C2 (2-h) levels are not superior to trough levels as estimates of the area under the curve in tacrolimus-treated renal-transplant patients

Kaj Jørgensen1,, Johan Povlsen1, Søren Madsen1, Melvin Madsen1, Hans Hansen1, Asger Pedersen2, Else-Marie Heinsvig3 and Jørgen Poulsen3

1 Department of Renal Medicine C, Skjeby Sygehus, Århus University Hospital, 2 Department of Biostatistics, University of Aarhus and 3 Department of Clinical Biochemistry, Århus Kommunehospital, Århus, Denmark



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Recently, 2-h samples (C2) have been found superior to trough levels for therapeutic monitoring of transplanted patients receiving cyclosporin. The present study was undertaken to see if the same was the case for tacrolimus.

Methods. Blood tacrolimus levels were determined in 21 consecutive patients at 3 and 14 days after renal transplantation, and before and 1, 2, 3, 4, and 6 h after oral intake of 0.1 mg/kg tacrolimus. The area under the blood concentration/time curve (AUC) was determined by the trapezoidal method, association between blood concentration at each sampling time and AUC by Pearson's correlation coefficient, and pairs of correlation coefficients were compared by an asymptotic Wald-type test.

Results. AUC varied five-fold despite near-equal dosing. Pearson's correlation coefficient for trough level, 1, 2, 3, 4, and 6 h were 0.84, 0.60, 0.81, 0.95, 0.95, and 0.94 on day 3 and 0.94, 0.69, 0.92, 0.96, 0.94, 0.92 on day 14. Three-, 4- and 6-h levels had significantly higher correlation coefficients compared to trough, 1- and 2-h levels. One-hour samples had significantly lower correlation coefficients compared to all other sampling times on day 14. The patient with the highest AUC developed nephrotoxicity despite trough levels in the desired range.

Conclusions. Two-hour levels are not superior to trough levels in tacrolimus-treated renal transplant patients. Despite good correlation between trough level and AUC, some patients may still receive nephrotoxic doses despite trough levels in the desired range. Sampling 3–6 h after oral intake may be at least as good as trough levels.

Keywords: area under the curve; C2 (2-h) levels; nephrotoxicity; renal-transplant patients; tacrolimus; trough levels



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The variable pharmacokinetics and narrow therapeutic index of tacrolimus and cyclosporin (CsA) have made therapeutic drug monitoring (TDM) necessary. For the CsA formulation Neoral®, it has recently been postulated that blood sampling 2 h after oral drug intake (C2) is superior to trough levels sampled 12 h after drug intake [1], which is currently the most widespread method of TDM for both drugs. Since the time window for C2 monitoring is much narrower than that for trough-level monitoring, a shift to C2 monitoring implies a great increase in patient and staff discipline. This would be even more difficult if the monitoring procedures for the two drugs were different. We therefore decided to find out if the C2 values were superior to trough-level values in the case of tacrolimus. It has also been postulated that low systemic exposure to tacrolimus correlates with acute rejection [2]. We were therefore interested to see if this could be confirmed in our patients.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The study was approved by the local ethics committee (Den videnskabsetiske Komité for Århus Amt, journalnumber 1999/4636) and the Danish drug authorities (Lægemiddelstyrelsen).

Patients
Twenty-one consecutive patients undergoing renal transplantation between 1 January 2000 and 30 June 2000 at the Department of Renal Medicine C, Skejby Sygehus, Århus University Hospital, participated in the study after giving their written informed consent. One patient who had the graft removed on day 10 after transplantation because of artery thrombosis only participated in the day 3 part of the study. Nineteen patients received a kidney from a cadaveric donor and two from a living related donor. Fourteen patients were transplanted for the first time, while four had been transplanted once before and three had received their third renal allograft. All patients received basiliximab, steroids, and tacrolimus. All patients received tacrolimus (Prograf®) orally at an initial dose of 0.1 mg/kg twice daily. The dose was adjusted according to trough level determined twice weekly, aiming at trough levels between 10 and 20 ng/ml. The patients did not receive any drugs known to significantly alter tacrolimus pharmacokinetics. Steroids were administered as 500 mg of methylprednisolone just after surgery and then 40 mg prednisolone daily for patients with a body weight of 60 kg and above, while patients under 60 kg were given 35 mg daily. On day 8 after surgery, the prednisolone dose was reduced by 5 mg. Eighteen patients also received azathioprine, while three patients considered to be immunologically at ‘high risk’ due to panel-reactive antibodies were treated with mycophenolate mofetil (MMF) 1000 mg twice daily. On day 3 post-transplant, careful registration of the gastrointestinal conditions of all patients was done, especially focusing on intestinal paralysis, flatus, and bowel movement. On day 14 all patients were without complaints of nausea, vomiting, diarrhoea, or constipation. The patients were followed until 3 months after transplantation.

