAT activity. The pharmacokinetics of MPL showed bi-exponential disposition with two first-order absorption components from the injection site and bioavailability was 21%. Plasma CST was reduced after MPL dosing, but resumed its daily circadian pattern within 36 h. Cytosolic receptor density was significantly suppressed (90%) and returned to baseline by 72 h resuming its biphasic pattern. Hepatic GR mRNA follows a circadian pattern which was disrupted by MPL and did not return during the study. MPL caused significant down-regulation (50%) in GR mRNA which was followed by a delayed rebound phase (60-70 h). Hepatic TAT mRNA and activity showed up-regulation as a consequence of MPL, and returned to their circadian baseline within 72 and 24 h of treatment. A mechanistic receptor/gene-mediated pharmacokinetic/pharmacodynamic model was able to satisfactorily describe the complex interplay of exogenous and endogenous corticosteroid effects on hepatic GR mRNA, cytosolic free GR, TAT mRNA, and TAT activity in normal rats.     

Modeling of Corticosteroid Effects on Hepatic Low-Density Lipoprotein Receptors and Plasma Lipid Dynamics in Rats

Purpose This study examines methylprednisolone (MPL) effects on the dynamics of hepatic low-density lipoprotein receptor (LDLR) mRNA and plasma lipids associated with increased risks for atherosclerosis. Materials and methods Normal male Wistar rats were given 50 mg/kg MPL intramuscularly (IM) and sacrificed at various times. Measurements included plasma MPL and CST, hepatic glucocorticoid receptor (GR) mRNA, cytosolic GR density and hepatic LDLR mRNA, and plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), high density lipoprotein cholesterol (HDLC), and triglycerides (TG). Results MPL showed bi-exponential disposition with two first-order absorption components. Hepatic GR and LDLR mRNA exhibited circadian patterns which were disrupted by MPL. Down-regulation in GR mRNA (40–50%) was followed by a delayed rebound phase. LDLR mRNA exhibited transient down-regulation (60–70%). Cytosolic GR density was significantly suppressed but returned to baseline by 72 h. Plasma TC and LDLC showed increases (55 and 142%) at 12 h. A mechanistic receptor/gene pharmacokinetic/pharmacodynamic model was developed to describe CS effects on hepatic LDLR mRNA and plasma cholesterols. Conclusions Our PK/PD model was able to satisfactorily capture the MPL effects on hepatic LDLR, its relationship to various plasma cholesterols, and builds the foundation to explore this area in the future. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Receptor-mediated methylprednisolone pharmacodynamics in rats: steroid-induced receptor down-regulation.

Several approaches to receptor down-regulation were examined to extend previous receptor/gene-mediated pharmacokinetic/dynamic models of corticosteroids. Down-regulation of the glucocorticoid receptor was considered as an instantaneous event or as a gradual steroid-receptor-mediated process. Concentrations of plasma methylprednisolone, free hepatic cytosolic receptors, and the activity of hepatic tyrosine aminotransferase (TAT) enzyme were measured for 16 hr following administration of 0, 10, and 50 mg/kg methylprednisolone sodium succinate to 93 adrenalectomized rats. Receptor down-regulation was best described by a fractional decrement in the rate of return of free cytosolic glucocorticoid receptor. Predicted values for free receptor, bound receptor, nuclear bound receptor, and transfer compartments were in accord with the expected rank order values based on the high and low steroid doses. Model parameter estimates were independent of dose and described the rapid depletion of free cytosolic receptor, late-phase return of cytosolic receptor to a new baseline level that was 20-40% lower than control, and the TAT induction/dissipation pattern following steroid dosing. The microscopic association and dissociation constants describing the steroid-receptor interaction were 0.23 L/nmole per hr (k(on)) and 4.74 hr-1 (k(off)) for methylprednisolone compared to previously obtained values of 0.20 L/nmole per hr and 15.7 hr-1 for the related steroid prednisolone. The time course of TAT induction was similar to that observed previously for prednisolone. Efficiency of TAT induction was more closely related to steroid receptor occupancy than plasma methylprednisolone concentrations due to receptor saturability and receptor recycling.     

