Comparative Study of the Cellular Pharmacodynamics of Calcineurin Inhibitors between Patients with Chronic Renal Failure Awaiting Renal Transplantation and Cirrhosis Patients Awaiting Liver Transplantation

Abstract

The in vitro response of peripheral blood mononuclear cells (PBMCs) to the suppressive effects of calcineurin inhibitors is known to correlate with the clinical efficacy of drugs used in renal transplantations. The present study was conducted to examine the differences of PBMC responses to calcineurin inhibitors between chronic renal failure (CRF) patients awaiting renal transplantation and cirrhosis patients awaiting liver transplantation. The study included 99 CRF patients awaiting renal transplantation and 27 cirrhosis patients awaiting liver transplantation. Twenty milliliters of venous blood was taken 1–7 days before transplantation. The in vitro drug concentrations giving 50% inhibition of PBMC blastogenesis stimulated with concanavalin A (IC₅₀s) were calculated. The suppressive effects of tacrolimus against PBMC blastogenesis were more than 10–100 times stronger than those of cyclosporine. The median IC₅₀ value for cyclosporine against the CRF PBMCs was not significantly different from the median IC₅₀ value against the cirrhosis PBMCs. In contrast, tacrolimus sensitivity in cirrhosis PBMCs is approximately seven times higher than that in CRF PBMCs. The median IC₅₀ value for tacrolimus against cirrhosis PBMCs was significantly lower and therefore the effect was stronger in comparison to the CRF PBMCs (p < 0.001). These data suggest that the PBMCs of cirrhosis patients, in comparison to those of CRF patients, are highly sensitive to the suppressive effect of tacrolimus. However, PBMC sensitivity to cyclosporine was not significantly different between the CRF and cirrhosis patients. These observations raise the possibility that treatment with tacrolimus, rather than cyclosporine, may therefore be a better choice to reduce the risks of allograft rejection in liver transplantation.

Introduction

Immunosuppressive drugs, including calcineurin inhibitors, are indispensable in the treatment of organ transplantations (2,5,19,21,25–27,31). Therefore, the clinical outcome of organ transplant recipients is affected by successful immunosuppressive drug therapy. However, variations in the clinical efficacy of immunosuppressive drugs in individual patients has been observed and patients with poor responses to their drug therapy have required long-term or relatively high doses and thus have experienced rejection episodes and/or serious side effects (4,5,17,21,25,27,31).

One of the attractive ways to predict the clinical efficacy of calcineurin inhibitors in individual patients is the cellular pharmacodynamics of drugs using the patients' peripheral blood mononuclear cells (PBMCs) (13–17). The in vitro response of PBMCs to the suppressive effects of glucocorticoids and calcineurin inhibitors is correlated with the clinical efficacy of the drugs in treating renal transplantation (6,14,15,17,30), asthma (8), minimal change nephrotic syndrome (16), psoriasis (15,17), rheumatoid arthritis (18), and ulcerative colitis (11). Calcineurin inhibitors, cyclosporine and tacrolimus, are the key drugs for successful organ transplantation; however, there is limited evidence available to justify the selection of either cyclosporine or tacrolimus in renal and liver transplantations. The alternative use of these drugs based on individual PBMC sensitivity before transplantation should thus be considered for the improvement of graft outcome in these recipients. In addition, the best calcineurin inhibitor for the treatment of renal and liver transplantations, respectively, on the basis of recipient drug sensitivity has not been considered. However, no comparative study has been carried out for PBMC sensitivities to calcineurin inhibitors between renal transplant recipients and liver transplant recipients.

The present study was thus conducted to examine the differences in the in vitro response of PBMCs to calcineurin inhibitors between CRF patients awaiting renal transplantation and cirrhosis patients awaiting liver transplantation.

