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PHARMACOKINETICS AND PHARMACODYNAMICS

A Comparative Study of Sirolimus Tablet Versus Oral Solution for Prophylaxis of Acute Renal Allograft Rejection

Timothy H. Mathew, MBBS, FRACP, Charles Van Buren, MD, Barry D. Kahan, MD, Khalid Butt, MD, Sundaram Hariharan, MD, James J. Zimmerman, PhD for the Rapamune 309 Study Group

From the Queen Elizabeth Hospital, Woodville, Australia (Mr Mathew); the University of Texas School of Medicine, Houston (Dr Van Buren, Dr Kahan); Westchester County Medical Center, Valhalla, New York (Dr Butt); Froedtert Memorial Lutheran Hospital, Milwaukee, Wisconsin (Dr Hariharan); and Clinical Pharmacology, Wyeth Research, Collegeville, Pennsylvania (Dr Zimmerman).

Address for reprints: James J. Zimmerman, PhD, Clinical Pharmacology Department, A3042, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 ACKNOWLEDGEMENTS
 REFERENCES
 
This multicenter, open-label study compared the efficacy, safety, and pharmacokinetic parameters of sirolimus (rapamycin) tablet and liquid formulations for prevention of efficacy failure. A total of 477 renal allograft recipients were randomly assigned (1:1) to receive either tablet or solution formulations of sirolimus for 12 months, plus cyclosporine (CsA) and steroids. Pharmacokinetic parameters were analyzed based on trough concentrations and 24-hour pharmacokinetic profiles. There were no significant differences in efficacy failure at 3 or 12 months between tablet and solution groups. Graft survival, patient survival, rate of first biopsy-confirmed acute rejection, time to and severity of acute rejection, and laboratory parameters were not significantly different between groups. Mean steady-state sirolimus and CsA pharmacokinetic parameters on days 30 and 90 were not significantly different by formulation, except for longer sirolimus tmax after tablet administration. Multivariate logistic regression analysis indicated that low sirolimus Cmin,TN and more human leukocyte antigen mismatches were predictors of acute rejection. The tablet and solution formulations of sirolimus demonstrated therapeutic equivalence.

Key Words: Sirolimus • pharmacokinetics • pharmacodynamics • immunotherapy • efficacy failure

The prevalence of acute rejection in renal allograft recipients has greatly declined since the introduction of cyclosporine (CsA) in 1983.1 Currently, acute rejection rates using double- or triple-combination regimens range from 20% to 30%, and 1-year graft survival in this patient population exceeds 90%.1,2 However, several challenges remain for the transplantation community, including optimizing combination therapy and therapy for high-risk patients and reducing the rate of chronic rejection, which is the primary cause of graft loss in all solid organ transplants.3 Studies have demonstrated that effective prophylaxis of acute rejection may have long-term benefits on graft function and survival.4,5 Sirolimus (rapamycin), a macrocyclic lactone and a natural fermentation product of Streptomyces hygroscopicus, is an immunosuppressant with a unique mechanism of action.6 It exerts potent immunosuppressive and antiproliferative effects by inhibiting the mammalian target of rapamycin (mTOR).7 Inhibition of mTOR blocks several cytokine-mediated signal transduction pathways and suppresses interleukin-2-(IL-2-) and IL-4-driven progression of T cells from the G1 to the S phase of the cell cycle.7 In contrast, the calcineurin inhibitors, CsA and tacrolimus, inhibit cytokine gene expression and block progression of T cells from the G0 to the G1 phase of the cell cycle.8

In a phase II clinical study, sirolimus in combination with either full- or reduced-dose CsA and steroids was found to be superior to CsA and steroids alone in reducing acute rejection rates in renal transplant recipients.9 In 2 large, randomized phase III clinical studies, sirolimus was shown to significantly reduce the rate of acute renal allograft rejection compared with placebo or azathioprine when combined with full-dose CsA and steroids.10,11 These clinical studies used an oral liquid formulation of sirolimus. A tablet formulation has been developed, and comparative bioavailability studies in healthy volunteers showed that the 2 formulations were not strictly bioequivalent.12 However, recent trials of CsA elimination and sirolimus maintenance immunotherapy with the tablet formulation demonstrated excellent rates of patient and graft survival and a significant improvement in renal function in renal transplant patients who had CsA withdrawn.13-16 Therefore, instead of conducting a simple bioequivalence trial in renal transplant patients, the current study was designed to determine the therapeutic equivalence, long-term safety, and pharmacokinetic profiles of the tablet and solution formulations in de novo renal allograft recipients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 APPENDIX
 ACKNOWLEDGEMENTS
 REFERENCES
 