Receptor-mediated methylprednisolone pharmacodynamics in rats: Steroid-induced receptor down-regulation

Several approaches to receptor down-regulation were examined to extend previous receptor/ genemediated pharmacokinetic/dynamic models of corticosteroids. Down-regulation of the glucocorticoid receptor was considered as an instantaneous event or as a gradual steroid-receptor-mediated process. Concentrations of plasma methylprednisolone, free hepatic cytosolic receptors, and the activity of hepatic tyrosine aminotransferase (TAT) enzyme were measured for 16 hr following administration of 0, 10, and 50 mg/kg methylprednisolone sodium succinate to 93 adrenalectomized rats. Receptor down-regulation was best described by a fractional decrement in the rate of return of free cytosolic glucocorticoid receptor. Predicted values for free receptor, bound receptor, nuclear bound receptor, and transfer compartments were in accord with the expected rank order values based on the high and low steroid doses. Model parameter estimates were independent of dose and described the rapid depletion of free cytosolic receptor, latephase return of cytosolic receptor to a new baseline level that was 20–40% lower than control, and the TAT induction/dissipation pattern following steroid dosing. The microscopic association and dissociation constants describing the steroidreceptor interaction were 0.23 L/nmole per hr (k_on and 4.74 hr^–1 (k_off) for methylprednisolone compared to previously obtained values of 0.20 L/nmole per hr and 15.7 hr^–1 for the related steroid prednisolone. The time course of TAT induction was similar to that observed previously for prednisolone. Efficiency of TAT induction was more closely related to steroid receptor occupancy than plasma methylprednisolone concentrations due to receptor saturability and receptor recycling.Supported in part by Grant No. 24211 from the National Institutes of General Medical Sciences, NIH, and by a Fellowship from the American Foundation for Pharmaceutical Education for D.H.     

Pharmacokinetics of methylprednisolone after intravenous and intramuscular administration in rats

Methylprednisolone (MPL) pharmacokinetics was examined in adrenalectomized (ADX) and normal rats to assess the feasibility of intramuscular (i.m.) dosing for use in pharmacodynamic studies. Several study phases were pursued. Parallel group studies were performed in normal and ADX rats given 50 mg/kg MPL (i.v. or i.m.) and blood samples were collected up to 6 h. Data from studies where normal rats were dosed with 50 mg/kg MPL i.m. and killed over either 6 or 96 h were combined to determine muscle site and plasma MPL concentrations. Lastly, ADX rats were dosed with 50 mg/kg MPL i.m. and killed over 18 h to assess hepatic tyrosine aminotransferase (TAT) dynamics. MPL exhibited bi-exponential kinetics after i.v. dosing with a terminal slope of 2.1 h(-1). The i.m. drug was absorbed slowly with two first-order absorption rate constants, 1.26 and 0.219 h(-1) indicating flip-flop kinetics with overall 50% bioavailability. The kinetics of MPL at the injection site exhibited slow, dual absorption rates. Although i.m. MPL showed lower bioavailability compared with other corticosteroids in rats, TAT dynamics revealed similar i.m. and i.v. response profiles. The more convenient intramuscular dosing can replace the i.v. route without causing marked differences in pharmacodynamics.     

Integrated QSPR--pharmacodynamic model of genomic effects of several corticosteroids

The results from a quantitative structure-property relationship (QSPR) model was integrated into a fifth-generation pharmacokinetic/pharmacodynamic (PK/PD) model of corticosteroid receptor/gene-mediated effects. The proposed model was developed using previously reported tyrosine aminotransferase (TAT) activity data following a 50 mg/kg intravenous dose of methylprednisolone in male adrenalectomized (ADX) rats. Induced TAT activity is a classical measure of corticosteroid genomic effects and the typical time course shows an initial lag-time, a slow rise to peak response, and a gradual return toward baseline values. The TAT activity profiles were subsequently predicted for two additional steroids (dexamethasone and hydrocortisone), which were confirmed experimentally. Two groups of male ADX Wistar rats (n = 18 each) were given either 0.1 mg/kg dexamethasone or 50 mg/kg hydrocortisone by penile vein injections. Plasma drug concentrations and liver TAT activity were measured at various time points. Baseline TAT activity was significantly lower in this study as compared to previous reports. Model simulations well captured the pharmacodynamic data once initial conditions were corrected for observed baseline values. Additional TAT profiles reported in the literature for prednisolone were also reasonably predicted using the final model. This study serves as a demonstration of how in vitro pharmacologic data and QSPR modeling results may be incorporated into existing mechanistic PK/PD models to anticipate the effects of other chemically related compounds.     

Receptor-mediated prednisolone pharmacodynamics in rats: model verification using a dose-sparing regimen

