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Recently, we established a pharmacodynamic assay to monitor immunosuppressive effectiveness of cyclosporine A (CsA) in patients on standard CsA regimen. The aim of the present study was to extend this correlation to reduced CsA regimen and to compare pharmacodynamic and kinetic parameters to allow prediction of rejections and infections. In 53 heart allograft recipients, nuclear factor of activated T cells (NFAT)-regulated gene expression was quantified at trough (C0) and 2-h post-CsA dose (C2). Gene expression at C2 was calculated relative to C0 (residual gene expression, RGE) or relative to a healthy reference group (absolute gene expression, AGE). RGE correlated with CsA C2-levels in bimodal fashion: above 575 ng/ml correlation was seen with flat regression gradient. Below 575 ng/ml, correlation was excellent with markedly steeper gradient. At C0 in the low-C2 group (<575 ng/ml), AGE remained unchanged, whereas in the high-C2 group (>575 ng/ml) AGE was markedly reduced. In both groups, AGE at C2 was strongly inhibited. In patients contracting infection during follow-up, RGE was lower than in those without infections independent of CsA levels. CsA-monitoring by quantitation of NFAT-regulated gene expression is feasible with standard and reduced CsA regimens. It correlates better with the incidence of infections than measurement of CsA concentrations and might help in avoiding over-immunosuppression.

Background: At present it is unclear which dose and consecutive blood levels of cyclosporine A (CsA) are optimal with respect to immunosuppressive efficacy and drug specific side effects at the level of individual patients. Several pharmacodynamic measures of CsA effects have been proposed, but have not become clinical routine yet. Besides the lack of practicability, the biological relevance of these assays has not been determined so far., Methods: Residual expression of nuclear factor of activated T-cells (NFAT)-regulated genes two hours after drug intake was used as molecular pharmacodynamic marker to assess CsA effects on lymphocytes and correlated with the frequency of recurrent infections and malignancies in patients with five or more years of follow-up posttransplantation., Results: Recurrent infectious complications were observed in 44% and malignancies in 20% of the 133 patients studied. Patients with a strong suppression of NFAT-regulated genes by CsA--as judged by a residual level of transcription of less than 15% after drug intake--develop more frequent infections (53% vs. 29%; P = 0.005) and malignancies (22% vs. 4%; P = 0.002). The lack of correlation between the incidence of these complications and CsA blood concentration might point to the interindividual differences in the sensitivity towards calcineurin inhibition., Conclusion: The data presented here reveal a clear relation between the frequency of infectious and malignant complications and the degree of suppression of NFAT-regulated genes by CsA in transplanted patients. Therefore, pharmacodynamic monitoring of CsA efficacy in transplanted patients might be a useful tool to adjust immunosuppressive therapy in individual patients.

