Impact of the variability of cyclosporin A trough levels on long-term renal allograft function

Johannes Waiser1,, Torsten Slowinski1, Andrea Brinker-Paschke2, Klemens Budde1, Matthias Schreiber2, Torsten Böhler1, Ingeborg Hauser3 and Hans-Hellmut Neumayer1

1 Department of Internal Medicine-Nephrology, University Hospital Charité, Campus Charité Mitte, Humboldt-University, Berlin, 2 4th Medical Clinic, University of Erlangen-Nürnberg, Nürnberg and 3 Medical Clinic IV, University Frankfurt/Main, Frankfurt am Main, Germany



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Among renal allograft recipients, there is a considerable variability in cyclosporin A (CsA) trough levels. Some of the CsA metabolites are pharmacologically active. The variability of polyclonal CsA trough levels may contribute to the fact that long-term renal allograft survival is still not satisfactory. In a retrospective, single-centre study, we investigated the influence of the variability of polyclonal CsA trough levels on long-term renal allograft function.

Methods. Patients (n=381) received double immunosuppression consisting of CsA and methylprednisolone (MP). For each patient the CsA coefficient of variation (CCV) and the mean CsA trough level during the observation period (5 years) were calculated. Based on receiver operating characteristic (ROC) analysis, patients were divided into two groups: group I, CCV <28.05%, n=231; group II, CCV >28.05%, n=150. Additionally, patients were divided into three groups according to their mean CsA trough level: group A, <270 ng/ml, n=50; group B, 270–370 ng/ml, n=282; group C: >370 ng/ml, n=49.

Results. Compared to group I, patients in group II experienced a higher incidence of acute rejection episodes (40.7% vs 29.4%, P=0.02), reduced 5-year graft survival (81.1% vs 93.3%, P=0.002), and higher serum creatinine levels (1.7±1.2 mg/dl vs 1.4±0.5 mg/dl, P=0.03). In patients with low mean CsA trough levels, the incidence of acute rejection episodes was elevated (group A vs B, 50.0% vs 30.9%, P=0.008) and 5-year graft survival was reduced (group A vs B, 79.8% vs 89.5%, P=0.005). Multiple logistic regression analysis confirmed that the risk of graft failure within 5 years after transplantation was markedly elevated in group II (RR: 6.2, P=0.013) and in group A (RR: 8.9, P=0.008). Whereas the effect of CCV on 5-year graft survival was still evident in patients with normal or high mean CsA trough levels (>270 ng/ml, 81.9% vs 94.8%, P=0.0005), graft survival was independent from CCV in patients with low mean CsA trough levels (<270 ng/ml, 77.0% vs 81.7%, P=NS).

Conclusions. Both, the intra-individual variability and the mean of polyclonal CsA trough levels influence long-term renal allograft survival. Targeting at sufficiently high mean CsA levels with a low intra-individual variability may help to further improve long-term renal allograft survival.

Keywords: CsA trough level; cyclosporin A; graft function; kidney transplantation; single-centre study; variability



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Besides other well-described factors, renal allograft survival is mainly dependent on the efficacy of the immunosuppressive therapy. The introduction of cyclosporin A (CsA) resulted in substantial progress in this field. For more than a decade CsA has been the mainstay of most immunosuppressive standard protocols. The effect of CsA is strictly dose dependent. Because its pharmacokinetic profile is difficult to predict and because of its nephrotoxic side-effects, CsA is not an easy drug to use. Its water insolubility results in a variable gastrointestinal absorption [1]. Functional polymorphisms of the multi-drug resistance (MDR)-1 gene [2] as well as the effects of food [3] and drugs [4] on the activity of the MDR-1 gene product P-glycoprotein (P-gp) may affect absorption and tissue concentrations of orally administered P-gp substrates like CsA. Distribution is affected by the lipoprotein concentration in plasma and by the haematocrit [5]. Inter-patient heterogeneity in gut cytochrome P-450 3A4 (CYP3A4) gene expression also explains some of the wide inter-patient variability in CsA kinetics [6]. In addition, the CYP3A4-dependent metabolism is influenced by drugs that induce or inhibit CYP3A4 [7]. Finally, demographic factors, such as age, gender, and race contribute to the variability of CsA pharmacokinetics [8].

These characteristics, combined with the dangers inherent in either over- or under-immunosuppression, result in the narrow therapeutic range that characterizes CsA as a critical-dose drug. There is consensus that trough level monitoring throughout the whole post-transplantation period is indispensable, in order to determine dosage requirements. However, despite regular trough-level monitoring and consecutive dose adjustments, a considerable intra- and inter-individual variability of CsA trough levels can be observed [9]. This variability may contribute to the fact that long-term renal allograft survival is still not satisfactory [10].

