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Declining intracellular T-lymphocyte concentration of cyclosporine a precedes acute rejection in kidney transplant recipients.

Background. We investigated cyclosporine A (CsA) concentrations at the site of action, inside T-lymphocytes, to evaluate its applicability as a new supplementary therapeutic drug monitoring method after renal transplantation. Method. In this prospective single-center study, 20 kidney transplant recipients, mean age 54 (range 21–74) years, on CsA-based immunosuppression were included within 2 weeks posttransplant and followed for 3 months. Nine patients also had one full 12-hour pharmacokinetic profile performed. T-lymphocytes were isolated from 7 ml whole blood using Prepacyte and intracellular CsA concentrations were determined using a validated liquid chromatography double mass spectrometry method. Results. Seven patients (35%) experienced acute rejections (all biopsy verified) during the first three months posttransplantation. Intracellular CsA concentrations tended to decline 1 week prior to acute rejection and the decrease was significant (−27.1±14.6%, P=0.014) three days before the rejection episodes were recognized clinically. In addition, the intracellular CsA area under the curve 0–12 measured during stable phase was 182% higher in the rejection-free patients (P=0.004). There was no difference between patients experiencing rejection and the rejection-free patients with respect to CsA C2-levels, dose (mg/kg), human leukocyte antigen mismatch, donor age, recipient age, or ABCB1 genotyping. Conclusion. Intracellular CsA T-lymphocyte concentrations declined significantly 3 days prior to a rejection episode and there was a general lower intracellular exposure of CsA in recipients experiencing rejection. Intracellular measurement of CsA therefore seems to have a potential to further improve individualization of therapeutic drug monitoring. Larger studies are needed to elucidate the role for intracellular T-lymphocyte measurements in ordinary clinical care, for both CsA and other immunosuppressive drugs.

Reduced elimination of cyclosporine A in elderly (>65 years) kidney transplant recipients.

Background. Physiologic functions that may affect pharmacokinetics of drugs are altered in elderly patients. The current study was performed to elucidate the effect of age on cyclosporine A (CsA) pharmacokinetics in renal transplant recipients. Method. Twenty-five renal transplant recipients on CsA treatment were included in the study. CsA doses were adjusted by C2 monitoring. The patients were divided into two groups based on age; elderly: more than 65 years (n=11, mean 73 years) and younger: 18 to 64 years (n=14, mean 43 years). A full 12-hr pharmacokinetic profile was performed during stable phase. CsA whole blood and intracellular T-lymphocytes concentrations (first 6 hr) were measured. Genotyping of the CYP3A5*1/*3 and ABCB1 (C1236T, G2677T, C3435T) polymorphisms and quantification of whole blood mRNA ABCB1 expression were performed in all patients. Results. Elderly patients achieved target C2 levels with lower CsA doses than the younger patients (4.3±0.8 vs. 6.1±2.1 mg/day/kg, P=0.025) because of lower clearance of CsA (22.7±5.1 vs. 30.5±11.1 L/hr, P=0.031). Elderly patients also showed 44% higher intracellular-to-whole blood CsA ratio than younger patients (P=0.02). Neither the CYP3A5*1, the ABCB1 genotypes nor mRNA ABCB1 expression revealed any significant influence on CsA pharmacokinetics. Conclusion. The clearance of CsA decreased with increasing age. In addition, elderly patients had a significant larger proportion of the whole blood CsA concentration located at the site of action (within T lymphocytes). This indicates that in elderly recipients it might be safe to aim for an even lower whole blood target levels than current guidelines propose.

The concentration of cyclosporine metabolites is significantly lower in kidney transplant recipients with diabetes mellitus.

Background: Diabetes mellitus is prevalent among kidney transplant recipients. The activity of drug metabolizing enzymes or transporters may be altered by diabetes leading to changes in the concentration of parent drug or metabolites. This study was aimed to characterize the effect of diabetes on the concentration of cyclosporine (CsA) and metabolites. Methods: Concentration–time profiles of CsA and metabolites (AM1, AM9, AM4N, AM1c, AM19, and AM1c9) were characterized over a 12-hour dosing interval in 10 nondiabetic and 7 diabetic stable kidney transplant recipients. All patients were male, had nonfunctional CYP3A5*3 genotype, and were on combination therapy with ketoconazole. Results: The average daily dose (±SD) of CsA was 65 ± 21 and 68 ± 35 mg in nondiabetic and diabetic subjects, respectively (P = 0.550). Cyclosporine metabolites that involved amino acid 1 (AM1, AM19, AM1c) exhibited significantly lower dose-normalized values of area under the concentration–time curve in patients with diabetes. Moreover, during the postabsorption phase (≥3 hours after dose), metabolite–parent concentration ratios for all metabolites, except AM4N, was significantly lower in diabetic patients. The pharmacokinetic parameters of ketoconazole were similar between the 2 groups thus excluding inconsistent ketoconazole exposure as a source of altered CsA metabolism. Conclusions: This study indicates that diabetes mellitus significantly affects the concentration of CsA metabolites. Because CsA is eliminated as metabolites via the biliary route, the decrease in the blood concentration of CsA metabolites during postabsorption phase would probably reflect lower hepatic cytochrome P450 3A4 enzyme activity. However, other mechanisms including altered expression of transporters may also play a role. Results of cyclosporine therapeutic drug monitoring in diabetic patients must be interpreted with caution when nonspecific assays are used.

