Simultaneous simple and fast quantification of three major immunosuppressants by liquid chromatography--tandem mass-spectrometry.

OBJECTIVES: The aim of the current study was to develop a simple, fast and universal method for quantification of any combination of the three major immunosuppressants sirolimus, tacrolimus and cyclosporin in whole blood, using a LC-tandem mass spectrometer (API-2000, SCIEX, Toronto, Canada). METHODS: 250 microL whole blood was spiked with internal standard (ritonavir), and protein precipitated with 350 microL acetonitrile. The sample was centrifuged and 30 microL aliquot was injected onto the HPLC column, where it underwent an online extraction with ammonium acetate. After that the automatic switching valve was activated, changing the mobile phase to methanol and thereby eluting the analytes into the tandem mass spectrometer. The high selectivity of a tandem mass analyzer allows determination of any combination of the three drugs within a 5 min run. RESULTS: Between-day precision was between 2.4% and 9.7% for all analytes at the concentrations tested. Accuracy ranged between 98.8% and 103.2% (n = 20). The method was linear over the measuring ranges of all analytes. Within-run precision was below %CV = 6% for all analytes. Good correlation with other analytical methods was observed. CONCLUSIONS: The simplicity, universality and high throughput of the method make it suitable for application in a clinical laboratory. The method has been implemented in our laboratory for a routine use.

Effect of low dose cyclosporine and sirolimus on hepatic drug metabolism in the rat1.

BACKGROUND: We examined the effect cyclosporine (CsA) and sirolimus (SRL) alone and in combination on hepatic cytochrome P450-mediated metabolism in rats. METHODS: Rats were given 1 mg/kg of CsA or 0.4 mg/kg of SRL alone or in combination via constant intravenous infusion. Renal function was evaluated at the end of treatment. Blood samples were obtained to estimate CsA and SRL concentrations. Hepatic microsomes were prepared for immunoblotting and catalytic assays. RESULTS: CsA alone did not alter serum creatinine levels. SRL given alone or in combination with CsA produced a significant increase in urine output without changes in fluid balance. Although CsA and SRL administered alone caused damage to renal proximal tubules, the two-drug combination dramatically increased the renal structural damage. CsA alone suppressed cytochrome P450 (CYP) 3A2 protein levels by 39% (P=0.012) and catalytic activity by 30% (P=0.042). SRL alone reduced catalytic activity by 38% (P=0.012). Combination therapy reduced both CYP3A2 levels by 55% (P<0.001) and catalytic activity by 55% (P=0.001). CYP2C11 protein expression or catalytic activity were not changed in any group. CYP2A1 protein expression and catalytic activity were both significantly reduced in rats given CsA or/and SRL. Steady-state CsA levels were increased during concurrent SRL dosing, however, SRL concentrations were not changed by CsA coadministration. CONCLUSIONS: Concurrent SRL dosing increases CsA concentrations due to inhibition of hepatic CYP3A2 protein expression. Nephrotoxicity caused by combination therapy is due to CsA elevating levels of SRL or by SRL itself. Concurrent administration of CsA and SRL in transplant patients should be performed with caution.

Cytochrome P450 3A9 catalyzes the metabolism of progesterone and other steroid hormones.

The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein. The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b5. P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone (DHEA). No activity of P450 3A9 toward cortisol was detectable under our reconstitution conditions. Among all the steroid hormones examined, female-specific P450 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6beta-, 16alpha-, and 21-hydroxyprogesterone. Our data also showed that P450 3A9 can catalyze the formation of a dihydroxy product, 4-pregnen-6beta, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P450. Based on the Vmax/Km values for P450 3A9 using either 21-hydroxprogesterone or 6beta-hydroxyprogesterone as a substrate, 4-pregnen-6beta, 21-diol-3, 20-dione may be formed either by 6beta-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6beta-hydroxyprogesterone. As a major isoform of CYP3A expressed in rat brain, the activities of P450 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P450 3A9 in the metabolism of neurosteroids.

A practical guide to the analysis of sirolimus using high-performance liquid chromatography with ultraviolet detection.