Our receptor/gene-mediated model of corticosteroid action was tested and extended by examining the pharmacokinetics/dynamics of multiple low doses vs. a single higher dose of intravenously administered prednisolone in adrenalectomized male Wistar rats. Low-dose rats received 3 bolus doses (5 mg/kg) of prednisolone at 0, 0.5 and 1.0 hr. High-dose animals were given a single 25 mg/kg dose of prednisolone. Both regimens were expected to produce equivalent net responses based on model predictions. Control rats were not dosed. The profiles of free hepatic cytosolic glucocorticoid receptors and the hepatic tyrosine aminotransferase (TAT) enzyme were examined. Plasma prednisolone concentrations showed bi-exponential decline for both doses using pooled animal data. Clearance of total plasma prednisolone was 4.16 and 3.21 L/hr per kg in low- and high-dose groups. Volume of distribution at steady state (approximately 1.50 L/kg) and central volume (approximately 0.6 L/kg) were similar for both groups. Receptor levels from 5-16 hr stabilized at 64% of the 0-hr control value. Receptor and TAT profiles were essentially superimposable for both dosing groups. Our previous model was used to simultaneously describe prednisolone plasma concentrations, hepatic receptors, and TAT activity. The ability of total plasma prednisolone (Cp), corticosteroid binding globulin (CBG)-free plasma prednisolone (CCBG), and free plasma prednisolone (CF) to describe the kinetics/dynamics were examined. The CF values produced optimum fitting of all receptor data. The similarity of the two dosing groups supports the view that appropriately timed doses of a steroid can be used in an optimally efficacious manner by first filling all receptor sites and then replacing steroid as receptors are expected to recycle from nuclear/DNA binding sites as the steroid is eliminated.     

Regulation by chronic drug administration of neuronal and cardiac calcium channel, beta-adrenoceptor and muscarinic receptor levels

Chronic administration of atropine (40-100 mg/kg, 23 days) produced a 29-33% increase in muscarinic receptors, measured by [3H]quinuclidinyl benzilate binding, in rat brain. Diisopropyl phosphorofluoridate (0.9 mg/kg, 14 days) produced a 35% decrease in muscarinic receptors. Propranolol administration (800 micrograms/kg/hr, 10 days) increased beta-adrenoceptors, measured by [3H]dihydroalprenolol binding, by 69 and 50% in brain and heart respectively. Isoproterenol administration (800 micrograms/kg/hr, 10 days) produced a 50% reduction in cardiac beta-adrenoceptors but did not alter brain receptors. These drug treatments were without effect on binding of the Ca2+ channel ligands, [3H]nimodipine and [3H]nitrendipine, to brain or heart respectively. However, chronic administration of nifedipine for 20 days (36 and 360 micrograms/kg/hr) did produce down-regulation of both cardiac and neuronal Ca2+ channels and a similar down-regulation of beta-adrenoceptors. Co-regulation of Ca2+ channels and neurotransmitter receptors may occur but may not be an automatic consequence of either receptor or channel regulation.     

Suppression by delta 9-tetrahydrocannabinol of induction of hepatic tyrosine aminotransferase and tryptophan oxygenase.

The present study characterizes the action of Δ⁹-THC on enzyme induction by studying its effects on the induction of hepatic tyrosine aminotransferase (TAT) by steroids. In none of our studies did Δ⁹-THC inhibit TAT activity in the absence of steroid. Although treatment with hydrocortisone (HC, 150 mg/kg, 2 hr prior to sacrifice) caused a 2.1-fold induction of enzyme activity, pretreatment with Δ⁹-THC (200 mg/kg, 2 hr prior to sacrifice) decreased this induction to 1.3-fold. When mice were treated with Δ⁹-THC 1 hr prior to HC induction, TAT activity was induced only 1.1-fold over control while HC alone induced TAT activity 2.5-fold. Even when steroid treatment preceded Δ⁹-THC administration by 3 hr, there was significant inhibitory activity. Enzyme activity at 0, 3, and 6 hr after steroid was 18.7, 41.4, and 55.5 μmol of PPA/g of liver/hr, respectively. When Δ⁹-THC was administered at 3 hr after steroid and mice killed 3 hr later, enzyme activity was reduced to 36.2 μmol PPA/g liver/hr. Inhibition of steroid induction was dose-related over a range of 50–400 mg/kg of Δ⁹-THC. Δ⁹-THC had little effect on induction of TAT or tryptophan oxygenase in mouse liver by tryptophan and had no effect on tryptophan induction of tryptophan oxygenase in rat liver.     

Icariin attenuates social defeat-induced down-regulation of glucocorticoid receptor in mice

Icariin is a major constituent of flavonoids isolated from the herb Epimedium. It displays antidepressant-like activity in mice behavioral despair models and chronic mild stress models. In this study, a chronic social defeat protocol is used as a mouse model for depression, and the social avoidance effects of icariin administration are investigated. The data indicate that social defeat significantly reduces mice social interaction time and that icariin administered at 25 mg/kg and 50 mg/kg for 28 consecutive days produce remarkable increases in social interaction time. Impaired glucocorticoid receptor (GR) function is related to depression and normalization of GR function is closely associated with the recovery from depression. In this study, GR binding affinity and protein expression were evaluated by radioactive ligand and western blot, respectively. Our results demonstrate that both GR binding affinity and protein expression in the social defeat model are remarkably decreased and that icariin administration attenuates social defeat-induced GR down-regulation. In the present study, our data also show that icariin administration significantly inhibits social defeat-induced increases of corticosterone and IL-6 levels. The potential mechanisms of icariin induced GR modulation, such as effects on HPA-axis function, proinflammatory signaling pathway and membrane steroid transporters, need further study.     