Study design
This was an open, prospective, non-randomized, single-centre study. The patients were investigated on day 3 and day 14 after renal transplantation. A blood sample was collected in the morning in fasting patients, 12 h after the last dose of tacrolimus and just before the morning dose. Blood samples were then collected at 1, 2, 3, 4, and 6 h after intake of about 0.1 mg/kg tacrolimus orally. Blood was collected in tubes containing EDTA and frozen at -20°C until analysis, which was performed using the IMX method utilizing monoclonal antibodies [3].

Statistics
The area under the blood level/time curve (AUC) between oral doses was calculated by the trapezoidal method [4] assuming that 0- and 12-h values were identical. The association between blood levels sampled at different times and AUC was estimated by Pearson's correlation coefficient. Pairs of correlation coefficients were compared by an asymptotic Wald-type test. The elimination rate K was estimated by the method of residuals [5] and subsequently analysed by a mixed model with systematic difference between days and random subject levels in the PROC MIXED procedure of the SAS statistical software [6]. Comparison of inter-individual and intra-individual K estimates was performed using an asymptotic Chi square test (REML-likelihood, 1 degree of freedom). Significance of the estimated systematic difference between days 3 and 14 was tested by approximate F-test based on the REML-likelihood with Satterthwaite approximation of the denominator number of degrees of freedom [6]. Renal biopsies were taken in all cases of suspected rejection and graded according to the Banff 97 nomenclature [7].



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
On day 3 the mean tacrolimus dose was 0.097 (SE: 0.002) mg/kg and 0.108 (SE: 0.003) mg/kg on day 14. This small increase in mean dose was primarily due to a decrease in body weight, since the patients were kept intentionally overhydrated in the first days after renal transplantation.

Despite this near equal dosing of the patients, the range of the AUC was very wide as seen in Figure 1Go. The mean AUC increased from 226 to 240 ng/h/ml.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.  The AUC of tacrolimus on day 3 (open columns) and on day 14 (black columns) after renal transplantation. Shown are the AUC of the patients who received MMF and patients who experienced acute rejection during the first 3 months after transplantation.

 
The mean blood tacrolimus levels at the different sampling times are shown in Figure 2Go. Table 1Go shows the Pearson's correlation coefficients between the blood levels at each sampling time and AUC, while Table 2Go shows the P values of the differences between the correlation coefficients. The highest correlation was found with samples harvested 3 h after oral intake on both day 3 and day 14. The 3-, 4-, and 6-h correlation coefficients were significantly different from the 0 (trough level), 1-, and 2-h correlation coefficients on day 3, while we only found the 1-h correlation coefficient to be different from all the other coefficients on day 14 (Table 2Go). Two-hour values were not better than trough levels (0 h). The correlation of AUC to serum albumin and to haematocrit was poor. The mean K was 0.1789 (SE 0.0175) 1/h on day 3 and 0.1508 (SE 0.0135) 1/h on day 14. There were no significant differences between the inter-patient and the intra-patient variation (P=0.3711). The estimated systematic difference between day 3 and day 14 was 0.0250 (SE 0.0197). This difference was not significant (P=0.2191). All but the patient with graft-artery thrombosis had functioning grafts at the end of the 3-months observation period, but four had experienced acute rejection episodes. The AUC of the patients receiving MMF did not differ from the patients receiving azathioprine. Two patients had intestinal paralysis on day 3. Their AUCs were 108 and 230 ng/h/ml. AUC from 10 patients with flatus had a mean value of 225 ng/h/ml. The rest of the patients, with bowel movement, had a mean AUC of 240 ng/h/ml.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.  Mean (and SE) whole-blood tacrolimus concentration at different times after oral intake of approximately 0.1 mg/kg tacrolimus on day 3 and day 14 after renal transplantation.

 

View this table:
[in this window]
[in a new window]
 
Table 1.  The Pearson correlation coefficients of blood levels sampled at different times (h) after oral intake of tacrolimus compared to 12-h trapezoidal AUC

 

View this table:
[in this window]
[in a new window]
 
