The present pilot study does not support the hypothesis of decreased intracellular T-lymphocyte concentration of CsA prior to rejection episodes. The main finding, however, was that there were no correlations between CsA concentrations in whole blood, T-lymphocytes, or endomyocardial tissue.
Gustafsson and colleagues are, to our knowledge, the only group who previously has measured intralymphocyte CsA concentration in heart transplant recipients . The study discovered a close association between whole blood CsA C2 concentrations and lymphocyte CsA AUC0-12 in MMF treated patients. This is contradictory to our findings where no correlation between CsA in whole blood and T-lymphocytes was found. A possible explanation to this discrepancy could be the fact that Gustafsson et al. performed measurement of whole blood CsA concentration in C2 samples and determined lymphocyte CsA AUC0-12, while in the present study CsA concentration were measured in C0 samples. C2 monitoring leads to an improvement in the clinical outcomes in heart transplant recipients [20, 21] and measuring whole blood C2 concentrations could perhaps more precisely predict the CsA concentration and, in turn AUC, within lymphocytes. Nevertheless, our results are in agreement with previous studies reporting of no correlation between CsA concentration in whole blood and lymphocytes [22, 23]. Although these studies were performed in different patient populations (renal transplant recipients and healthy volunteers), the findings demonstrate that whole blood CsA concentrations may not be a good predictor of the target site concentration of CsA. To the best of our knowledge, the present pilot study is the first to report of CsA concentration in endomyocardial tissue and to show the absence of correlation with both whole blood and intralymphocyte CsA concentrations in heart transplant recipients. In a recent study, Capron et al. evaluated the correlation of intrahepatic, peripheral mononuclear cells (PBMC) and blood concentrations of tacrolimus (Tac), another calcineurin inhibitor, in liver transplant recipients. In this study, no correlation was found between mean Tac blood concentration and PBMC or intrahepatic concentration of Tac. However, it was discovered that intrahepatic Tac concentration significantly correlated with Tac PBMC concentrations . Capron et al. have earlier showed that hepatic tissue concentrations of Tac correlated with early acute rejection after liver transplantation, this in contrast to blood concentrations . These findings also suggest that direct drug measurement at the target sites (lymphocytes and graft tissue) could be a better approach than measuring whole blood concentration to predict the efficacy of immunosuppressive drugs.
The present pilot study failed to show correlation between intracellular CsA concentration in T-lymphocytes and acute rejection episodes. Several other groups have however shown a possible correlation between low intracellular CsA concentration and rejection episodes in renal transplant recipients. A study conducted by Barbari et al. demonstrated that rejecting patients exhibited a low CsA lymphocyte content despite a higher or similar CsA blood concentration . Similarly, we have shown that renal transplant recipients experiencing a rejection episode had a lower intracellular exposure of CsA several days before clinical diagnosis of acute rejection episodes . The difference observed between renal and heart transplant recipients in this respect have no obvious explanation. However, as mentioned before C2 concentrations are known to correlate better with acute rejections compared to trough concentrations  and it was C2 concentrations that were used in our previous study . Further, it cannot be ruled out that the renal transplant recipients experiencing an acute rejection episode had a stronger immune response compared to rejecting patients in the present study.
Since CsA is both a substrate and an inhibitor of P-gp, the patients’ genotype for this efflux pump was determined as it is expressed in T-lymphocytes. The ABCB1 haplotype TTT (1236T, 2677T and 3435T) has previously been associated with impaired functional transport activity . In the present study only three patients experienced an acute rejection episode. Two of the three rejection patients were homozygote ABCB1 TTT haplotypes, but all patients included in the study were potential TTT haplotypes. This makes the interpretation of the data difficult, but if the hypothesis that acute rejection episode are associated with lower intracellular CsA concentrations should hold true, it would be expected that rejection patients have high transport activity of P-gp, contradictory to our findings [7, 27].
Renal failure is a frequent and recognized complication following heart transplantation and CsA has been implicated as a potential risk factor [28–31]. Previous studies indicate that elevated blood and urine concentrations of the secondary metabolites AM19, AM1c, and AM1c9 may be associated with renal dysfunction in CsA treated patients [31–35], and that CYP3A5 expressers have higher formation of the secondary metabolites AM19 and AM1c9 . Contrary, in renal transplant recipients on Tac-based immunosuppression, a protective role of CYP3A5 expression in the kidney has been observed . By contrast to previous findings [31–35, 38], the present study did not show any tendencies of a reduced renal function by an increased concentration of the secondary metabolites or functional CYP3A5 genotypes. This should however be carefully interpreted as the power is relatively low as outlined below.
The main limitation of this pilot study is the low sample size and only three patients experienced acute rejection episodes. This clearly limits the conclusion that could be drawn. In addition, CsA concentrations were measured in trough samples and not in C2 samples. The intralymphocyte CsA concentration displayed a high intra- and interindividual variation, and this could partly be explained by the complex isolation procedure and the low level of automatization of the T-lymphocyte isolation method.