Materials and Methods

Subjects

The present study was approved by the Ethical Committees of Tokyo Medical University and Tokyo University of Pharmacy and Life Sciences and informed consent was obtained from all patients. The study included 99 CRF patients awaiting kidney transplantation (51 male and 48 female; 55.2 ± 10.1 years of age) and 27 cirrhosis patients awaiting liver transplantation (11 male and 16 female; 53.1 ± 8.2 years of age). Liver or kidney transplantation was applicable to all of these patients, respectively, and blood sampling was carried out before transplantation. None of the patients included in this study had a history of taking immunosuppressive drugs. Cirrhosis patients examined in this study included 15 with hepatitis C virus (HCV) infection (11 of which had hepatocellular carcinoma), five with hepatitis B virus (HBV) infection (three of which had hepatocellular carcinoma), four with primary biliary cirrhosis, two with autoimmune hepatitis, and one unknown. The Child-Pugh classification of these patients was grade A (n = 4), grade B (n = 9), and grade C (n = 14), respectively.

Cell Culture and Evaluation of Immunosuppressant Pharmacodynamics

Twenty milliliters of venous blood was taken from patients 1–7 days before transplantation between 0930 and 1100 h in the morning and heparinized. This 20-ml sample size was the smallest possible to carry out drug sensitivity tests for two calcineurin inhibitors, but occasionally two of these agents could not be tested from each subject. The heparinized blood was loaded on 3 ml of Ficoll-Hypaque (Nakarai Co., Japan), centrifuged at 1300 × g for 20 min, and PBMCs were separated as described previously. The cells were washed and resuspended in an RPMI-1640 medium containing 10% fetal calf serum, 100,000 IU/L penicillin, and 100 mg/L streptomycin to a final density of 1 × 10⁶ cells/ml. Concanavalin A, as the mitogen, was added to each well to a final concentration of 5.0 μg/ml. Subsequently, 4 μl of an ethanol solution containing cyclosporine or tacrolimus were added to give final agent concentrations of 0.001–10,000 ng/ml. Four microliters of ethanol was added to the control wells. The plate was incubated for 96 h in 5% CO₂/air at 37°C. The cells were pulsed with 18.5 KBq/well of [³H]thymidine for the last 16 h of incubation and then collected on a glass-fiber filter paper using a multiharvester device and dried. The radioactivity retained on the filter was further processed for liquid scintillation counting. The mean of the counts for a duplicate or triplicate of each sample was determined. A PBMC stimulation index was calculated from the formula: [³H]thymidine incorporated in the presence of concanavalin A (dpm)/ [³H]thymidine incorporated in the absence of stimulant (dpm). Agent concentrations that would give 50% PBMC blastogenesis inhibition (IC₅₀) were determined from the dose-response curve.

Materials

RPMI-1640 medium and fetal calf serum were purchased from Gibco Co. (USA). Concanavalin A was obtained from Seikagaku Kogyo Co. (Japan). Cyclosporine A and tacrolimus were kindly provided by Novartis Pharma Co. (Japan) and Astellas Co. (Japan), respectively. [³H]Thymidine (5.55 × 10¹¹Bq/mmol) was purchased from New England Nuclear Corporation (USA). All other reagents were of the best available grade.

Statistics

Two-tailed unpaired t-tests and a variance analysis were used for comparisons of the mean ages between CRF patients awaiting renal transplantation and cirrhosis patients awaiting liver transplantation. Fisher's exact probability tests were used to compare the proportion of males and females in the two subject groups. IC₅₀ values for immunosuppressive drugs are reported as the mean (SD) or median (range). For the comparison of IC₅₀ values between PBMCs of CRF patients and those of cirrhosis patients, the data were first checked with a variance analysis. Subsequently, the data were analyzed with Mann-Whitney's U-test. Correlations between the IC₅₀ values of two calcineurin inhibitors were analyzed with Pearson's correlation coefficient test. These analyses were performed with Statview (10). In each case, two-sided values of p < 0.05 were considered to be significant.