Pharmacodynamics of glucose regulation by methylprednisolone. I. Adrenalectomized rats

Mechanisms related to the adverse effects of corticosteroids on glucose homeostasis were studied. Five groups of adrenalectomized (ADX) rats were given methylprednisolone (MPL) intravenously at 10 and 50 mg/kg, or a continuous 7 day infusion at rates of 0, 0.1, 0.3 mg/kg/h via subcutaneously implanted Alzet mini‐pumps. Plasma concentrations of MPL, glucose and insulin were determined at various time points up to 72 h after injection or 336 h after infusion. The pharmacokinetics of MPL was captured with a two‐compartment model. The Adapt II software was used in modeling. Injection of MPL caused a temporary glucose increase over 6 h by stimulating gluconeogenesis. The glucose changes stimulated pancreatic β‐cell secretion yielding a later insulin peak at around 10 h. In turn, insulin can stimulate glucose disposition. However, long‐term MPL treatment caused continuous hyperglycemia during and after infusion. Insulin was increased during infusion, and immediately returned to baseline after the infusion was terminated, despite the almost doubled glucose concentration. A disease progression model incorporating the reduced endogenous glucose disposition was included to capture glucose homeostasis under different treatments. The results exemplify the importance of the steroid dosing regimen in mediating pharmacological and adverse metabolic effects. This mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model quantitatively describes the induction of hyperglycemia and provides additional insights into metabolic disorders such as diabetes. Copyright © 2009 John Wiley & Sons, Ltd.     

Mu-opioid receptor down-regulation and tolerance are not equally dependent upon G-protein signaling

In the present study, the contribution of pertussis toxin (PTX)-sensitive G(i/o)-proteins to opioid tolerance and mu-opioid receptor down-regulation in the mouse were examined. Mice were injected once intracerebroventricularly and intrathecally with PTX (0.1 microg/site). Controls were treated with saline. On the 10th day following PTX treatment, continuous subcutaneous infusion of etorphine (150 or 200 microg/kg/day) or morphine (40 mg/kg/day+25 mg slow-release pellet) was begun. Control mice were implanted with inert placebo pellets. Pumps and pellets were removed 3 days later, and mice were tested for morphine analgesia or mu-opioid receptor density was determined in the whole brain, spinal cord, and midbrain. Both infusion doses of etorphine produced significant tolerance (ED50 shift=approximately 4-6-fold) and down-regulation of mu-opioid receptors (approximately 20-35%). Morphine treatment also produced significant tolerance (ED50 shift= approximately 5-8-fold), but no mu-opioid receptor down-regulation. PTX dramatically reduced the acute potency of morphine and blocked the further development of tolerance by both etorphine and morphine treatments. However, PTX had no effect on etorphine-induced mu-opioid receptor down-regulation in brain, cord, or midbrain. These results suggest that PTX-sensitive G-proteins have a minimal role in agonist-induced mu-opioid receptor density regulation in vivo, but are critical in mediating acute and chronic functional effects of opioids such as analgesia and tolerance.     

Down-regulation of phenobarbital-induced cytochrome P4502B mRNAs and proteins by endotoxin in mice: independence from nitric oxide production by inducible nitric oxide synthase

Multiple hepatic cytochrome P450 enzymes are down-regulated at the mRNA and protein levels during inflammation and infection. A body of evidence suggests that nitric oxide (NO) produced from inducible NO synthase (NOS2) is responsible for some of these effects. The current study was designed to examine the NO dependencies of the down-regulation of phenobarbital-induced CYP2B mRNAs and proteins by bacterial endotoxin (lipopolysaccharide, LPS) treatment in vivo, using an NOS2-null mouse model. Treatment of C57/BL6 mice with 0.3 mg/kg of LPS maximally suppressed phenobarbital-induced CYP2B9 and 2B10 mRNAs measured 12 hr after injection, whereas 1-10 mg/kg of LPS was required to elevate NO production. Down-regulation of CYP2B mRNAs by 1 mg/kg of LPS was equivalent in wild-type and NOS2-null mice. No effect of LPS in the dose range of 0.3 to 10 mg/kg was observed on microsomal CYP2B protein levels measured 12 hr after treatment, whereas 1 mg/kg of LPS suppressed CYP2B proteins 24 hr after treatment in both wild-type and NOS2-null mice. We conclude that the main mechanism for the down-regulation of CYP2B proteins in mouse liver following moderate- or high-dose LPS treatment is via NO-independent suppression of CYP2B9 and 2B10 mRNAs. Unlike rat hepatocytes, the contribution of a rapid, NO-dependent mechanism of CYP2B protein suppression in mouse liver appears to be minor or non-existent.