Despite development of a wide range of novel drugs, calcineurin inhibitors (CNIs) remain the major agents for immunosuppression in liver transplantation. Tacrolimus (Tac) has been widely accepted for immunotherapy, whereas for patients unable to tolerate Tac, cyclosporine A (CYA) has been described as a valuable rescue therapy (1–3). One meta-analysis demonstrated that as a primary immunosuppressive agent, Tac was superior to CYA (including both the original oil-based formulation and the newer microemulsion formulation) in terms of mortality, graft loss, and rejection at 1 year (4). Nonetheless, all but one study included in this meta-analysis measured CYA trough levels to attain adequate levels of exposure (4).
Recently, Levy et al. (5) reported a randomized, multicenter study indicating decreased overall incidence of and statistically less severe acute cellular rejection in liver transplant recipients on an oral microemulsion formulation of CYA (CYA-ME) when 2-hr postdose levels were monitored, as a surrogate marker of CYA peak, instead of conventional CYA trough level. More importantly, a subset of patients in the 2-hr postdose monitoring group who reached the minimum target CYA peak range by day 3 demonstrated a significantly lower incidence of acute cellular rejection compared with patients who only achieved the target peak level by days 7 and 10, suggesting that reaching target levels at an early stage after transplant is crucial when a 2-hr postdose monitoring strategy is to be implemented (5). Subsequently, several randomized trials including the LIS2T study have reported promising results in terms of efficacy, toxicity, and pharmacoeconomics with CYA-ME, showing equivalent results in patient groups receiving CYA-ME with 2-hr postdose monitoring or Tac for patient and graft survivals and the overall incidence of acute rejection (6–8).
Nevertheless, data collected for the previous studies were generally based on deceased donor liver transplantations, and these results cannot be simply applied to adult living donor liver transplantation (LDLT). The reduced size of graft livers (usually hemi-liver), prolonged intestinal paralysis because of lengthy operation, and posttransplant external bile diversion described in recent publications from high-volume LDLT centers mostly in Japan (9–12) are the distinctive features of adult LDLT, contributing to delayed graft functional recovery and poor enteral absorption, which in turn substantially interfere with achieving and maintaining the therapeutic CYA peak blood concentration mentioned earlier.
To overcome these problems, intravenous CYA infusion may become a promising option because of its ability to ensure sufficient CYA exposure to exert immunosuppressive effects regardless of enteral absorption and biliary drainage. Clinical evidence of the efficacy and safety of intravenous CYA in liver transplantation is scarce, and appropriate therapeutic drug monitoring remains to be elucidated (13, 14). Moreover, no study has compared the clinical outcomes of intravenous infusion of CYA (CYA-IV) with CYA-ME regimens in LDLT to date. In the era of individually tailored immunosuppression, establishing a standard intravenous CYA protocol in LDLT is paramount, as an alternative CNI-based immunotherapy with potential advantages over Tac with regard to posttransplant new-onset diabetes mellitus and in the treatment of transplant patients with hepatitis C virus (HCV) or primary biliary cirrhosis and as a salvage immunosuppressive regimen in cases of Tac-related side effects (15–17). In this study, we evaluated the feasibility and efficacy of 4-hr intravenous CYA immunotherapy for LDLT, focusing on its therapeutic drug monitoring in comparison with a CYA-ME regimen.
The CYA-ME (Neoral, Novartis Pharma K. K., Tokyo, Japan, n=23) group and the 4-hr continuous intravenous infusion of CYA (4-hr CYA-IV; Sandimmun, Novartis Pharma K. K., n=27) groups were comparable for age, indications, Child-Pugh grade, model for end-stage liver disease scores, preoperative conditions, Eastern Cooperative Oncology Group performance status, graft lobe, graft:recipient weight ratio, graft volume/recipient standard liver volume, donor age, and blood loss (Table 1). The number of males in the CYA-ME group was higher compared with that in the 4-hr CYA-IV group (P=0.035; Table 1). Regarding surgical factors, the proportion of patients who underwent duct-to-duct reconstructions was higher (P=0.044), and cold and warm ischemia times were longer (P=0.002 and P<0.001, respectively) in the 4-hr CYA-IV group compared with those in the CYA-ME group (Table 1).
Pharmacokinetic Profiles of 4-hr CYA-IV and CYA-ME Groups
For the CYA-ME group, 9 of 15 patients (60%) who were not switched to other CNIs completed full pharmacokinetic evaluations on day 3. Of these nine patients, only two (22%) reached the target peak range of 700 to 1000 ng/mL (2-hr postdose CYA level 484±272 ng/mL; Fig. 1A). In contrast, adequate and stable blood CYA trough/peak levels were successfully achieved at posttransplant day 3 in all 27 patients (100%) of the 4-hr CYA-IV group (4-hr postdose CYA level 856±129 ng/mL; Fig. 1B).