Investigating a cohort of 204 renal transplant recipients under a dual-drug immunosuppressive regimen (conventional CsA plus prednisolone) Kahan et al. [11] recently showed that a high intra-individual variability of monoclonal CsA trough concentrations (>=36%) was associated with an increased incidence of chronic rejection episodes (41% vs 23%) and elevated health-care costs. The incidence of acute rejection episodes, as well as graft survival and graft function were not evaluated. Today, more than 25 CsA metabolites are known, some of which are pharmacologically active [12,13]. The clinical relevance of these metabolites is still a controversial issue [14]. In a retrospective single-centre analysis, we addressed the question of whether the variability and the mean of polyclonal CsA trough levels influence (i) 5-year graft survival, (ii) the incidence of acute rejection episodes, and (iii) renal allograft function.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and immunosuppression
Data from 381 consecutive renal allograft recipients, transplanted between 1990 and 1995, were analysed. Only patients who received a standard dual-drug immunosuppressive regimen consisting of the olive oil-based liquid or corn oil-based capsule formulation of CsA (Sandimmun®; Novartis, Basel, Switzerland) and methylprednisolone (MP) throughout the whole observation period, were included. Paediatric recipients (<16 years) and patients with early graft failure (<1 year after transplantation) were excluded. The mean follow-up was 62.1±25.2 months. CsA was administered at concentration-controlled doses based on whole-blood trough levels, measured by polyclonal fluorescence polarization immunoassay (pFPIA) (TDx kit, Abbott Diagnostics, Abbott Park, IL, USA). The target range for polyclonal CsA trough levels throughout the whole post-transplantation period was 270–370 ng/ml. Routinely, CsA trough levels were measured daily during the first 3 weeks after transplantation, monthly during the first 6 months after transplantation, and every 3 months during the following period of time. During the observation period, we participated in the national quality assessment program for CsA measurements, conducted by the German Society for Clinical Chemistry. The coefficient of variation for the TDx pFPIA method at our centre was below 3.0%. MP was given as a bolus of 250 mg i.v. before transplantation, 100 mg on day 1, 60 mg for the next 3 days, and 40 mg within the first week. Thereafter the drug was reduced in a stepwise fashion to 4 mg/day, 6 months after transplantation.

Diagnosis and treatment of rejection
Acute allograft rejection was suspected by the presence of clinical signs, such as increased serum creatinine, decreased urine output, or graft tenderness. All rejection episodes were confirmed by core biopsy. Acute rejection episodes were primarily treated with i.v. MP boluses (250 mg/day) for 3 days. Steroid-resistant rejections were re-biopsied after 1 week and treated with antithymocyte globulin (5 mg/kg/day i.v. for 10 days) (ATG, Fresenius, Bad Homburg, Germany).

Statistical analysis
The data set included a total of 12605 CsA trough levels from 381 patients. The intra-patient CsA coefficient of variation (CCV) was calculated according to the following algorithm: standard deviation of the polyclonal CsA whole-blood trough levels measured by pFPIA, divided by the mean, converted to the percentage ((SD/mean)x100). The CCV of each patient was corrected for the number of CsA measurements by linear regression analysis. A receiver operating characteristic (ROC) curve was calculated, using CCV as predictor of graft loss within 5 years after transplantation. In addition, the mean CsA trough level of each patient during the entire observation period was assessed. Renal allograft survival was defined as the interval between transplantation and either resumption of dialysis or re-transplantation. Death with a functioning graft was considered as a censoring event. Functional graft survival was analysed according to Kaplan–Meier plots with a log-rank test. The unadjusted relationship of other patient characteristics with graft survival (donor age, recipient age, HLA-mismatches, donor serum creatinine, panel reactive antibodies, previous transplants, warm ischaemia time, cold ischaemia time, CMV status, primary graft function, and diabetes mellitus) was assessed by Chi-square test. Before testing, all continuous variables were transformed into binary endpoints as indicated in Table 3Go. All parameters that were predictive (P<0.20) for 5-year graft survival in the univariate analysis were included into subsequent multivariate analysis. Controlling for these predictive parameters, graft survival according to CCV and the mean CsA trough level was analysed by multiple logistic regression analysis. Differences between groups in continuous variables were assessed using the unpaired Student's t-test after testing for normal distribution. Differences between groups in categorical variables were assessed using the Chi-square test. All statistical analyses were performed using the Statistical Program of Social Sciences (SPSS 9.0 for Windows, SPSS Inc., Chicago, IL). Differences between groups were considered significant for P<0.05. All continuous data are expressed as means±SD.