BACKGROUND: Sirolimus, a new immunosuppressive agent, has recently been approved in the United States for use in combination with cyclosporine and corticosteroids in renal allograft transplantation. Therapeutic drug monitoring (TDM) of sirolimus is advocated by the drug's manufacturer in certain patient populations. Given the known pharmacokinetic interaction of sirolimus with cyclosporine and the requirement for patient compliance, physicians may wish to monitor steady-state trough levels of this agent. Several types of analytical methods have been investigated for use in TDM of sirolimus: immunoassay, high-performance liquid chromatography with ultraviolet detection (HPLC-UV), and HPLC with mass-spectrometric detection (HPLC-MS or HPLC/MS/MS). OBJECTIVE: This review identifies analytical parameters that are critical to clinical TDM of sirolimus when HPLC-UV is used. METHODS: Extraction of sirolimus from whole blood was performed using either liquid-liquid or solid phase techniques, whereas liquid chromatography was performed on reverse-phase analytical columns. The drug was detected at its UV extinction maximum. Calculated analytical parameters were evaluated according to current industry standards. RESULTS: HPLC-UV methods for the quantification of sirolimus meet or exceed industry standards of performance for clinical TDM. Comparison of the sirolimus levels in clinical samples analyzed by both HPLC-UV and HPLC/MS/MS indicated that the methods provided similar results. CONCLUSION: HPLC-UV methods, although they use a less-sophisticated mode of detection than HPLC-MS or HPLC/MS/MS methods, provide an alternative means of performing TDM without sacrificing the analytical performance required for clinical monitoring of sirolimus.

Therapeutic drug monitoring of sirolimus: correlations with efficacy and toxicity.

UNLABELLED: We sought to examine the potential benefits of therapeutic drug monitoring of sirolimus, a potent immunosuppressive agent that displays a pleiotropic array of side effects. METHODS: A high-performance liquid chromatography (LC) procedure combined with ultraviolet detection (UV) was used to measure serial concentrations of parent compound sirolimus in 150 renal transplant recipients over a period of 4 yr. Drug concentrations in whole blood at trough time, as well as within pharmacokinetic profiles, were correlated with clinical events using contingency tables, logistic regression analysis, and receiver operating characteristic (ROC) curves. RESULTS: The LC/UV method showed an excellent correlation with detection of LC-resolved components by tandem mass spectrometry, demonstrating that the LC/UV method selectively detected parent compound. Sirolimus displayed the characteristics of a critical-dose drug: Its concentration could not be predicted by a standard body or demographic measure, or by dose, and it showed high degrees of intra- and inter-individual variability. However, there was a good correlation between trough and area-under-the-curve measurements. There was a significant association between trough values expressed as either observed ( < 5 ng/mL) or dose-corrected parameter ( < 1.7 ng/mL per mg administered drug) and the occurrence and severity of acute rejection episodes - despite the low overall incidence of 23 episodes among the cohort of 150 patients. Similarly, ROC functions showed a correlation of the occurrence of hypertriglyceridemia, thrombocytopenia, and leukopenia, but not hypercholesterolemia, with trough concentrations above 15 ng/mL. CONCLUSION: Due to its behavior as a critical-dose drug, therapeutic monitoring to measure sirolimus concentrations by a LC/UV method may provide clinicians with a tool to optimize outcomes.

The FTY720 story.

The chemical 2-amino-2[2-(4-octylphenyl)ethyl]-1,3,propane diol is one of a class of small-molecule immunosuppressive agents. Better known as FTY720, this compound was chemically synthesized in an effort to minimize the toxic in vivo properties of a structurally related and highly potent immunosuppressive agent, myriocin. FTY720's mechanism of action, although not fully characterized, appears to be unique among immunosuppressants. Whereas the most well known biochemical characteristic of myriocin is its ability to inhibit serine palmitoyl transferase, the enzyme that initiates the biosynthetic pathway that leads to sphingosine, FTY720 is ineffective in this regard. In vivo, FTY720 induces a significant reduction in the number of circulating lymphocytes. It is thought to act by altering lymphocyte trafficking/homing patterns through modulation of cell surface adhesion receptors and ligands in a manner that has yet to be elucidated. Although much research has yet to be done to unravel the nature of the mechanism of action of FTY720, its efficacy has been sufficiently proven in numerous animal models, especially when administered in combination with cyclosporine. The agent is now progressing through human clinical trials, with the results of phase 1 clinical trials showing safety and tolerability in adult recipients of renal transplants. It is hoped that FTY720 will eventually prove to be an efficacious new weapon in the immunosuppressive armamentarium.

Comparison of steady-state trough sirolimus samples by HPLC and a radioreceptor assay.