Study Design and Patients
This multicenter, randomized, open-label study was conducted at 30 centers in the United States, Australia, and Canada between November 1997 and December 1999. Enrolled patients were ≥13 years old, weighed ≥40 kg, and received primary or secondary cadaveric, living-unrelated, or living-related (excluding zero-antigen mismatch) donor renal allografts. Patients were randomly assigned before transplantation (1:1) to receive either the tablet or the solution formulation of sirolimus (Rapamune) as a 6-mg initial loading dose and 2-mg daily maintenance doses. Planned treatment duration was 12 months, with 3 months of follow-up. Approval of independent ethics committees and institutional review boards was obtained before the study began, and informed consent was obtained from all patients at the time of enrollment. The study was conducted according to the Declaration of Helsinki and its amendments.

Treatments
Sirolimus tablets (1 mg/tablet) or oral solution (1 mg/mL concentrate, diluted with water or orange juice) were administered at an initial loading dose of 6 mg within 24 to 48 hours after transplantation, followed by a 2-mg/d maintenance dose for 12 months. The rationale for dose selection was based on the safety and efficacy of the 2-mg sirolimus dose in phase II studies. Phase III clinical studies, which were blinded at the time this study was initiated, confirmed the excellent risk/benefit of the 2-mg dose. The use of equivalent doses of the tablet and solution formulations was based on their similar bioavailabilities.12

Sirolimus dose adjustments were permitted for suspected toxicity and after acute rejection. If sirolimus was withheld for >21 consecutive days, patients were discontinued from treatment but continued with follow-up. Concomitant therapy with CsA microemulsion (Neoral; Novartis, Basel, Switzerland) was administered according to standard center practice. Sirolimus maintenance doses were given 4 hours after the morning CsA dose. Steroid therapy was initiated within 24 hours before or after transplantation according to local practice and adjusted to achieve a maintenance dose of 5 to 10 mg/d by the end of month 3. At the investigator's discretion, steroids could be tapered and discontinued at the end of month 6.

Prophylaxis for Pneumocystis carinii pneumonia was required for 12 months in all patients. Patients who received a kidney from a cytomegalovirus (CMV)-positive donor were required to receive prophylaxis for CMV infection with standard therapy for 3 months after transplantation. Countermeasures against oral candidiasis and hyperlipidemia were recommended. Patients were excluded if they received other investigational therapies, immunosuppressive therapies, or planned antilymphocyte antibody therapy beyond that required to treat acute tubular necrosis, delayed graft function, or acute rejection episodes.

Efficacy and Safety Assessments
The primary end point was the rate of efficacy failure, which was defined as a composite of the first occurrence of biopsy-confirmed acute rejection, graft loss (nephrectomy or loss of function requiring >56 days of dialysis), or death in the first 3 months. In addition, safety and efficacy outcomes at 12 months are reported. A stratified analysis of the primary end point (by human leukocyte antigen [HLA] mismatch, donor origin, age, race, gender, and primary vs secondary transplants) was performed to reflect the weighted averages of the strata. Secondary end points, including the incidence of biopsy-confirmed acute rejection, patient survival, graft survival, graft function (ie, serum creatinine and calculated glomeruler filtration rate [GFR; Nankivell method]), the incidence of infection (documented or presumed), and the incidence of histologically confirmed posttransplant lymphoproliferative disease or other malignancies, were assessed at 3, 6, and 12 months after transplantation.

Statistical Analyses
The primary intent-to-treat analysis to assess the equivalence of the tablet and solution formulations of sirolimus included all randomized patients, whether or not they received study medication. Equivalence of the 2 formulations was established by 2-tailed 95% confidence intervals (CIs) around the difference in rates of the primary end point. For stratified analyses of the primary end point, the difference in rates was not the simple arithmetic difference but rather the weighted average of the differences of the strata; the Cochran-Mantel-Haenszel weights were used. To establish equivalence, the CI had to include zero, and the upper boundary of the CI had to be ≤10%, ≤15%, or ≤20%, if the rate of the primary end point was 0% to 10%, >10% to 20%, or >20% to 30%, respectively. Secondary variables were analyzed using the Kaplan-Meier method and log-rank tests to estimate time to event and patient survival and graft survival at 12 months. The 95% CI for the differences in rates was calculated to assess secondary variables such as treated rejection episodes, lymphoproliferative disease, and malignancy. Serum creatinine and calculated GFR were assessed at months 3 and 12 using analysis of variance (ANOVA).