Background: Mycophenolate mofetil (NIMF) pharmacokinetics vary widely, and enterohepatic recirculation of the drug and its metabolites may be altered by concurrently administered immunosuppressants, including the widely used agent cyclosporin A (CsA). A reliable method of achieving effective and well-tolerated levels of NIMF-based immunosuppression would be of eminent interest., Objective: This study compared the use of measured mycophenolic acid (MPA) trough levels (C0) and abbreviated AUC estimation by limited-sampling strategies for monitoring MPA exposure in stable heart transplant recipients (>1 year after transplantation) receiving a CsA-containing or CsA-free immunosuppressive regimen., Methods: The treatment groups were receiving chronic maintenance immunosuppressive regimens consisting of either CsA/MMF or rapamycin (RAPA)/MMF. An additional subgroup of patients was switched from the CsA-containing regimen to the RAPA-containing regimen. Fasting venous blood samples were obtained before dosing and at 40, 75, 120, and 240 minutes after administration of the morning dose of MME The validated Emit assay was used to measure MPA plasma concentrations. Dose adjustment of AUCs was performed by dividing the AUC by the morning NIMF dose in grams. Cmax after administration of the morning dose was determined from available MPA data points using curve-fitting analysis. The increase to Cmax (Cmax-C0) was calculated, and dose adjustment was performed as before. Abbreviated 12-hour MPA AUCs were estimated using a limited-sampling strategy (before dosing and 30 and 120 minutes after dosing) based on high-performance liquid chromatography data. Adverse events were monitored during routine follow-up visits., Results: The study included 47 patients receiving CsA/MMF, 15 receiving RAPA/MMF, and 9 who were switched from CsA/MMF to RAPA/MME The population included 55 men and 7 women, with a mean age of 58.94 years and a mean weight of 81.85 kg. The only significant differences in baseline clinical characteristics between groups were the mean number of years since heart transplantation (3.62 CsA/MMF vs 8.53 RAPA/MMF; P<0.01) and the proportions of patients still receiving corticosteroids (44.7% vs 13.3%, respectively; P<0.01). Reported adverse events were generally mild, including leukopenia (8.1%), diarrhea (6.5%), and abdominal pain (4.8%), and did not require drug discontinuation. In patients receiving CsA/MMF, MPA AUCs ranged from 19.67 to 81.80 mg/h.L (mean [SD], 41.92 [14.14] mg/h.L). MPA Co levels were poorly correlated with total AUC (r2=0.36). MPA Co levels of 0.5 and 1.6 mg/L were correlated with AUCs of <30 and <40 mg/h.L, respectively. In patients receiving RAPA/MMF, MPA AUCs ranged from 34.40 to 87.60 mg/h.L (mean, 51.07 [15.80] mg/h.L). The correlation between Co and total AUC was better than in the CsA/MMF group (r2=0.61). MPA C0 levels of 1.0 and 2.3 mg/L were correlated with AUCs of 30 and 40 mg/h.L, respectively. Statistically significant differences between RAPA/MMF and CsA/MMF were noted in the mean MMF dosage (1.90 [0.71] vs 2.87 [0.78] g/d, respectively; P<0.001), the mean dose-adjusted MPA AUC (60.95 [27.42] vs 31.92 [16.12] mg/h.L.g MMF; P<0.001), and mean dose-adjusted MPA C0 levels (5.10 [3.41] vs 1.41 [0.95] mg/L.g; P<0.001). The dose-adjusted increase to Cmax after morning dosing was comparable between groups, and there was no difference in the frequency distribution of Cmax. In the group switched from the CsA-containing regimen to the RAPA-containing regimen, the changes in MMF dose, dose-adjusted AUC, and MPA C0 levels were similar to those in the CsA/MMF and RAPA/MMF groups., Conclusions: In this comparison of measured MPA C0 levels and 12-hour MPA AUCs estimated by a limited-sampling strategy in stable heart transplant patients receiving chronic maintenance immunosuppressive therapy with CsA/MMF or RAPA/MMF, abbreviated AUC estimation predicted drug exposure more accurately than did measured C0 levels. Thus, MPA AUCs obtained by limited sampling may be useful in guiding clinical management and dosing. However, further study is required, including validation of these findings in clinical outcome studies.

Aims: This comparative prospective multi-centre study evaluated efficacy and safety of cyclosporine A downtitration in heart transplant recipients with chronic renal dysfunction potentially attributable to cyclosporine (n=161)., Methods: In the intervention arm (n=109, recruited from 9 centres), mycophenolate mofetil was introduced de novo or substituting azathioprine, followed by cyclosporine reduction (target trough levels 2-4 microg/ml and 50 ng/ml, respectively). In controls (n=52, recruited from 1 centre), immunosuppression remained unchanged. Renal function was recorded twelve, six, and three months before, and throughout the eight-month study period., Results: At study entry, cyclosporine trough levels and renal function parameters were comparable. At study end, mean+/-SD cyclosporine in the intervention arm was 57+/-24 vs. 116+/-36 ng/ml in controls. During the study, creatinine decreased by 23.3+/-50.7 micromol/l (P<0.0001) in the intervention arm but increased by 7.3+/-46.9 micromol/l (P=0.992) in controls (P=0.0001 for comparison between groups). A creatinine reduction of at least 20% was found in 35% of subjects of the intervention arm but only in 4% in the control arm (P<0.0001 for comparison between groups). Improvement in renal function was not weakened after adjustment for baseline characteristics in multiple regression analysis. Renal function improved in strata of creatinine entry values from 150 to 310 micromol/l, regardless of the presence of diabetes. Myocardial biopsies at target levels for cyclosporine and mycophenolate mofetil showed three reversible subclinical rejection episodes., Conclusions: Cyclosporine downtitration improved renal dysfunction in diabetic and non-diabetic heart transplant recipients across a wide range of creatinine levels. The long-term benefit of this strategy deserves further study.