Results

Typical dose-response curves of calcineurin inhibitors against mitogen-induced blastogenesis of PBMCs obtained from one CRF patient and one cirrhosis patient are presented in Figure 1. The suppressive effects of tacrolimus were more than 10–100 times stronger than those of cyclosporine. In addition, the dose-response curve of tacrolimus against the PBMCs of the cirrhosis patients was shifted to low concentration ranges, in comparison to the dose-response curve against PBMCs of the CRF patients.

Figure 1.

Dose–response curves of calcineurin inhibitors against the concanavalin A-stimulated blastogenesis of PBMCs of one CRF patient (a) and one cirrhosis patient (b).

Next, the IC₅₀ values of the PBMCs of cyclosporine were compared between the PBMCs of the 93 CRF patients and those of the 26 cirrhosis patients. The IC₅₀ values for tacrolimus were also compared between the PBMCs of 39 CRF patients and those of 19 cirrhosis patients. The IC₅₀ values for cyclosporine against the blastogenesis of PBMCs from the CRF patients showed wide individual variations and were not significantly different from those against the PBMCs of the cirrhosis patients (p = 0.771) (Fig. 2a). The tacrolimus IC₅₀ values against the blastogenesis of both the CRF patients' PBMCs and cirrhosis patients' PBMCs also showed large individual variations (Fig. 2b). However, the median IC₅₀ value for tacrolimus against the blastogenesis of cirrhosis PBMCs was significantly lower than that against the blastogenesis of the CRF PBMCs (p < 0.001) (Fig. 2b). The means (SD) and medians (range) of these calcineurin inhibitors are presented in Table 1. The difference in the median cyclosporine IC₅₀ value between the CRF PBMCs and cirrhosis PBMCs was small and not statistically significant, whereas the difference in the tacrolimus IC₅₀ values between these patient groups came to approximately seven times or more.

Figure 2.

Comparison of the IC₅₀ values of calcineurin inhibitors between the CRF patients and cirrhosis patients. The IC₅₀ values (ng/ml) of calcineurin inhibitors against concanavalin A-stimulated blastogenesis in vitro were estimated in each subject as described in Materials and Methods. (a) Cyclosporine, (b) tacrolimus.

Table 1.

Data for IC₅₀ Values (ng/ml) of Calcineurin Inhibitors in PBMCs of CRF Patients and Cirrhosis Patients

	CRF	Cirrhosis	p-Value
Cyclosporine
Mean (SD)	13.3 (32.2)	11.63 (17.12)
Median (range)	4.9 (0.03–290.0)	4.84 (0.01–75.0)	NS
Tacrolimus
Mean (SD)	0.5 (1.1)	0.09 (0.16)
Median (range)	0.2 (0.01–6.8)	0.03 (0.001–0.49)	< 0.001

In addition, the relationship of IC₅₀ values between cyclosporine and tacrolimus in both CRF patients (n = 33) and cirrhosis patients (n = 18) was examined. The IC₅₀ values for cyclosporine correlated significantly with the IC₅₀ values for tacrolimus (Fig. 3a, b). However, this correlation in the CRF PBMCs (p = 0.024) was weaker than that in the cirrhosis PBMCs (p < 0.001). In the case of CRF and cirrhosis, some of the patients (e.g., numbered patients in Fig. 3) deviated largely from each correlation curve to either the ordinate or the horizontal line.

Figure 3.

The correlation of IC₅₀ values for cyclosporine and tacrolimus in (a) the CRF patients (n = 33) and (b) the cirrhosis patients (n = 18).

Discussion

This study compared the median IC₅₀ values of cyclosporine and tacrolimus between cirrhosis PBMCs and CRF PBMCs and showed the tacrolimus sensitivity in the cirrhosis PBMCs to be approximately seven times higher than that in CRF PBMCs. However, PBMC sensitivity to cyclosporine was not significantly different between the CRF and cirrhosis patients. These findings suggest that the PBMCs of cirrhosis patients, in comparison to those of CRF patients, are vulnerable to the immunosuppressive effects of tacrolimus in vitro. Therefore, the results obtained in the PBMCs of cirrhosis patients are different from those obtained from the PBMCs of CRF patients, in regard to tacrolimus sensitivity.