Table 2.  P values of differences between Pearson correlation coefficients of blood levels sampled at different times (h) after oral intake of tacrolimus compared to AUC. Day 3 values are to the upper right of the blank diagonal in the Table, day 14 values to the lower left.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The aim of the study was to determine if samples harvested 2 h after oral intake of tacrolimus (C2) would be a better estimate of AUC than trough levels. This was not the case for tacrolimus as it is for CsA. The Pearson correlation coefficients were almost the same and there were no significant differences between C2 and trough levels. The highest correlation to AUC was seen at 3 h. Wong et al. [8] described a two-point sampling method of 2- and 4-h values as good estimates of AUC in renal transplant patients. Ku and Min [9] found 4-h concentrations to be the best predictors of AUC in liver-transplant patients. The correlation of trough levels to AUC at day 14 was identical to that reported earlier [10,11], while the correlation coefficient was lower on day 3. Attempts were made to determine AUC to infinity by curve fitting, assuming first-order kinetics, but these results are omitted for the following reasons. First, we find the value of integrating to infinity for patients taking medication every 12 h very questionable. If one does integrate to infinity, the last determined value (in this case 6 h) would have a relatively large impact on the AUC. Fifteen patients on day 3 and 12 patients on day 14 had values to which curve fitting could be reasonably applied, but this leaves quite a number of patients in whom the curves were difficult to explain by first-order kinetics. We therefore believe that the non-parametric trapezoidal sums represent the most reliable estimates of AUC. First-order models for kinetics could be used in some patients, but it was difficult to describe the uptake of the drug in a substantial number of patients, while elimination was more predictable. A significant correlation between high trough levels and toxicity has been found in both kidney- and liver-transplant patients, while low levels have only been shown to correlate with rejection in kidney-transplant patients [12]. It has been demonstrated in a recent trial, that patients having rejections have lower AUC than rejection free patients [2]. The present study did not give any significant information on AUC and rejection. Two of the four rejections occurred in patients with AUC under 200 ng/h/ml on both days, while the other two patients experienced rejection later after the tacrolimus dose had been significantly reduced. Tacrolimus, like CsA, has been shown to decrease renal function [13] and to be nephrotoxic [14] in transplant recipients. Individual patients did experience nephrotoxicity and high AUC despite trough levels in the target range. We were unable to demonstrate any correlation between gastrointestinal mobility and AUC in this study.

In conclusion, we found that C2 blood tacrolimus levels were not superior to trough levels as estimates of the area under the blood concentration/time curve in tacrolimus-treated renal-transplant patients. We found a great variability in AUC despite nearly equal dosing in mg/kg.



   Acknowledgments
 
This study was supported by the Danish Kidney Association, the Danish Society of Nephrology, and the Danish Transplantation Society.



   Notes
 
Correspondence and offprint requests to: Kaj Jørgensen, Research laboratory, Renal Medicine C, Skjeby Sygehus, Århus University Hospital, DK-8200 Århus N, Denmark. Email: kaj{at}dadlnet.dk Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Belitsky P, Levy GA, Johnston A. Neoral absorption profiling: an evolution in effectiveness. Transplant Proc2000; 32: 45S–52S[ISI][Medline]
  2. Undre NA, van Hoof J, Christiaans M et al. Low systemic exposure to tacrolimus correlates with acute rejection. Transplant Proc1999; 31: 296–298[ISI][Medline]
  3. Grenier FC, Luczkiw J, Bergman M et al. A whole blood FK 506 assay for the IMx® analyzer. Transplant Proc1991; 23: 2748–2749[ISI][Medline]
  4. MacKichan JJ, Comstock TJ. General Pharmacokinetic Principles. In: Taylor WJ, Caviness MHD, eds. A Textbook for the Clinical Application of Therapeutic Drug Monitoring. Irving, Texas, 1988
  5. Gibaldi M, Perrier D. Pharmacokinetics, 2nd edn. Marcel Dekker Inc., New York, 1982
  6. SAS/STAT Software. Changes and Enhancements through release 6.12, SAS Institute Inc., Cary, NC, 1997
  7. Racusen LC, Solez K, Colvin RB et al. The Banff 97 working classification of renal allograft pathology. Kidney Int1999; 55: 713–723[ISI][Medline]
  8. Wong KM, Sheck CC, Chau KF, Li CS. Abbreviated tacrolimus area-under-the-curve-monitoring for renal transplant recipients. Am J Kidney Dis2000; 35: 660–666[ISI][Medline]
  9. Ku YM, Min DI. An abbreviated area-under-the-curve monitoring for tacrolimus in patients with liver transplants. Ther Drug Monit1998; 20: 219–223[ISI][Medline]
  10. Undre NA, Stevenson P, Schäfer A. Pharmacokinetics of tacrolimus: clinically relevant aspects. Transplant Proc1999; 31: 21S–24S
  11. Venkataramanan R, Swaminathan A, Prasad T et al. Clinical pharmacokinetics of tacrolimus. Clin Pharmacokinet1995; 29: 404–430[ISI][Medline]
  12. Kershner RP, Fitzsimmons WE. Relationship of FK506 whole blood concentrations and efficacy and toxicity after liver and kidney transplantation. Transplantation1996; 52: 920–926
  13. Japanese FK 506 Study Group. Clinicopathological evaluation of kidney transplants in patients given a fixed dose of FK 506. Transplant Proc1991; 23: 3111–3115[ISI][Medline]
  14. Tauxe WN, Mochizuki T, McCauley J et al. A comparison of the renal effects (ERPF, GFR, and FF) of FK 506 and cyclosporine in patients with liver transplantation. Transplant Proc1991; 23: 3146–3147[ISI][Medline]
Received for publication: 24.11.01
Accepted in revised form: 13. 3.02