OBJECTIVES: We have previously identified a minor immunophilin of 52 kDa molecular weight capable of binding tacrolimus and sirolimus. Because immunophilins are capable of binding both parent drug and metabolites and HPLC assays are typically used to assess parent drug in clinical situations, we used this immunophilin in a radioreceptor assay (RRA) to determine if any metabolites not included in the HPLC measurement would bind to the immunophilin and be associated with thrombocytopenia in patients receiving sirolimus. DESIGN AND METHODS: We tested 51 steady-state trough whole blood samples from non-thrombocytopenic patients and 51 steady-state trough samples from thrombocytopenic patients and compared them to HPLC measurements of parent drug in the same samples. We also tested whole blood samples spiked with authentic sirolimus metabolites using RRA to ascertain the effect these metabolites have on the technique. RESULTS: We found minimal cross-reactivity in this assay for sirolimus metabolites (binding ranged from <10% to 26%), and good correlation of the radioreceptor assay with HPLC (linear regression slope 0.92, y-intercept 0.79). There was no statistically significant difference between the RRA and HPLC results in two patient groups-thrombocytopenic and non-thrombocytopenic-using the paired t-test (p<0.005) and Bland-Altman analysis. CONCLUSIONS: These findings indicate that although the RRA could be substituted for HPLC in therapeutic drug monitoring, the 52 kDa immunophilin does not offer an advantage in terms of detecting metabolites associated with thrombocytopenia. However, the RRA offers the advantages of shorter turnaround time, smaller sample volume and potential for automation.

The development of sirolimus: The University of Texas-Houston experience.

The transplant team at The University of Texas-Houston has studied sirolimus from preclinical through pivotal Phase III trials to single-center Phase IV trials as we continue to refine algorithms for sirolimus therapy. The sirolimus/CsA combination produces a marked reduction in the occurrence and severity of acute allograft rejection episodes. A recently completed post hoc median effect analysis of drug blood concentrations displayed by patients in the 2 pivotal Phase III trials documented that the combination displays synergistic interactions. Patients in the sirolimus/CsA arms did not display an increased incidence of infectious or malignant complications. However, they did experience a range of nonimmune toxicities, including potentiation of putatively CsA-related adverse reactions, such as renal dysfunction and hypercholesterolemia, which appear to be mitigated by reduction or elimination of CsA. However, thrombocytopenia and to a lesser extent leukopenia and anemia appear to be sirolimus-related side effects. The occurrence and severity of these adverse reactions seem to be avoided or ameliorated in most patients by optimizing sirolimus exposure at concentrations (15 ng/mL or by dose reduction. Sirolimus thus appears to be a potent and unique agent for developing new immunosuppressive strategies in organ transplantation.

Conversion from liquid to solid rapamycin formulations in stable renal allograft transplant recipients.

Sirolimus (rapamycin, RAPA, Rapamunetrade mark) is a potent immunosuppressive agent currently being investigated for prophylaxis against acute rejection episodes in renal transplant recipients. In the present study, stable renal allograft recipients under maintenance therapy with RAPA and cyclosporine (CsA) were converted from the original oil-based liquid RAPA to a solid tablet formulation on a milligram-to-milligram basis, in order to evaluate the pharmacokinetics and safety of this new dosage form. Twelve-hour pharmacokinetic (PK) profiles of both RAPA and CsA were conducted with the final liquid RAPA dose, and at 2, 4, and 8 weeks postconversion to the solid tablet. In addition, the parameters of the PK profiles for the solid formulation were compared with those for liquid RAPA, which were performed prior to this study. Area under the concentration-time curve (AUC) values for the liquid formulation and for the solid tablet at 2, 4, and 8 weeks postconversion were 256.5, 205.8, 226.1, and 224.4 ng.h/mL, respectively (p=NS). Time to maximum RAPA concentration was longer at 4 weeks postconversion, but similar at 2 and 8 weeks. There were no differences observed between the liquid and solid tablet trough concentrations. The only significant differences observed among the PK parameters of the solid tablet versus those of the liquid formulation were the lower C(max) values of the solid, namely 25.3, 24.9, and 26.7 ng/mL versus 37.1 ng/mL (p<0.05). In addition, the dose corrected C(max) was lower in the solid tablet PK profiles compared with the prior PK profiles for the liquid (7.7 versus 10.2 ng/mL, p<0.02). Cyclosporine AUC values did not change appreciably during the study. Conversion from the liquid to the solid formulation was neither associated with episodes of acute rejection, nor changes in laboratory values, during the 8-week study. In summary, conversion from the liquid to the solid RAPA formulation resulted in similar PK profiles and appears to be both safe and well-tolerated in renal transplant recipients.

