Gender-related assessment of cyclosporine/prednisolone/sirolimus interactions in three human lymphocyte proliferation assays.

BACKGROUND: Cyclosporine (CsA), prednisolone (Pred), and sirolimus (Sir) are immunosuppressive compounds inhibiting lymphocyte proliferation at the cytokine gene transcription (CsA and Pred) or signal transduction (Sir) levels. METHODS: Double- and triple-drug interactions were simultaneously studied using lectin-induced proliferation of isolated cell lymphocytes (ICLP) and whole blood lymphocytes from men and women as well as two-way mixed lymphocyte reaction assays. Drug interactions were described with isobolograms and quantitated with the universal response surface approach by estimating the interaction parameter alpha. RESULTS: All compounds inhibited more than 89% of control proliferative responses. In each assay, CsA was less potent than Pred (3- to 14-fold) and Sir (5- to 11-fold). Sir was of similar or higher potency than Pred and 1.5-fold more potent in men than women. Pred was 1.4 times more potent in women but this was found only in the ICLP assay. All combinations were synergistic (alpha>0), with greater synergism found for combinations involving Sir, especially in the ICLP (alpha>13) and two-way mixed lymphocyte reaction (alpha>40) assays. Moreover, the Sir/Pred interaction in the ICLP assay was two to five times more synergistic in women, because their mean alpha was 56 compared with 13 in men. Double-combination alpha values were able to reasonably describe CsA/Pred/Sir triple-interaction effects. CONCLUSIONS: These studies indicate that CsA, Pred, and Sir act and synergistically interact in vitro, with gender and assay as additional factors, and that whole blood lymphocyte proliferation cultures are useful in assessing the nature and intensity of drug interactions.

Pharmacokinetic and pharmacoimmunodynamic interactions between prednisolone and sirolimus in adrenalectomized rats.

Prednisolone (Pred) and sirolimus (SIR) are immunosuppressive compounds acting through different mechanisms with moderate synergism found in vitro. Both drugs are metabolized partly by CYP3A enzymes. After i.v. administration of placebo, Pred (5 mg/kg), SIR (1 mg/kg), or Pred with SIR (5 and 1 mg/kg doses) to adrenalectomized male rats, Pred plasma and SIR whole blood concentrations were followed for 48 hr along with circulating T-helper and T-cytotoxic cell counts. Ex vivo whole blood lymphocyte proliferation marked host responsiveness. An extended indirect PK/PD model was used to describe responses to these drugs, alone or combined. An interactive two-stage population analysis showed no modification in drug PK. Mean Pred plasma clearance was 0.655 L/hr (interrat++ variability: 11%) and significantly increased with weight. Mean SIR whole blood volume of distribution and clearance were 5.6 L (62%) and 0.28 L/hr (32%), and animal scaling showed weight-power proportionality. In vitro metabolism studies showed no significant inhibition of Pred or prednisone CYP3A metabolism by SIR (50 microM), but this pathway accounted for less than 5% of Pred metabolism. Pred decreased numbers of T-helper lymphocytes with a mean IC50 of 37.8 nM (21%) alone or 12.3 nM (130%) with SIR. Results for T-cytotoxic lymphocytes were similar. SIR increased lymphocyte numbers with a mean IC50 of 52.2 nM (24%) for T-helper and 28.8 nM (51%) for T-cytotoxic cells. Taking into account drug effects on lymphocyte trafficking, Pred directly inhibited ex vivo lymphocyte proliferation with a mean IC50 of 1.08 nM (38%). SIR, after a transduction step, inhibited proliferation with a mean IC50 of 1.00 nM (26%). Responses measured after drug coadministration were reasonably quantitated by addition of single drug effects. Since, at pharmacologic concentrations in rats, Pred and SIR did not interact in their PK but synergistically or additively interact in their dynamics, their joint therapeutic use is promising. The adrenalectomized rat may be a suitable animal model to characterize drug effects on lymphocyte trafficking and reactivity.

Pharmacokinetics and pharmacodynamics of prednisolone in obese rats.

The pharmacokinetics and pharmacodynamic response to prednisolone were examined in dietary-induced obese rats and matched controls. Pharmacokinetic parameters were examined in absolute and weight normalized terms. After an i.v. dose (range, 4.0-6.3 mg/kg) of prednisolone adjusted to achieve similar initial prednisolone plasma concentrations, the time course of glucocorticoid receptors in hepatic cytosol and hepatic tyrosine aminotransferase (TAT) activity were examined. Plasma prednisolone concentrations declined biexponentially with time. Mean (S.D.) for prednisolone plasma clearance normalized for total body mass (TBM) was 2.3 (0.9) liters/hr/kg in normal rats and 2.7 (0.7) liters/hr/kg in obese rats. The volume of distribution at steady-state averaged 0.82 (0.46) liters/kg of TBM in normal rats vs. 1.08 (0.40) liters/kg of TBM in obese rats. Base-line receptor levels for obese rats were 53% higher than control levels. A model to describe simultaneously kinetics and receptor-mediated dynamics was used to analyze the data and obtain estimates for the efficiency of TAT induction. This efficiency parameter in obese rats was 22% of controls, reflecting the innate degree of diminished TAT response. This decreased response in obese animals may indicate a need for joint pharmacokinetic/dynamic considerations in dosing obese individuals with corticosteroids.