Adjustability of Immunosuppressive Agents
To evaluate the effort needed to adjust the immunosuppressive agents between the two groups, dose variance was calculated for each case as follows:
A Student's t test showed that patients in the 4-hr CYA-IV group had a significantly smaller dose variance (18.3%±13.9%) compared with those in the CYA-ME (35.3%±21.9%; P=0.017) group, indicating minor interpatient and intrapatient variability (Fig. 2).
Effect of External Bile Diversion on Oral CYA Absorption
In the CYA-ME group, daily bile output and blood CYA peak (2 hr after dose)/trough levels were measured in 14 of 15 patients (93%) at posttransplant weeks 2 to 3 when bowel functions were deemed to have returned to their baselines, and patients could tolerate regular diet. Patients with CYA peak:trough ratios more than or equal to 2.0, demonstrating adequate exposure to CYA, had a significantly lesser amount of biliary drainage (173±50 mL/day) compared with those with ratios less than 2.0 (403±46 mL/day, P=0.006; Fig. 3).
No patients (0%) in the 4-hr CYA-IV group suffered acute cellular rejections, and this rate was significantly lower compared with that observed in the CYA-ME group (17%, P=0.038; Table 2). All four episodes of acute cellular rejection in the CYA-ME group occurred within 1 month posttransplant (range 7–21 days) while the patients were on oral CYA. The incidences of posttransplant comorbidities, including dialysis-dependent renal insufficiency, neurotoxicity, infection, and cytomegalovirus antigenemia, were similar between the two groups (Table 2). None of the patients in either group developed hypertension, severe electrolyte disturbance, or hyperlipidemia.
In the 4-hr CYA-IV group, 2 of 27 patients (7%) required a CNI switch to Tac because of seizures and severe antibody-mediated rejection. This rate was significantly lower than that observed in the CYA-ME group (8/23 patients, 35%, P=0.030; Table 2). The main reason for a CNI switch in the CYA-ME group was inability to achieve target trough levels, which occurred for five patients. The other causes included acute cellular rejection, seizures, and acute pancreatitis. In-hospital deaths occurred in 2 of 23 patients (9%) in the CYA-ME group (chronic rejection and multiple organ failure) and 4 of 27 patients (15%) in the CYA-IV group (multiple organ failures, posttransplant lymphoproliferative disease, and recurrent pneumonia). Mortality rates were comparable between the two groups (P=0.67; Table 2).
Optimal Initial Dose of Oral CYA After 4-hr CYA-IV
In the 4-hr CYA group, 4 cases of in-hospital mortalities were excluded, and the remaining 23 patients underwent trials of oral CYA conversion from 4-hr CYA-IV. Conversions were successful for 18 of 23 patients (78%) with a median posttransplant day of 27 (range 10–82 days). For the other 5 of 23 patients (22%), target trough/peak levels were not achieved, and their CNI was switched to Tac. The median doses of 4-hr CYA-IV before oral CYA conversion and initial oral CYA were 30 mg (range 10–65 mg) and 83 mg (range 25–200 mg), respectively. The median dose ratio of 4-hr CYA-IV before conversion to initial oral CYA was 1:3 (range 2.0–3.8). The median difference between the oral CYA dose at conversion and at discharge was 20% (range 0%–50%). From oral CYA conversion to time of discharge, none of the 18 patients experienced acute cellular rejections or adverse events, and adequate trough CYA levels were well maintained throughout the study period.
During a median follow-up period of 52 months (range 5–108 months), the 5-year overall survival rates for patients in the CYA-ME and 4-hr CYA-IV groups were 78% and 81%, respectively (P=0.88).
This is the first series to demonstrate the feasibility and efficacy of 4-hr CYA-IV immunotherapy after LDLT in comparison with the CYA-ME regimen, a milestone in CNI-based immunotherapy. Our 4-hr CYA-IV protocol demonstrated excellent immunosuppressive potency (acute cellular rejection rate of 0%) with a similar toxicity profile and mortality rate to those of the CYA-ME regimen.