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Table 3.  Influence of patient characteristics on 5-year graft survival: univariate analysis

 


   Results
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
Graft survival
The CCV of each patient during the observation period was calculated. Figure 1Go shows the distribution of CCV in the study population. The accuracy of CCV as a predictor of graft loss within 5 years after transplantation was assessed by ROC analysis (Figure 2Go). For a given CCV, the y-axis shows the proportion of true-positive results (representing the percentage of patients with graft loss within 5 years after transplantation), and the x-axis shows the proportion of false-positive results (representing the percentage of patients with functioning graft at 5 years after transplantation). According to the ROC curve, the optimal cut-off value to distinguish between graft loss within 5 years after transplantation and graft survival was 28.05% of CCV (area under the ROC curve, 0.667; sensitivity, 67.7%; specificity, 59.3%). Based on this result, two groups of patients were defined: group I, CCV <28.05%, n=231, and group II, CCV >28.05%, n=150. Comparison between these groups revealed that 5-year graft survival was significantly better in patients with a low CCV as compared to patients with a high CCV (group I vs group II, 93.3% vs 81.1%, P=0.002) (Figure 3Go). The incidence of death with functioning graft was about equal in both groups (group I vs group II, 3.0% vs 4.0%, P=NS). CCV, mean CsA trough level and mean CsA dose according to both patient groups are shown in Table 1Go.



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Fig. 1.  Distribution of CCV (cyclosporin A coefficient of variation) in the study population (5% intervals).

 


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Fig. 2.  Receiver operating characteristic (ROC) curve showing the CCV as predictor of graft loss within 5 years after transplantation. For a given %CCV, the ordinate values show the corresponding true-positive rate (sensitivity, representing the percentage of patients with graft loss), and the abscissa values show the corresponding false-positive rate (1-specificity, representing the percentage of patients with functioning graft). The inflection point (indicated by the dot: 28.05%) was chosen as the optimal cut-off value.

 


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Fig. 3.  Graft survival according to CCV; Kaplan–Meier plot with log-rank test. Group I (CCV <28.05%, n=231) vs group II (CCV >28.05%, n=150).

 

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Table 1.  CCV, mean CsA trough level and mean CsA dose according to patient groups

 
Additionally, patients were divided into groups according to their mean CsA trough level during the observation period. Based on our target range (270–370 ng/ml) patients were subjected to three different groups: group A, mean <270 ng/ml, n=50; group B, mean 270–370 ng/ml, n=282; and group C, mean >370 ng/ml, n=49. Five-year graft survival was significantly reduced in patients with low mean CsA trough levels (group A vs group B, 79.8 vs 89.5%, P=0.005) (Figure 4Go). In group C, 5-year graft survival was comparable to group B (88.0%, P=NS). CCV, mean CsA trough level, and mean CsA dose within the three patient groups are shown in Table 1Go.



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Fig. 4.  Graft survival according to the mean CsA trough level; Kaplan–Meier plot with log-rank test. Group A (mean <270 ng/ml, n=50) vs group B (mean 270–370 ng/ml, n=282) vs group C (mean >370 ng/ml, n=49).

 
All relevant patient characteristics are shown in Table 2Go. The unadjusted relationship of these parameters with 5-year graft survival was assessed by univariate analysis (Table 3Go). This analysis revealed that the presence of HLA-A mismatches, the presence of HLA-DR mismatches, the presence of panel reactive antibodies, warm ischaemia time (>=35 vs <35 min), donor CMV status, and primary graft function were all predictive for 5-year renal allograft survival (P<0.20). Including these parameters, we performed a multiple logistic regression analysis in which the impact of both CCV and mean CsA trough levels on 5-year graft survival was assessed (Table 4Go). Because graft survival in groups B and C was almost equal (Figure 4Go), both groups were combined in this analysis. We found that the risk of graft loss within 5 years after transplantation was 6.2-fold higher (95% CI, 1.5–25.7, P=0.013) in patients with a high CCV (group II, CCV >28.05%) than in patients with a low CCV (group I, CCV <28.05%). Concerning the impact of the mean CsA trough level on renal allograft survival, it transpired that patients with low mean CsA trough levels (<270 ng/ml) had an 8.9-fold increased risk of graft loss (95% CI, 1.8–45.2, P=0.008) within 5 years after transplantation as compared to patients with normal or high mean CsA trough levels (>270 ng/ml).