Correlation between pretransplantation test dose cyclosporine pharmacokinetic profiles and posttransplantation sirolimus blood levels in renal transplant recipients.

We sought to determine whether pretransplantation test dose pharmacokinetic measurements of cyclosporine (CsA) concentrations would forecast the posttransplantation blood concentrations of sirolimus in renal transplant patients treated de novo with CsA, sirolimus, and prednisone. All 44 renal transplant recipients enrolled in Phase I/II studies of de novo posttransplantation therapy with sirolimus, CsA, and prednisone underwent pretransplantation pharmacokinetic profiling after having received paired intravenous (i.v.) and oral test doses of CsA. After transplantation, all patients were treated with CsA on a once- or twice-daily schedule (according to a concentration-controlled regimen), with tapering doses of prednisone, and with fixed doses of sirolimus on a once-daily schedule. Patients were divided into four cohorts based on the deviation of their pretransplantation CsA clearance or bioavailability values from the mean. Patients with high pretransplantation CsA clearance rates displayed a significantly lower mean posttransplantation value of sirolimus trough concentrations than patients with low pretransplantation CsA clearance rates. In contrast, values for pretransplantation absolute oral CsA bioavailability failed to correlate with the mean posttransplantation concentration of sirolimus but did predict posttransplantation CsA bioavailability. Therefore, pretransplantation CsA clearance rate estimates may forecast posttransplantation sirolimus concentrations, possibly guiding use of sirolimus therapy to achieve an optimal ratio of concentration-dependent immunosuppressive versus toxic effects.

Metabolism of cyclosporine by cytochromes P450 3A9 and 3A4.

The ability of P450 3A9 to transform cyclosporine was studied and compared to that of human P450 3A4. Purified P450 3A4 and P450 3A9 proteins were reconstituted in a system containing potassium phosphate buffer, lipids, NADPH-P450 reductase, and glutathione with NADPH added to initiate the reaction. Cyclosporine was added alone and with or without the inhibitors, ketoconazole or troleandomycin. High performance liquid chromatography with ultraviolet (HPLC/UV) techniques were used to analyze for cyclosporine metabolites. Both P450 3A4 and P450 3A9 transformed cyclosporine to three metabolites: AM1, AM9, and AM4n. P450 3A4 predominantly formed AM1 (63% of metabolites formed) while P450 3A9 formed AM4n (59% of metabolites formed). Ketoconazole (0.5 microM) completely inhibited P450 3A9 catalyzed formation of AM1 and AM9 and reduced AM4n formation to 28% of control. AM4n, AM1, and AM9 formation catalyzed by P450 3A4 was reduced to 50%, 30%, and 10% of control, respectively, by 0.5 microM ketoconazole. Troleandomycin (> 10 microM) inhibited the formation of AM4n by P450 3A4 and P450 3A9 to 60-70% of control, while the production of AM1 by P450 3A4 was increased to 120% of control and the production of AM1 by P450 3A9 was inhibited to 50% of control. Inhibition of P450 3A4 by troleandomycin (> 10 microM) reduced the formation of AM9 to 40% of control, but only reduced P450 3A9 formation of AM9 to 80% of control. This study shows that rat P450 3A9 is capable of transforming cyclosporine to multiple metabolites similar to those generated by human P450 3A4.

Immunosuppressive effects and safety of a sirolimus/cyclosporine combination regimen for renal transplantation.