Bioanalysis
Whole-blood samples from the pharmacokinetic subgroups were analyzed for sirolimus concentrations by either Taylor Technology Inc (Princeton, NJ) using a validated high-performance liquid chromatographic tandem mass spectrometry (LC/MS/MS) method17 or by Australian Bioanalytical Services Pty Ltd (Woolloongabba, Australia) using a validated high-performance electrospray liquid chromatographic tandem mass spectrometry (LC-E-MS/MS) method.18 The 2 methods were cross-validated, and both used 32-desmethoxyrapamyin as the internal standard. The LC/MS/MS method had a linear range of 0.1 to 100 ng/mL (1 mL), with quality control (QC) samples showing a bias of –2.7% to +3.1% of the theoretical concentrations; the LC-E-MS/MS method had a linear range of 0.2 to 100 ng/mL (0.5 mL), with a bias of –4.0% to +1.6%. The QCs for both assays showed an imprecision of <10%.

Trough whole-blood samples were analyzed for sirolimus by either Wyeth Research (Collegeville, Penn) or Australian Bioanalytical Services Pty Ltd, using a validated investigational automated microparticle enzyme immunoassay (Imx; Abbott Laboratories, Abbott Park, Ill).19 The 2 sites were cross-validated. The method had a linear range of 3 to 30 ng/mL (0.15 mL). The QC samples at the Pennsylvania site showed an imprecision of <18% and a bias of –8.0% to 1.8%, while the QC samples at the Australia site showed an imprecision <11% and bias of –6.6% to +6.4%.

Whole-blood samples from the pharmacokinetic subgroups were analyzed for CsA concentrations by the Cyclosporine Laboratory at the University of Texas, Houston, using an automated fluorescence polarization method (TDx/FLx; Abbott Laboratories, Abbott Park, Ill). The quantification range was 100 to 1500 ng/mL. The mean bias for the QC samples quantified during the sample analysis was +1.45% for the 150-ng/mL sample, +2.22% for the 400-ng/mL sample, and +0.87% for the 800-ng/mL sample. Whole-blood CsA trough concentrations were determined by individual investigators.

Pharmacokinetic and Pharmacodynamic Analyses
The pharmacokinetic analysis included trough concentrations collected from all patients before dosing at protocol-designated study visit times and complete 24-hour pharmacokinetic profiles on days 1, 30, and 90 from selected patients at some clinical sites. Whole-blood sirolimus and CsA trough concentrations were parameterized as time-normalized (TN) values, which were estimated from the relationship Cmin,TN = AUC0-t/t, where AUC0-t is the area under the trough concentration-time curve to the time of the patient's final blood sample. Sirolimus and CsA pharmacokinetic parameters (Cmax, tmax, C24h, AUC0-12h [CsA only], AUC0-24h [sirolimus only], and CL/F) were estimated by noncompartmental methods.

The statistical analyses for TN trough concentrations and pharmacokinetic parameters were made using ANOVA. Before statistical analysis, sirolimus and CsA trough concentrations were dose normalized using the relationship DN-Cmin,TN = Cmin,TN • DoseTN/NDOSE, where DoseTN = AUD0-t/t and NDOSE = 2 mg for sirolimus and 100 mg for CsA. The parameter AUD0-t is the area under the dose curve up to the final blood sample. The estimates for Cmax, AUC0-12h, and AUC0-24h were also dose normalized before statistical analysis, except for the values of sirolimus Cmax and AUC0-24h on day 1. The latter estimates were not dose normalized because the whole-blood sirolimus concentrations on day 1 did not reflect a large fraction of the loading dose due to extensive tissue distribution. Linear regression analysis was used to determine the relationship between sirolimus AUC0-24h and C24h on study days 30 and 90.