Our 4-hr CYA-IV regimen allowed effortless achievement of target CYA trough and peak levels in all patients (100%) with small interindividual and intraindividual dose variation by posttransplant day 3, which is considered to be the critical period for preventing acute cellular rejection (5). In LDLT, intravenous CYA infusion for 4 hr facilitates adequate and stable CYA exposure to reproduce the unique area under the concentration-time curve of CYA-ME exhibited in deceased donor liver transplantation recipients, characterized by a rapid increase in blood CYA concentrations usually within 2 hr after drug administration (18, 19). This CYA peak level correlates well with area under the concentration-time curve and shows strong association with freedom from graft rejection (19). On the contrary, dose adjustment in the CYA-ME group was demanding in our series with only 22% of patients reaching the minimum target CYA peak level by day 3. Marked dose disparity among CYA-ME patients was also observed, along with a significantly higher incidence of acute cellular rejection and an increased risk for switch to Tac compared with the 4-hr CYA-IV group. Published evidence demonstrating successful adoption of CYA-ME with 2-hr postdose monitoring in deceased donor liver transplantation recipients cannot be extrapolated to LDLT patients, whose CYA exposure levels are unpredictable with inferior outcomes when oral administration is used. Thus, when CYA is used as the primary immunosuppressive agent in LDLT, our 4-hr CYA-IV protocol provides ideal therapeutic drug monitoring for optimization of CYA dosing and effect in the early stage after transplant.
In contrast to a recent report (14), we did not identify factors (including graft:recipient weight ratio) that significantly correlated with initial blood CYA trough levels in the 4-hr CYA-IV group. The timing of measurement and the dissimilarity in patient backgrounds may explain this difference. However, we did not extensively investigate this subject because reaching target peak CYA level within several days posttransplant is a matter of utmost importance (5). We successfully achieved an ideal concentration-time curve at posttransplant day 3 in all patients of the 4-hr CYA-IV group with no acute cellular rejection. This is one of the striking advantages of intravenous CYA infusion.
We also investigated the optimal initial dose and time for converting from 4-hr CYA-IV to oral CYA administration. Data are limited for determining the conversion dose ratio of CYA-IV:oral CYA, ranging from 1:2 to 1:9 (14, 20, 21). Our current policy, based on clinical data, is to administer initial oral CYA at a dose 3-fold greater than that of intravenous CYA. This is in accordance with previous reports that described the absolute bioavailability of oral CYA as 38%±10% in healthy volunteers and the use of 4-hr CYA-IV for LDLT (14, 22). Regarding the time, although we sought to convert CYA from intravenous to oral administration at posttransplant weeks 2 to 3, considerable interindividual discrepancies in patient and graft recoveries hindered prompt conversion, and only 6 of 23 patients (26%) were successfully changed to oral administration by day 21. Because the amount of external biliary drainage obviously affected CYA-ME absorption (Fig. 3), intermittent tube clamping in stable LDLT recipients with more than or equal to 300 mL/day bile output, or simply increasing the initial oral CYA dose at the time of oral CYA conversion, may be a reasonable strategy to provide sufficient trough and peak CYA levels. Our goal is to start oral CYA once the patient's condition is stabilized, ideally around posttransplant day 7 to 10, and a safe and rapid oral conversion protocol has yet to be determined.
The present series has several limitations. It was a retrospective analysis of data collected in a single center, and the number of patients was small. Era bias may exist. A significant number of patients in the CYA-ME group required CNI switching. The selection of antimetabolites and biliary reconstruction techniques were not standardized. A prospective study might be required to validate the proposed 1:3 ratio of intravenous CYA:oral CYA dose at the time of conversion.
In conclusion, our 4-hr CYA-IV protocol enables accurate therapeutic drug monitoring and provides safe and effective immunosuppression for LDLT. Excellent patient compliance is expected because of the minor interindividual variances. A 4-hr CYA-IV regimen is superior to CYA-ME and would be a potent alternative strategy for primary immunosuppression in LDLT. Conversion to oral CYA is affected by external biliary drainage and is occasionally demanding, in which case establishing optimal dose and time is warranted.
MATERIALS AND METHODS
We undertook a single-center, retrospective cohort analysis of 50 adult patients (older than 18 years) who underwent primary ABO-compatible LDLTs between April 2001 and December 2009. Written informed consent was obtained from all patients. This study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was conducted under the approval of the Institutional Review Board of Keio University School of Medicine (2010-075).
Regardless of the type of biliary reconstruction (duct-to-duct vs. Roux-en-Y), external bile diversion was performed in all patients. Biliary drainage tubes were routinely left open during the first 2 weeks and then clamped according to the general condition of the patient. Study subjects were divided into the following two groups based on the primary CNI administered as an immunosuppressive agent: CYA-ME and 4-hr CYA-IV. We introduced CYA-based immunotherapy in 2001, and CYA-ME was employed as the primary immunosuppression until 2004. A transition was made in 2005 to the current protocol of 4-hr CYA-IV. Patient backgrounds, CNI dose adjustability, and clinical outcomes were compared between the two groups.