Enhancement of tissue delivery and receptor occupancy of methylprednisolone in rats by a liposomal formulation.

A liposomal formulation of methylprednisolone (L-MPL) was developed to improve localization of this immunosuppressant in lymphatic tissues. Liposomes containing MPL were formulated from a mixture of phosphatydylcholine and phosphatydylglycerol (molar ratio, 9:1) and sized by extrusion through a 0.1-micron membrane. Male Sprague-Dawley rats received a bolus dose of 2 mg/kg of L-MPL or free MPL in solution (control). Samples of blood, spleen, liver, thymus, and bone marrow were collected at intervals over a 66-hr period. Concentrations of MPL in plasma and organs and free cytosolic glucocorticoid receptors (GCR) in spleen and liver were determined. The plasma MPL profiles for free and L-MPL were bi- and triexponential. Although the alpha phase kinetics of both dosage forms were similar, L-MPL showed a substantially slower elimination phase than did free drug. Incorporation of MPL into liposomes caused the following increases: terminal half-life, from 0.48 (MPL) to 30.13 hr (L-MPL); MRT, from 0.42 to 11.95 hr, Vss, from 2.10 to 21.87 L/kg; and AUC, from 339 to 1093 ng x hr/mL. Uptake of liposomes enhanced significantly the delivery of drug to lymphatic tissues and liver; AUC tissue:plasma ratios for spleen increased 77-fold; for liver, 9-fold; and for thymus, 27-fold. The duration of GCR occupancy was extended 10-fold in spleen and 13-fold in liver by the liposomal formulation. Lymphatic tissue selectivity and extended receptor binding of liposome-delivered MPL offer promise for enhanced immunosuppression.

Dose-dependence and repeated-dose studies for receptor/gene-mediated pharmacodynamics of methylprednisolone on glucocorticoid receptor down-regulation and tyrosine aminotransferase induction in rat liver.

Dose-dependent and repeated-dose effects of methylprednisolone (MPL) on down-regulation of glucocorticoid receptor messenger RNA (GR mRNA) and GR density, as well as tyrosine aminotransferase (TAT) mRNA and TAT induction by receptor/gene-mediated mechanisms in rat liver were examined. A previously developed pharmacokinetic/pharmacodynamic (PK/PD) model was used to design these studies which sought to challenge the model. Three groups of male adrenalectomized Wistar rats received MPL by i.v. injection: low-dose (10 mg/kg at Time 0), high-dose (50 mg/kg at Time 0), and dual-dose (50 mg/kg at Time 0 and 24 hr). Plasma concentrations of MPL, and hepatic content of free GR, GR mRNA, TAT mRNA, and TAT activity were determined. The P-Pharm program was applied for population analysis of MPL PK revealing low interindividual variation in CL and Vc values (3-14%). Two indirect response models were applied to test two competing hypotheses for GR mRNA dynamics. Indirect Pharmacodynamic Response Model I (Model A) where the complex in the nucleus decreases the transcription rate of GR mRNA better described GR mRNA/GR down-regulation. Levels of TAT mRNA began to increase at 1-2 hr, reached a maximum at 5-6 hr, and declined to the baseline at 12-14 hr after MPL dosing. The induction of TAT activity followed a similar pattern with a delay of about 1-2 hr. The low-dose group had 50-60% of the TAT mRNA and TAT induction compared to the high-dose group. Since the GR density returned to about 70% of the baseline level before the second 50 mg/kg dose at 24 hr, tolerance was found for TAT mRNA/TAT induction where only 50-60% of the initial responses were produced. Our fourth-generation model describes the dose dependence and tolerance effects of TAT mRNA/TAT induction by MPL involving multiple-step signal transduction controlled by the steroid regimen, free GR density, and GR occupancy. This model may provide the foundation for studying other induced proteins or enzymes mediated by the similar receptor/nuclear events.

Fifteen years of operation of a high-performance liquid chromatographic assay for prednisolone, cortisol and prednisone in plasma.

A high-performance liquid chromatographic (HPLC) assay first described in 1979 has been modified and revalidated for the simultaneous determination of prednisone, cortisol and prednisolone in human plasma using betamethasone as an internal standard. Revisions include: mobile phase composition; use of a precolumn, automated injector, integrator, and computer software; improved sensitivity and quantitation; thorough investigation of stability, variation, and specimen type; and inclusion of suggested quality control criteria. Plasma-based drug standards are extracted with methylene chloride and washed with sodium hydroxide followed by a water wash. After evaporation of solvent and reconstitution with mobile phase, the extracts are then injected onto a silica gel column (Zorbax SIL) for chromatography with UV absorbance at 254 nm. Calculated limits of quantitation are 10 ng/ml and limits of detection are less than 5 ng/ml. Intra- and inter-day coefficients of variation for quality control samples for all three corticosteroids are less than 11.2%. Recovery and stability data are also provided. Several drugs that may be coadministered do not interfere with the analysis.