Pharmacodynamic analyses were conducted primarily to assess formulation effects. These evaluations included the use of multiple mixed-effect regression analysis and logistic regression analysis. Multiple linear mixed-effect regression analysis that incorporated repeated-measurement analysis was used to elucidate the relationship between laboratory parameters and sirolimus and CsA Cmin,TN values, while adjusting for other covariates (including formulation). Laboratory parameters obtained during months 2 through 6 after transplantation were used for the analysis, and these consisted of changes from baseline in platelet count, lactate dehydrogenase (LDH), white blood cell (WBC) count, fasting triglycerides, fasting cholesterol, creatine kinase, and aspartate aminotransferase (AST/SGOT), together with actual values of blood urea nitrogen (BUN), hemoglobin, serum creatinine, calculated GFR, and potassium.

Logistic regression analysis was used to analyze the influence of drug concentrations, demography, immunologic characteristics, and formulation on biopsy-confirmed acute rejection up to 105 days after transplantation. Development of the final logistic regression model consisted of sequentially conducting univariate logistic regression and multivariate stepwise logistic regression analysis. During univariate analysis, each independent variable was tested against acute rejection without controlling for the other variables. The independent variables tested by logistic regression analysis included Cmin,TN parameters (sirolimus, CsA), gender (female, male), race (black, nonblack), age (recipient, donor), donor type (cadaveric, living), HLA mismatch, ischemia time, and formulation (solution, tablet). Explanatory variables shown to be significant by the univariate analysis or known to be of biologic importance were further evaluated by stepwise multivariate logistic regression. The results of the multivariate logistic regression analysis gave the intercept, together with the regression coefficient, odds ratio, and associated 95% CI for significant independent variables in the model, corrected for other variables. The criterion for the entry and removal of variables during the stepwise procedure was a P value <.15. The appropriate scale (linear vs nonlinear) was identified for significant continuous variables, and finally, interaction-term testing was conducted on the preliminary final model. The SAS statistical software was used for all statistical analyses.20


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 APPENDIX
 ACKNOWLEDGEMENTS
 REFERENCES
 
Demographics and Baseline Characteristics
A total of 477 patients were enrolled in the study; 239 received the sirolimus tablet formulation, and 238 received the sirolimus oral solution. Of these patients, 457 received at least 1 dose of study medication, 228 in the tablet group and 229 in the solution group. Twenty patients withdrew from the study before receiving the study drug, 11 and 9 from the tablet and solution groups, respectively. The most common reason for withdrawal from the study before receiving study medication was protocol violation. Demographic and baseline criteria were not significantly different between the tablet and solution groups (Table I).


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  		Table I Demographic Characteristics

 

Discontinuations and Protocol Deviations
The numbers of patients who discontinued treatment during the first 12 months after transplantation were not significantly different between the tablet (n = 74, 32%) and solution (n = 76, 33%) treatment groups. Reasons for discontinuation in the tablet and solution groups, respectively, were adverse events (18%, 17%), lack of efficacy (11%, 9%), patient request (2%, 3%), failure to return (<1%, 1%), nonmedical event (<1%, 2%), and protocol violation (<1%, 0%).

Rate of Efficacy Failure
In the efficacy analysis of the intent-to-treat population, the incidence of the primary composite end point (ie, efficacy failure at 3 months) in the tablet treatment group (24.7%) was equivalent to that in the solution group (23.5%; 95% CI = –6.5 to 8.8; Table II). The criteria for equivalence were met because the 95% CI for the difference in rates included zero and the upper boundary of the CI was less than 20%. The criteria for equivalence were also met in the 12-month efficacy analysis, in which the rates of efficacy failure were 31.4% for the tablet group and 30.3% (95% CI = –7.2 to 9.4) for the solution formulation (Table II). None of the stratified analyses showed any statistically significant differences between groups. When stratified by HLA mismatch, the rate of efficacy failure at 3 and 12 months was greater in patients with more than 3 HLA mismatches after administration of both the oral solution and tablet formulations. Equivalence of the tablet and solution formulations was also demonstrated after stratification of the rates of efficacy failure by HLA mismatch (≤3 vs >3), donor origin (cadaver vs living donor), age (≤45 years, >45 years), race (black vs other), gender (female vs male), and transplant type (primary vs nonprimary). The percentage formulation differences (95% CIs) at 3 months for HLA mismatch, donor origin, age, race, gender, and transplant type were 1.4 (–6.2 to9.0), 1(–6.8 to 8.7), 1.5(–6.2 to 9.2), 1(–6.7 to 8.7), 1.2 (–6.5 to 8.9), and 1.1 (–6.6 to 8.8), respectively. Similarly, the formulation differences (95% CIs) at 12 months for HLA mismatch, donor origin, age, race, gender, and transplant type were 1.4 (–6.8 to 9.6), 0.9 (–7.4 to 9.2), 1.4(–7.0 to 9.7), 1(–7.3 to 9.3), 1.2(–7.1 to 9.5), and 1.1 (–7.2 to 9.5), respectively.