BACKGROUND: Sirolimus, a novel immunosuppressant that inhibits cytokine-driven cell proliferation and maturation, prolongs allograft survival in animal models. After a phase I trial in stable renal transplant recipients documented that cyclosporine and sirolimus have few overlapping toxicities, we conducted an open-label, single-center, phase I/II dose-escalation trial to examine the safety and efficacy of this drug combination. METHODS: Forty mismatched living-donor renal transplant recipients were sequentially assigned to receive escalating initial doses of sirolimus (0.5-7.0 mg/m2/day), in addition to courses of prednisone and a concentration-controlled regimen of cyclosporine. We conducted surveillance for drug-induced side effects among sirolimus-treated patients and compared their incidence of acute rejection episodes as well as mean laboratory values with those of a historical cohort of 65 consecutive, immediately precedent, demographically similar recipients treated with the same concentration-controlled regimen of cyclosporine and tapering doses of prednisone. RESULTS: The addition of sirolimus reduced the overall incidence of acute allograft rejection episodes to 7.5% from 32% in the immediately precedent cyclosporine/prednisone-treated patients. At 18- to 47-month follow-up periods, both treatment groups displayed similar rates of patient and graft survival, as well as morbid complications. Although sirolimus-treated patients displayed comparatively lower platelet and white blood cell counts and higher levels of serum cholesterol and triglycerides, sirolimus did not augment the nephrotoxic or hypertensive proclivities of cyclosporine. The degree of change in the laboratory values was more directly associated with whole blood trough drug concentrations than with doses of sirolimus. CONCLUSIONS: Sirolimus potentiates the immunosuppressive effects of a cyclosporine-based regimen by reducing the rate of acute rejection episodes.

Relative tissue distributions of cyclosporine and sirolimus after concomitant peroral administration to the rat: evidence for pharmacokinetic interactions.

The authors sought to determine the effect of concomitant peroral (PO) administration of cyclosporine (CsA) and sirolimus (SRL, rapamycin) on the tissue distributions of CsA and SRL in the rat. Groups of four adult male Wistar-Furth rats were treated for 14 days with 2.5, 5.0, or 10.0 mg CsA/kg x day. Other groups of four adult male Wistar-Furth rats were treated for 14 days with a 1-to-6.25 weight-to-weight ratio of SRL to CsA at SRL doses of 0.4, 0.8, or 1.6 mg/kg x day. Concentrations of CsA and SRL in homogenates of heart, intestinal, kidney, liver, lung, muscle, spleen, and testes were compared to those in whole blood (WB). There was a large, dose-dependent, distinctive distribution of CsA among rat tissues, as has previously been well documented. At a constant molar dose ratio, concomitant oral administration of SRL produced an approximately two-fold increase in the concentrations of CsA in rat tissues, although SRL did not change the CsA tissue-to-WB partition coefficients. Concomitant oral CsA administration produced dose-dependent increases in SRL tissue concentrations and decreases in the SRL tissue-to-WB partition coefficients. The increases in tissue and WB concentrations on coadministration of both agents may be explained either by an increase in absorption caused by competition between the two agents for binding sites on P-glycoprotein in the gut, a reduced rate of metabolism, or to an as yet unidentified elimination mechanism. The dose-independent and unchanged CsA tissue-to-WB partition coefficients suggest that SRL does not affect the equilibrium of CsA between the central and tissue compartments, namely the tissue uptake or intracellular binding. Altered values of the SRL tissue-to-WB partition coefficients suggest that, under the conditions studied, CsA disturbs the equilibrium of SRL between the central and tissue compartments.

The effects of relative timing of sirolimus and cyclosporine microemulsion formulation coadministration on the pharmacokinetics of each agent.

INTRODUCTION: Sirolimus and cyclosporine (INN, ciclosporin) share the same cytochrome P4503A metabolic pathways, and both drugs are substrates for p-glycoprotein countertransport. These factors most likely affect the bioavailability of both drugs. This study served to examine whether the timing of the administration of cyclosporine and sirolimus might significantly affect the relative bioavailability of one or both of these drugs. METHODS: In this randomized crossover trial, 24 stable kidney transplant recipients who were treated after transplant with a combination of sirolimus, cyclosporine, and prednisone for at least 3 months before study initiation, received individualized doses of sirolimus once daily and cyclosporine in the microemulsion formulation twice a day. A total of 20 patients completed the full crossover design. Patients were randomized to one of two dosing schedules: 10 of the patients who completed the study received the morning doses of sirolimus and cyclosporine concomitantly for the first 7 days (schedule I), and from days 8 to 14 the sirolimus dose was administered 4 hours after the morning cyclosporine dose. Ten of the patients received the sirolimus dose 4 hours after the morning cyclosporine dose for the first 7 days (schedule II), and from days 8 to 14 they received concomitant morning doses of cyclosporine and sirolimus. Pharmacokinetic profiles of sirolimus and cyclosporine performed on all patients on days 7, 14, and 15 yielded the area under the concentration-time curve (AUC), peak concentration (Cmax), time to Cmax (tmax), trough cyclosporine concentration (Co), and trough sirolimus concentration (Co) values. RESULTS: Sirolimus AUC and sirolimus trough levels were consistently and significantly higher when both cyclosporine and sirolimus were administered concomitantly than when they were administered 4 hours apart. No effect attributable to timing of drug administration could be found on cyclosporine pharmacokinetic parameters. CONCLUSION: This study shows that the relative timing of administration of cyclosporine and sirolimus significantly affects the blood concentrations of sirolimus.