A Population Pharmacokinetic Model of Ciclosporin Applicable for Assisting Dose Management of Kidney Transplant Recipients

Background and ObjectiveThe pharmacokinetic disposition of ciclosporin shows great intra- and interpatient variability, and that combined with a narrow therapeutic window makes therapeutic drug monitoring of ciclosporin necessary. The nonlinear mixed-effects population pharmacokinetic program NONMEM® predicts individual pharmacokinetic parameters based not only on individual patient observations but also on population characteristics and the patient’s covariates. The aim of this model development is to potentially use it in the clinical setting to optimize ciclosporin dosing in renal transplant recipients.MethodsA population pharmacokinetic model of ciclosporin has been developed with NONMEM® using full 12-hour pharmacokinetic profiles from 29 renal transplant recipients, 3 months of daily follow-up data from an additional 11 recipients, and both 3 months of follow-up data and full 12-hour pharmacokinetic profiles from nine patients. The internal validation of the model was based on data splitting and jack-knife methods. In addition, the model was validated for its clinical applicability on standard trough and 2-hour post-dose concentration data from 12 additional patients with 3 months of follow-up.ResultsThe model that best described the ciclosporin data was a two-compartment model with first-order absorption process with lagged time. The population pharmacokinetic parameters were oral clearance (CL/F) = 26.9 L/h; central volume of distribution after oral administration (V1/F) =24.4 L; absorption rate constant (ka) = 0.544 h-1; lag time =0.460 h; peripheral volume of distribution = 1119 L and intercompartmental clearance after oral administration (Q/F) =19.6 L/h. Three covariates had significant effect on a total of six pharmacokinetic parameters. These were bodyweight on V1/F and ka, time after transplantation on ka, and age on CL/F, ka and V1/F. Cytochrome P450 3A5 genotype was also a significant covariate but was not included in the final model since such information is not available in clinical practice. The external validation showed that the model was able to predict ciclosporin concentrations in the 12 new patients with an average predictive error of 17.4 ± 14% when the standard sample concentrations from the previous week were given.ConclusionA NONMEM® pharmacokinetic model for ciclosporin in renal transplant recipients was successfully developed and validated for the first 3 months post-transplantation. The model showed good predictability in a new patient cohort. After further clinical validation, the model may be applicable as a clinical tool for optimizing ciclosporin dosing in renal transplant recipients in the early post-transplant period.

Computer-Assisted Cyclosporine Dosing Performs Better Than Traditional Dosing in Renal Transplant Recipients: Results of a Pilot Study

Cyclosporine A (CsA) is widely used after organ transplantation. Its narrow therapeutic window and large pharmacokinetic variability makes therapeutic drug monitoring (TDM) demanding and frequent dose adjustments are needed, especially early after transplantation. The aim of the present pilot study was to compare accuracy of CsA TDM by experienced clinicians against a computer-assisted dosing model. Renal transplant recipients on CsA, prednisolone, and mycophenolate were included 2 weeks after transplantation, randomized (1:1) to either computer dosing (MAP-BE) or control (CONTR) and followed for at least 8 weeks. A maximum a posteriori probability Bayesian estimation method, applying a population pharmacokinetic model and the POSTHOC option in nonlinear mixed effects modeling, was used to individualize CsA doses in the MAP-BE group. Forty patients (31 men, 27.5% living donor) between 28 and 80 years were included. A total of 798 CsA concentration measurements and adherent dosing evaluations/adjustments were performed. During the entire study, blood concentrations were on average 10% ± 5% from the predefined therapeutic target range in the MAP-BE group, as compared with 13% ± 8% in the CONTR group (P = 0.042). However, there was no significant difference between groups regarding the percentage of CsA concentrations truly within the therapeutic windows [MAP-BE: 37% ± 17%, CONTR: 33% ± 15% (P = 0.57)] or in CsA dose [MAP-BE: 3.55 ± 0.8, CONTR: 3.90 ± 0.9 mg/kg/d (P = 0.26)]. Acute rejections were present in 4 and 3 patients, respectively (P = 1.00). The computer-assisted TDM-targeted CsA blood concentrations significantly better than experienced transplant physicians. A possible favorable effect on short- and long-term outcome needs to be verified in further, properly powered, clinical trials.