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  		Table II Rate of Efficacy Failure and Biopsy-Proven Acute Rejection by Treatment Group at 3 and 12 Months

 

Secondary Efficacy End Points
At 3, 6, and 12 months after transplantation, there were no statistically significant differences in patient survival between the tablet (98.7%, 97.9%, and 96.2%) and solution (97.5%, 97.1%, and 95.8%) treatment groups, respectively. Similarly, there was no significant difference in graft survival between the tablet (91.6%, 90.8%, and 88.7%) and solution (94.5%, 94.1%, and 92.0%) treatment groups at these time points, respectively.

There was no statistically significant difference in the incidence of biopsy-confirmed acute rejection between treatment groups at 3 months and 12 months (Table II) or in the time to first biopsy-confirmed acute rejection during the first 12 months after transplantation (log-rank P = .639). In addition, the distribution of severity of biopsy-confirmed acute rejections was similar between treatment groups (Table II).

Adverse Events
All patients who received at least 1 dose of sirolimus were evaluated for safety and included 228 patients in the tablet group and 229 in the solution group. There were no significant differences in the incidence of clinically important infections between treatment groups. At 12 months, the rates of sepsis (8.4%, 6.3%), generalized CMV (3.3%, 2.1%), tissue-invasive CMV (1.3%, 1.3%), pneumonia (10%, 11.8%), herpes simplex (5.4%, 5.5%), herpes zoster (4.6%, 3.4%), urinary tract infection/pyelonephritis (26.4%, 23.5%), wound infection (12.1%, 15.5%), and Epstein-Barr virus infection (0.4%, 0%) were similar in the tablet and solution groups, respectively.

The incidences of clinically important adverse events at 12 months were similar between treatment groups except for hypertonia (P = .037), which occurred more frequently in the solution group (6.6% vs 2.2% of patients), and abnormal liver function tests (P = .017), which occurred more frequently in the tablet group (12.7% vs 6.1% of patients). At 12 months, there was no significant difference in the incidence of death in the tablet (n = 9, 3.8%) and solution (n = 10, 4.2%) groups. The most common causes of death were infection and cardiovascular events. There was also no significant difference in the incidence of graft loss between the tablet (n = 26, 10.9%) and solution (n = 14, 5.9%) treatment groups at 12 months. The primary etiologies of graft loss were death with a functioning graft (n = 9), acute tubular necrosis (n = 10), acute rejection (n = 7), renal vein thrombosis (n = 3), thrombotic microangiopathy (n = 1), proliferative arteriopathy (n = 1), prolonged hypotension (n = 1), and other (n = 8). There was no significant difference in the incidence of malignancies between the tablet (n = 12) and solution (n = 7) treatment groups. Posttransplant lymphoproliferative disease was reported in 2 patients in the tablet group and 3 patients in the solution group.

Laboratory Values
Mean serum values of creatinine, cholesterol, triglycerides, platelets, and calculated GFR rates at 1, 3, and 12 months after transplantation were not significantly different between groups. The values at 12 months are shown in Table III. When the World Health Organization (WHO) toxicity grades were applied to the creatinine values for each patient, most patients had grade 1 (<250 µmol/L; <2.8 mg/dL) elevations of serum creatinine at 12 months (Table III). The distribution of cholesterol and triglyceride elevations by severity (National Cholesterol Education Program guidelines) were also identical in the tablet and solution treatment groups at 12 months. Cholesterol levels were normal to borderline (≤6.19 mmol/L; ≤240 mg/dL) in 68.4% and 71.4% of patients in the tablet and solution groups, respectively. Similarly, triglycerides were normal to grade 1 (≤4.5 mmol/L; ≤400 mg/dL) in 90.8% and 78.7% of patients in the tablet and solution groups, respectively. Platelet levels were within the reference range (WHO grades of toxicity) in 98.6% of patients in both treatment groups at 12 months.