In addition, the relationship between the IC₅₀s of tacrolimus and those of cyclosporine in both cirrhosis patients and CRF patients were examined. These values are likely to correlate, because the immunosuppressive mechanisms of cyclosporine and tacrolimus are both based on calcineurin inhibition and they actually did so (Figs. 3a, b). However, some of the patients obviously deviated from the correlation curves to either the ordinate or horizontal line (patients 1–5 in Fig. 3). Accordingly, patient 1 in Figure 3a and patients 4 and 5 in Figure 3b, for instance, would be recommended to be treated with tacrolimus, whereas patients 2 and 3 in Figure 3a would be recommended to be treated with cyclosporine.

The in vitro response of PBMCs to the suppressive effects of immunosuppressive drugs correlates with the clinical efficacy of the drugs used in treating renal transplantation (13–16). Although the number of cases examined was not sufficient, a previous study raised the possibility that the response of PBMCs to the effects of immunosuppressive drugs correlated with the clinical efficacy of tacrolimus in liver transplantation (22). In addition, the current study showed large individual variations in the IC₅₀ values for calcineurin inhibitors in both CRF patients and cirrhosis patients. According to these observations, the dose of calcineurin inhibitors should be changed according to the individual sensitivity of PBMCs to the drugs used in treating liver transplantation, as well as in renal transplantation.

Many reports have suggested that the immune reaction of cirrhosis patients was impaired in comparison to that of healthy subjects (1,7,24). From these data, including the present observations, when immunosuppressive drugs are used for maintenance immunosuppressive therapy in liver transplant recipients, attention must be paid to the overdosing of the drugs for the prevention of infectious diseases. Moreover, immunosuppressive drugs, including calcineurin inhibitors, are generally expensive and an individualized dose-management of these drugs based on the drug sensitivity of PBMCs is also encouraging from this point of view. No significant difference in the incidence of recurrence of hepatitis from C hepatitis virus infection has been reported between the liver transplant recipients treated with tacrolimus and those treated with cyclosporine (3,12). The preferable clinical course of liver transplant recipients treated with either cyclosporine or tacrolimus is controversial (9,20,23), but a recent report by O'Grady et al. showed significant improvement of graft survival in tacrolimus-treated recipients, rather than cyclosporine-treated recipients (23).

The molecular basis for the difference in PBMC sensitivity to tacrolimus between CRF and cirrhosis was not clarified in the present study. However, an interesting observation was that anti-FKBP12 autoantibodies detected in liver transplant recipients may modify the tacrolimus immunosuppressive efficacy, thus influencing allograft survival (28). These antibodies were detected in the sera of the recipients even before liver transplantation and correlated with the occurrence of allograft rejection after transplantation (28). The anti-FKBP12 autoantibodies have also been detected in the sera of patients with several autoimmune diseases, including: systemic lupus erythematosus, rheumatoid arthritis, and autoimmune hepatitis (29). However, no information could be obtained concerning the appearance of these kinds of autoantibodies in renal failure patients or hemodialysis patients and thus the influence of these antibodies on the molecular basis for the different PBMC sensitivity to tacrolimus in CRF and cirrhosis remains to be elucidated.

In conclusion, based on the results of this analysis, treatment with tacrolimus, rather than cyclosporine, may substantially reduce the burden of graft loss in liver transplantation. It is thus possible to reduce the doses of tacrolimus after liver transplantation in highly sensitive patients, which may decrease the risks of drug side effects, including infection. In contrast, the individual selection of calcineurin inhibitors in renal transplant recipients is recommended to be carried out based on their in vitro PBMC sensitivities to the drugs.