Urinary proteomic shotgun approach for identification of potential acute rejection biomarkers in renal transplant recipients

BackgroundAcute rejection (AR) episodes in renal transplant recipients are suspected when plasma creatinine is elevated and other potential causes out ruled. Graft biopsies are however needed for definite diagnosis. Non-invasive AR-biomarkers is an unmet clinical need. The urinary proteome is an interesting source in the search for such a biomarker in this population.MethodsIn this proof of principle study, serial urine samples in the early post transplant phase from 6 patients with biopsy verified acute rejections and 6 age-matched controls without clinical signs of rejection were analyzed by shotgun proteomics.ResultsEleven proteins fulfilled predefined criteria for regulation in association with AR. They presented detectable regulation already several days before clinical suspicion of AR (increased plasma creatinine). The regulated proteins could be grouped by their biological function; proteins related to growth and proteins related to immune response. Growth-related proteins (IGFBP7, Vasorin, EGF and Galectin-3-binding protein) were significantly up-regulated in association with AR (P = 0.03) while proteins related to immune response (MASP2, C3, CD59, Ceruloplasmin, PiGR and CD74) tended to be up-regulated ( P = 0.13).ConclusionThe use of shotgun proteomics provides a robust and sensitive method for identification of potentially predictive urinary biomarkers of AR. Further validation of the current findings is needed to establish their potential clinical role with regards to clinical AR diagnosis.Trial registrationClinicalTrials.gov number NCT00139009

Endomyocardial, intralymphocyte, and whole blood concentrations of ciclosporin A in heart transplant recipients

BackgroundIn the early phases following heart transplantation a main challenge is to reduce the impact of acute rejections. Previous studies indicate that intracellular ciclosporin A (CsA) concentration may be a sensitive acute rejection marker in renal transplant recipients. The aims of this study were to evaluate the relationships between CsA concentrations at different target sites as potential therapeutic drug monitoring (TDM) tools in heart transplant recipients.MethodsTen heart transplant recipients (8 men, 2 women) on CsA-based immunosuppression were enrolled in this prospective single-center pilot study. Blood samples were obtained once to twice weekly up to 12 weeks post-transplant. One of the routine biopsies was allocated to this study at each sampling time. Whole blood, intralymphocyte, and endomyocardial CsA concentrations were determined with validated HPLC-MS/MS-methods. Mann–Whitney U test was used when evaluating parameters between the two groups of patients. To correlate whole blood, intralymphocyte, and endomyocardial CsA concentrations linear regression analysis was used.ResultsThree patients experienced mild rejections. In the study period, the mean (range) intralymphocyte CsA trough concentrations were 10.1 (1.5 to 39) and 8.1 (1.3 to 25) ng/106 cells in the rejection and no-rejection group, respectively (P=0.21). Corresponding whole blood CsA concentrations were 316 (153 to 564) and 301 (152 to 513) ng/mL (P=0.33). There were no correlations between whole blood, intralymphocyte, or endomyocardial concentrations of CsA (P >0.11).ConclusionsThe study did not support an association between decreasing intralymphocyte CsA concentrations and acute rejections. Further, there were no association between blood concentrations and concentrations at sites of action, potentially challenging TDM in these patients.

Individual Differences in Cyclosporine A Pharmacokinetics and Its Association with Acute Renal Function Following Heart Transplantation

Background: The secondary metabolites of cyclosporine A (CsA), AM19, AM1c and AM1c9, have been indi- cated to be nephrotoxic. The aim of the present pilot study was to investigate the relationship between acute renal failure and CsA metabolite levels, including relevant pharmacokinetic genotypes, following heart transplantation. Methods: Whole-blood samples were drawn the first posttransplant week in 22 patients (median 54 years, range from 27 to 65). Whole blood concentrations of CsA and its six main metabolites were analyzed with a validated HPLC-MS/MS method, and relevant CYP3A5 and ABCB1 genotypes determined. Renal function was monitored daily during the first posttransplant month and also at months 3, 6 and 12. Results: One patient died early posttransplant. Six patients were in need of dialysis directly after transplantation. Nine pa- tients developed sustained impaired renal function, while six had stable renal function. Sustained renal impairment tended to be associated with high levels of toxic metabolites (P=0.08). Six of the patients with possible ABCB1 TTT-haplotype developed renal impairment (P=0.12). Conclusion: The present study indicates that toxic CsA metabolites seems to be associated with development of impaired renal function and together with ABCB1 genotyping they might be promising biomarkers for optimalization of immuno- suppressive drug treatment in future studies.