Patients were principally treated with a standard triple regimen comprising CNI, corticosteroids, and an antimetabolite. The patients in the 4-hr CYA-IV group received a 4-hr continuous intravenous infusion of CYA at an initial dose of 0.8 mg/kg twice daily. In the CYA-ME group, oral administration of CYA was initiated at 2.5 mg/kg twice daily. In both groups, the trough CYA levels were measured twice daily. The peak CYA levels were measured frequently whenever the dose was modified, at the end of infusion in the 4-hr CYA-IV group and at 2 hr after oral administration in the CYA-ME group. For patients in the 4-hr CYA-IV group, a switch to oral CYA administration was attempted 2 to 3 weeks after transplant when the patients were considered clinically stable; the basic starting dose was 3-fold greater than that of intravenous CYA and was administered twice daily. The CYA doses were adjusted to maintain therapeutic levels according to the posttransplant period (target trough and peak ranges 300–400 and 700–1000 ng/mL, respectively, to month 1; 150–300 and 500–700 ng/mL, respectively, to month 3; and 80–150 and 300–500 ng/mL, respectively, thereafter). The decision to switch from CYA to Tac was made if a patient was unable to reach the target CYA range during the first 3 days after LDLT or whenever acute rejection or CYA toxicity occurred. Blood CYA concentrations were measured by fluorescence polarization immunoassay (Abbott Laboratories, Abbott Park, IL). Methylprednisolone was given intravenously to all patients at a dose of 10 mg/kg at the time of graft reperfusion, at 2 mg/kg/day for days 1 through 3, at 1 mg/kg/day for days 4 through 6, and at 0.5 mg/kg/day thereafter, and then tapered and terminated at approximately 6 months after LDLT. For stable patients, antimetabolites were added to supplement the immunosuppressive regimen at the discretion of the transplant team; mycophenolate mofetil (500–1500 mg/day) or mizoribine (2–3 mg/kg/day) was used in most patients. Because the antimetabolites were prescribed on an auxiliary basis, they were prone to switching to other agents or withdrawal if a patient suffered rejection, infection, or suspected drug-induced toxicities.
In 2004, for patients with liver failure because of HCV infection, basiliximab (antiinterleukin 2 receptor α chain monoclonal antibody) was introduced. They were maintained steroid-free throughout the posttransplant period, but the dosage of CNIs and antimetabolites was kept identical to that with non-HCV patients.
For the 4-hr CYA-IV group, a complete pharmacokinetic profile was obtained on day 3 by measuring blood CYA concentrations at 0, 4, and 10 hr after the start of infusion. For the CYA-ME group, the pharmacokinetic profile on day 3 was obtained by measuring blood CYA concentrations at 0, 1, 2, 4, 6, 10, and 12 hr after oral administration.
Rejection and Adverse Events
An acute cellular rejection episode was defined as a biopsy-proven, histologic diagnosis of moderate to severe rejection according to the Banff Schema that required a corticosteroid increment, including steroid pulse therapy, with or without CNI switches (23). Patients who received dialysis after transplant and who were not already known to have renal failure were classified as suffering from dialysis-dependent renal insufficiency. Neurotoxicity included convulsions, altered mental status, and leukoencephalopathy. Infection was identified whenever antimicrobial therapy was initiated separately from the routine prophylactic antibiotics. Cytomegalovirus antigenemia was checked twice weekly until discharge.
Demographic data were presented as means±standard deviations or medians (ranges). Categorical variables were compared using a chi-square test or a Fisher's exact test. Continuous variables were compared using a Student's t test. If variables were not normally distributed with unequal variances (Levene's test), a Wilcoxon Mann–Whitney U test was used, when appropriate. Overall survivals were determined by the Kaplan–Meier method and compared using a log-rank test. P less than 0.05 was considered statistically significant. All data analyses used SPSS version 17.0 (SPSS Inc., Chicago, IL).
1. Jain A, Brody D, Hamad I, et al. Conversion to neoral for neurotoxicity after primary adult liver transplantation under tacrolimus. Transplantation
2000; 69: 172.
2. Abouljoud MS, Kumar MS, Brayman KL, et al; OLN-452 Study Group. Neoral® rescue therapy in transplant patients with intolerance to tacrolimus. Clin Transplant
2002; 16: 168.
3. Tamura S, Sugawara Y, Kishi Y, et al. Conversion to cyclosporine provides valuable rescue therapy for living donor adult liver transplant patients intolerant to tacrolimus: A single-center experience at the University of Tokyo. Transplant Proc
2004; 36: 3242.