Background: With the introduction of cyclosporine A (CsA), long-term allograft function has significantly improved. Problems related to limited therapeutic margins and CsA toxicity remain unsolved. Until now there have been no reliable, practical markers to measure the biologic activity of CsA in vivo., Methods: Expression of NFAT (nuclear factor of activated T cells)-regulated genes (interleukin 2, interferon-gamma, and granulocyte-macrophage colony-stimulating factor) in phorbol myristate acetate/ionomycin-stimulated peripheral blood from healthy volunteers (n=34) and from stable renal (n=25), cardiac (n=26), and liver (n=14) transplant recipients receiving CsA therapy was measured by quantitative real-time reverse transcriptase-polymerase chain reaction before and 2 hr after drug intake. Gene expression and CsA plasma levels were correlated., Results: Two hours after oral CsA ingestion, the mean suppression of induced interleukin 2, interferon-gamma, and granulocyte-macrophage colony-stimulating factor gene expression was 85%. The individual decline of NFAT-regulated gene expression and the total drug exposure at this time point were closely related. Six hours after oral CsA uptake, gene expression levels reached predose values and subsequently increased further in some patients (rebound effect)., Conclusion: Quantitative measurement of the inhibition of NFAT-regulated gene expression 2 hr after CsA intake represents a novel approach to assess the biologic effectiveness of CsA therapy and has the potential to enable individualized immunosuppressive regimens.

Background: Cytolytic induction therapy with anti-thymocyte globulin (ATG) should induce effective immunosuppression, with a low rate of rejection in the initial phase after heart transplantation. Induction therapy with ATG allows post-operative renal recovery without the negative effects of highly nephrotoxic cyclosporine levels. An increased rate of infection is a common problem, however, and has been associated with “over-immunosuppression” early after transplantation. Therefore, we investigated whether reduced T-cell-adapted ATG induction therapy could be performed without increasing the risk of graft loss by rejection and whether reductions in infection rates and costs are possible. Methods: Between March 1999 and December 2002, T-cell-adapted ATG induction therapy with ATG (Sangstat) (1.5 mg/kg) was given to 62 heart transplant recipients (study group) starting on post-operative Days 1 to 6. T-lymphocyte sub-populations were screened daily using flow cytometry. If total lymphocytes were &#60;100/μl (reference 1,300 to 2,300/μl), T-helper lymphocytes (CD4+) &#60;50/μl (reference >500/μl) and T-suppressor cells (CD8+) &#60;50/μl (reference >300/μl), then no ATG was given. Further immunosuppression was continued with triple therapy consisting of methylprednisolone, azathioprine and cyclosporine. An historic group of heart transplant recipients given a full-dose ATG regimen for 8 days served as controls. These recipients were treated with ATG (Merieux 1.5 mg/kg) until reaching monoclonal cyclosporine levels of >300 mg/dl. Additional immunosuppressive treatment did not differ. Patients in both groups received systemic antibiotics (Imipenem) peri-operatively. Results of routine endomyocardial biopsies and rates of infections were examined. Results: Study group patients were older (52 ± 10 vs 49 ± 14 years). In the study group, mean cumulative ATG dose was reduced significantly to 596 ± 220 mg (p &#60; 0.05) for 3.9 ± 1.6 days compared with 1,159 ± 376 mg for 6.9 ± 1.1 days in the control group. The rate of cytomegalovirus (CMV) seroconversion was 23% in the study group compared with 13% in the control group. Rate of deep sternal infections was lower in the study group (1.6% vs 3.2%). The mean rejection rate in the first 3 months was 0.4 ± 0.7 for the study patients (185 biopsies) vs 1.1 ± 1.7 for controls (237 biopsies). All biopsies with ISHLT Grade >2 were treated successfully with 1,000 mg of methylprednisolone intravenously for 3 days. Both groups showed a similar 1-year survival rate (study 88%, control 89%). Conclusions: T-cell-adapted ATG induction therapy can be a helpful tool for individualized immunosuppression. It is not associated with an increased rate of rejection. Lower doses of immunosuppression help to minimize the rates of infection. In addition, cytolytic induction therapy combined with reduced ATG results in significant cost reduction. [Copyright &y& Elsevier]