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Table 2.  Relevant patient characteristics

 

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Table 4.  Influence of patient characteristics, CCV, and mean CsA trough level on 5-year graft survival: multivariate analysis

 
In order to further evaluate whether the effect of CCV on renal allograft survival was influenced by the mean CsA trough level, we separately investigated the effect of CCV on graft survival in patients with low mean polyclonal CsA trough levels (group A, mean <270 ng/ml) and in patients with normal or high mean CsA trough levels (groups B and C, mean >270 ng/ml). This subgroup analysis showed that graft survival was equally low in patients with low mean CsA trough levels (group A), no matter whether they had a low CCV (<28.05%) or a high CCV (>28.05%) (5-year graft survival, 81.7 vs 77.0%, P=NS) (Figure 5AGo). In contrast, 5-year graft survival was markedly influenced by CCV (CCV <28.05 vs >28.05%, 94.8 vs 81.9%, P=0.0005) in patients with normal or high mean CsA trough levels (groups B and C) (Figure 5BGo).



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Fig. 5.  Graft survival according to CCV and the mean CsA trough level; Kaplan–Meier plot with log-rank test. Panel A (mean <270 ng/ml), CCV <28.05% (n=29) vs CCV >28.05% (n=21). Panel B (mean >270 ng/ml), CCV <28.05% (n=202) vs CCV >28.05% (n=129).

 

Acute rejection episodes
Because the detrimental effect of high CCV levels and low mean CsA trough levels on renal allograft survival may have been mediated by an increased incidence of acute rejection episodes, we assessed the incidence of acute rejection episodes in each of the described groups. We found that the prevalence of acute rejection episodes was lower in patients with a low CCV (group I) as compared to patients with a high CCV (group II) (Figure 6Go). Concerning the influence of the mean CsA trough level on the occurrence of acute rejection episodes, we found that the prevalence of acute rejection episodes was higher in patients with low mean CsA trough levels (group A) as compared to patients with normal mean CsA trough levels (group B). No difference was found between patients with normal mean CsA trough levels (group B) and patients with high mean CsA trough levels (group C) (Figure 6Go).



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Fig. 6.  Incidence of acute rejection episodes according to patient group. Group I (CCV <28.05%, n=231) vs group II (CCV >28.05%, n=150). Group A (mean <270 ng/ml, n=50) vs group B (mean 270–370 ng/ml, n=282) vs group C (mean >370 ng/ml, n=49). Statistics: Chi-square test.

 

Graft function
The mean serum creatinine concentrations of each patient group are shown in Table 5Go. During the first year after transplantation, the mean serum creatinine concentration was not significantly different between patients with a high CCV (group I) as compared to patients with a low CCV (group II). However, during the following years serum creatinine was significantly elevated in patients with a high CCV. No differences were found between groups with different mean polyclonal CsA trough levels.


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Table 5.  Serum creatinine concentration (mg/dl) according to patient groups

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Polyclonal CsA trough levels were analysed using the TDx pFPIA method, which detects the parent molecule plus several metabolites [15] with a high intra- and inter-assay reproducibility [16]. Therefore the variability of polyclonal CsA trough levels may have been be influenced by varying formation of metabolites, all measured by the applied assay. On average, the results obtained by the TDx pFPIA method in whole-blood samples are 3–4 times higher than those obtained by high-performance liquid chromatography (HPLC) methods, which measure the parent drug specifically and are used as reference procedures [17].

In order to assess the influence of the intra-individual variability of polyclonal CsA trough levels on long-term renal allograft function, the CCV of each patient was calculated. Using ROC analysis, the optimal cut-off value to distinguish between graft loss within 5 years after transplantation and graft survival was defined (CCV, 28.05%). Comparison of the two resulting groups showed that a high CCV was associated with a significant reduction in 5-year graft survival. In addition, we found that the incidence of acute rejection episodes as well as serum creatinine levels was significantly elevated in this group. Multiple logistic regression analysis confirmed that the effect of CCV on graft survival was independent from other relevant patient characteristics. Subdivision of patients according to their mean polyclonal CsA trough levels revealed that the influence of CCV on renal allograft survival was reproducible in patients with normal (270–370 ng/ml) or high (>370 ng/ml) mean polyclonal CsA trough levels. In contrast, graft survival was equally low in patients with low mean polyclonal CsA trough levels (<270 ng/ml), no matter whether they had a high or a low CCV.