4. McAlister VC, Haddad E, Renouf E, et al. Cyclosporin versus tacrolimus as primary immunosuppressant after liver transplantation: A meta-analysis. Am J Transplant
2006; 6: 1578.
5. Levy G, Burra P, Cavallari A, et al. Improved clinical outcomes for liver transplant recipients using cyclosporine monitoring based on 2-hr post-dose levels (C2). Transplantation
2002; 73: 953.
6. Tanaka K, Lake J, Villamil F, et al. Comparison of cyclosporine microemulsion and tacrolimus in 39 recipients of living donor liver transplantation
. Liver Transpl
2005; 11: 1395.
7. Levy G, Grazi GL, Sanjuan F, et al. 12-month follow-up analysis of a multicenter, randomized, prospective trial in de novo liver transplant recipients (LIS2T) comparing cyclosporine microemulsion (C2 monitoring) and tacrolimus. Liver Transpl
2006; 12: 1464.
8. Shenoy S, Hardinger KL, Crippin J, et al. A randomized, prospective, pharmacoeconomic trial of neoral 2-hour postdose concentration monitoring versus tacrolimus trough concentration monitoring in de novo liver transplant recipients. Liver Transpl
2008; 14: 173.
9. Marubashi S, Dono K, Nagano H, et al. Biliary reconstruction in living donor liver transplantation
: Technical invention and risk factor analysis for anastomotic stricture. Transplantation
2009; 88: 1123.
10. Kasahara M, Egawa H, Takada Y, et al. Biliary reconstruction in right lobe living-donor liver transplantation: Comparison of different techniques in 321 recipients. Ann Surg
2006; 243: 559.
11. Soejima Y, Taketomi A, Yoshizumi T, et al. Biliary strictures in living donor liver transplantation
: Incidence, management, and technical evolution. Liver Transpl
2006; 12: 979.
12. Dulundu E, Sugawara Y, Sano K, et al. Duct-to-duct biliary reconstruction in adult living-donor liver transplantation. Transplantation
2004; 78: 574.
13. Lück R, Böger J, Kuse E, et al. Achieving adequate cyclosporine exposure in liver transplant recipients: A novel strategy for monitoring and dosing using intravenous therapy. Liver Transpl
2004; 10: 686.
14. Sato K, Iwane T, Sekiguchi S, et al. Management of living donor liver transplant patients using twice-daily 4-hour intravenous cyclosporine therapy. Transplant Proc
2009; 41: 229.
15. Selzner N, Renner EL, Selzner M, et al. Antiviral treatment of recurrent hepatitis C after liver transplantation: Predictors of response and long-term outcome. Transplantation
2009; 88: 1214.
16. Montano-Loza AJ, Wasilenko S, Bintner J, et al. Cyclosporine A
protects against primary biliary cirrhosis recurrence after liver transplantation. Am J Transplant
2010; 10: 852.
17. Kuo HT, Sampaio MS, Ye X, et al. Risk factors for new-onset diabetes mellitus in adult liver transplant recipients, an analysis of the Organ Procurement and Transplant Network/United Network for Organ Sharing database. Transplantation
2010; 89: 1134.
18. Cantarovich M, Barkun JS, Tchervenkov JI, et al. Comparison of neoral dose monitoring with cyclosporine trough levels versus 2-hr postdose levels in stable liver transplant patients. Transplantation
1998; 66: 1621.
19. Grant D, Kneteman N, Tchervenkov J, et al. Peak cyclosporine levels (Cmax) correlate with freedom from liver graft rejection
: Results of a prospective, randomized comparison of neoral and sandimmune for liver transplantation (NOF-8). Transplantation
1999; 67: 1133.
20. Parquet N, Reigneau O, Humbert H, et al. New oral formulation of cyclosporin A (Neoral) pharmacokinetics in allogeneic bone marrow transplant recipients. Bone Marrow Transplant
2000; 25: 965.
21. Ishizawa T, Sugawara Y, Ikeda M, et al. Optimal initial dose of orally administered cyclosporine following intravenous cyclosporine therapy. Transplant Proc
2005; 37: 4370.
22. Ku YM, Min DI, Flanigan M. Effect of grapefruit juice on the pharmacokinetics of microemulsion cyclosporine and its metabolite in healthy volunteers: Does the formulation difference matter? J Clin Pharmacol
1998; 38: 959.
23. International Panel. Banff schema for grading liver allograft rejection
: An international consensus document. Hepatology
1997; 25: 658.