Radioreceptor assay for sirolimus in patients with decreased platelet counts.

OBJECTIVE: Sirolimus (RAPA) is a new immunosuppressive drug currently in Phase III clinical trials in combination with cyclosporine A (CsA). The toxicity profiles for CsA and RAPA are only partially overlapping, with RAPA toxicity consisting primarily of hyperlipidemia and myelodepression but without the nephrotoxicity, neurotoxicity, and hepatotoxicity, which are seen with CsA. Patients in the clinical trial are being monitored using HPLC or LC/MS/MS assays; there is no immunoassay for RAPA reported to date. We have previously reported a radioreceptor assay (RRA) for RAPA, which has an excellent correlation with the HPLC assay (r = 0.997). The RRA has several advantages including excellent precision, sensitivity, rapid turnaround time, and a one-step extraction procedure. We report the evaluation of blood samples from patients who were exhibiting RAPA toxicity and comparison of the RRA results with the HPLC results. METHODS: EDTA whole blood specimens (n = 42) were obtained from six renal transplant recipients taking RAPA and CsA and exhibiting decreased platelet counts. Thirty-two samples from patients without decreased platelet counts were also received. The samples were analyzed with the RRA and the results were compared to those obtained with the HPLC assay. RESULTS: By HPLC, the results ranged from 3.2-72.6 micrograms/L RAPA with 43% of the results > or = 30 micrograms/L. With the RRA, the range was 7.7-83.0 micrograms/L RAPA equivalents, with 60% of the results > or = 30 micrograms/L. The RRA results are distinctly higher than the HPLC results all along the range. The correlation between the two assays was 0.861, with a slope of 0.966 and a Y-intercept of 11.1. CONCLUSION: Since the RRA is consistently higher than HPLC concentration in patients with decreased platelet counts, but correlates well in patients with no signs of toxicity, the RRA may be useful for monitoring patients for toxicity, by giving a better indication of increasing degree of immunosuppression than the HPLC assay.

Synergistic mechanisms by which sirolimus and cyclosporin inhibit rat heart and kidney allograft rejection.

The studies presented herein examined the mechanism(s) whereby sirolimus (SRL) and cyclosporin (CsA) act synergistically to block allograft rejection. Combination index (CI = 1 reflects additive, CI > 1 antagonistic, and CI < 1 synergistic, effects) analysis documented potent synergism between SRL and CsA to block allograft rejection. Combinations of the two drugs produced synergistic prolongation of heart (CI = 0.001-0.2) or kidney (CI = 0.03-0.5) allograft survival at SRL/CsA ratios ranging from 1:12.5 to 1:200. Pharmacokinetic analysis of the individual drugs showed that CsA does not affect the blood levels of SRL, and SRL mildly increases the levels of CsA in SRL/CsA-treated rats. Quantitative polymerase chain reaction analysis was used to document that both subtherapeutic (1.0 mg/kg) and therapeutic (2.0 or 4.0 mg/kg) CsA doses inhibited the expression of interferon-gamma (IFN-gamma) (P < 0.03) and IL-2 (P < 0.003) mRNA produced by T helper (Th) 1 cells, as well as IL-10 (P < 0.001), but not IL-4 (NS) mRNA produced by Th2 cells. Contrariwise, all tested SRL doses (0.02, 0.04 or 0.08 mg/kg) did not affect cytokine mRNA expression. However, heart allografts from rat recipients treated with synergistic SRL/CsA doses displayed reduced levels of IFN-gamma (P < 0.01), IL-2 (P < 0.001) and IL-10 (P < 0.001) mRNA. Thus, because subtherapeutic doses of CsA reduce Th1/Th2 activity, thereby facilitating the inhibition of signal transduction by low does of SRL, the two agents act synergistically to inhibit allograft rejection.