Acute rejection is the most important risk factor for chronic rejection [18], and chronic rejection is one of the main reasons for renal allograft loss [19]. Thus, the increased incidence of acute rejection episodes in patients with a high CCV seems to be a possible explanation for the deleterious effect of a high CCV on renal allograft survival. A recent study by Kahan et al. [11] supports this hypothesis. In patients receiving a similar double-immunosuppressive regimen (conventional CsA plus prednisolone) as the population described here within a comparable period of time (1988–1994), they observed that a high intra-individual variability of monoclonal CsA trough concentrations (>=36%) was associated with an increased incidence of chronic rejection episodes. Both studies together indicate that long-term graft survival in patients with a high CCV is reduced by an increased incidence of chronic rejection episodes, caused by an increased incidence of acute rejection episodes. In agreement with Kahan et al. [11], we speculate that the pharmacokinetic variability of CsA is due to episodic absorptive variations caused by co-administered over-the-counter medications and/or a variety of foods in the diet. In addition, non-compliance also seems to be a major reason for fluctuating CsA levels [20].

To date, there are only two other studies in which the influence of the intra-individual CsA variability on renal allograft survival was investigated. Inoue et al. [21] analysed the impact of fluctuations in CsA plasma trough levels during the first month after transplantation on the long-term outcome of living-related renal allograft recipients. They found that high fluctuations in CsA trough levels were associated with a higher incidence of acute rejection episodes, with higher serum creatinine concentrations and decreased graft survival. However, the results of this study are limited by the small number of patients investigated (n=37), and by the fact that two different assay systems (polyclonal RIA and polyclonal FPIA) were used. In addition, patients had been treated with different immunosuppressive protocols. Savoldi et al. [22] investigated the influence of the intra-patient variability of monoclonal CsA trough levels, measured by RIA (Cyclotrac, Incstar Corp.) after the 6th month post-transplant, on renal allograft survival in 157 patients. In agreement with our results, they observed that a high intra-patient variability (>31%) was an important risk factor for late graft loss.

In order to assess the influence of the mean CsA trough level on renal allograft survival, we calculated the mean polyclonal CsA trough level during the entire observation period for each patient. Based on our target range (270–370 ng/ml), patients were divided into three groups. As demonstrated by Oellerich et al. [14], there is a substantial variability among transplant centres concerning the therapeutic CsA ranges for kidney transplantation. In agreement with other centres [14], we aimed at relatively low target levels throughout the whole post-transplantation period. In patients with CsA trough levels below the target range (<270 ng/ml), graft survival was reduced as compared to patients with normal (270–370 ng/ml) or high mean CsA trough levels (>370 ng/ml). In addition, the incidence of acute rejection episodes was elevated. No differences between groups were found concerning serum creatinine concentrations. These results suggest that long-term graft survival in patients with low mean polyclonal CsA trough levels is reduced by an increased incidence of acute rejection episodes. In contrast, ‘normal’ CsA trough levels (270–370 ng/ml) provided an equivalent immunosuppressive effect as compared to high CsA trough levels (>370 ng/ml). Because the risk of side-effects is known to increase along with increasing CsA levels, aiming at ‘normal’ CsA trough levels seems to be advisable.

The influence of the inter-individual variability of CsA trough levels on renal allograft survival has been investigated in numerous studies. Although both, the immunosuppressive efficacy and the potential side-effects of CsA therapy are known to be dose-related, it is difficult to define an optimal CsA dosage, or even an optimal therapeutic range of blood concentrations. In several studies, a correlation between CsA trough levels and the occurrence of rejection episodes was found [23,24]. However, in other investigations such a dependency was not detectable [14]. The results obtained from studies on the long-term effects of CsA on renal structure and function are also conflicting [25]. Even patients displaying trough levels within a putative therapeutic range are not always spared from either rejection or nephrotoxicity [26]. Furthermore, the results of these studies are only partially comparable because organ- and time-specific therapeutic ranges vary widely between transplantation centres [14]. Differences relate mainly to the employed CsA assay, the concomitant immunosuppressive therapy, and the individual policy of each centre, including centre-specific preferences in the degree of initial exposure of patients to CsA.

In future, two strategies may help to reduce the pharmacological vagaries of CsA. First, the microemulsion CsA formulation Neoral® is less dependent on food intake and bile secretion [27], thus offering the advantage of a reduced variability in important pharmacokinetic parameters. Clinical reports show that intra- and inter-patient variability of CsA trough levels are reduced as compared to the conventional CsA formulation [28]. Secondly, new therapeutic drug monitoring strategies such as C2 level measurements and 2- or 3-point measurements offer the potential to predict the area under the curve more precisely and thus may provide a clinically important improvement over the use of CsA trough level measurements.



   Notes
 
Correspondence and offprint requests to: Dr med. Johannes Waiser, Medizinische Klinik mit Schwerpunkt Nephrologie, Universitätsklinikum Charité, Campus Charité Mitte, Schumannstraße 20/21, D-10117 Berlin, Germany. Email: johannes.waiser{at}charite.de Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 29. 8.01
Accepted in revised form: 14. 2.02