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  		Table III Renal Function and Lipid Analyses at 12 Months

 

Pharmacokinetics
Sirolimus pharmacokinetics. Mean whole-blood sirolimus trough concentrations reached steady state by approximately day 2 of therapy and remained at plateau levels over 12 months, as shown by a plot of the dose-normalized troughs in Figure 1. A comparison of the mean ± SD average dose-normalized sirolimus trough concentrations (DN-Cmin,TN) over 360 days suggested that DN-Cmin,TN was significantly increased (P = .03) by approximately 1 ng/mL after administration of the tablet formulation (9.74 ± 4.08, n = 210) compared with the solution (8.62 ± 3.96, n = 197). This small increase was not considered to be clinically significant because distribution frequency analysis of sirolimus DN-Cmin,TN for tablet versus solution produced similar values for the 10th (5.1 vs 4.4 ng/mL) and 90th (15.1 vs 14.3 ng/mL) percentiles. There was a large variability in sirolimus trough concentrations; interpatient and intrapatient coefficients of variation (CVs) were 41.9% and 38.8%, respectively, for the tablet and 46.0% and 47.7%, respectively, for the solution formulation.



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  		Figure 1. Whole-blood sirolimus trough concentrations (Cmin,TN) at study visit times during 1 year after transplantation normalized to a 2-mg daily dose.

 
Based on a statistical comparison of whole-blood sirolimus pharmacokinetic parameters for the solution and tablet formulations on days 1, 30, and 90 (Table IV), sirolimus peak concentration (Cmax) for the solution was significantly greater (P = .04) compared with that for the tablet, and Cmax for the solution on day 1 was significantly greater than on days 30 and 90 (P = .001). Furthermore, tmax was significantly lower for the solution (P = .02) among study days compared with the tablet, which suggests that sirolimus was more rapidly absorbed from the solution formulation than from the tablet. There were no statistically significant differences for AUC0-24h and CL/F among study days and formulations, although the intersubject variabilities were large, as reflected by CVs for the whole-blood sirolimus AUC0-24h of tablet versus solution on study days 30 (53.2% vs 26.0%) and 90 (34.6% vs 32.0%). The statistical power for detecting a 20% difference by formulation at an {alpha} level of .05 was low for all parameters except oral-dose clearance, CL/F (power = 0.81).


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  		Table IV Whole-Blood Sirolimus Pharmacokinetic Parameters Among Study Days and Formulations

 

There was a good correlation between sirolimus trough concentrations at 24 hours after dose administration (C24h) and total exposure (AUC0-24h) on study days 30 and 90 for both formulations, as shown in Figure 2. The regression equations for the solid lines in Figure 2 are represented by the linear regression equations Cmin,24h = 0.0270 x AUC0-24h + 0.265 (r2 = 0.854) for solution and Cmin,24h = 0.0348 x AUC0-24h – 0.540 (r2 = 0.840) for tablet. These results provide confidence that sirolimus trough concentrations can be used as a surrogate for sirolimus AUC0-24h measurements.



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  		Figure 2. The linear relationship between steady-state sirolimus area under the curve during 24 hours (AUC0-24h) and concentration at 24 hours (C24h) after administration of oral solution or tablets on combined days 30 and 90. Individual data, circles; linear regression, solid line; 95% prediction interval, dashed line.

 

CsA pharmacokinetics. Consistent with the standard practice of tapering CsA doses after renal transplantation, there was a parallel decline in CsA doses and trough concentrations over time after transplantation. Over the time intervals of 2 to 31 days, 32 to 91 days, and 92 to 390 days, CsA Cmin,TN showed mean ± SD values of 324 ± 139 ng/mL, 296 ± 126 ng/mL, and 216 ± 85 ng/mL, respectively, for the solution formulation. The corresponding mean ± SD CsA DoseTN values for the sirolimus solution formulation during the intervals of 2 to 31 days, 32 to 91 days, and 92 to 390 days were 509 ± 158 mg/d, 407 ± 167 mg/d, and 317 ± 138 mg/d, respectively. As expected, the CsA Cmin,TN values were significantly different among time intervals over 390 days (P = .001), but the CsA Cmin,TN for the sirolimus solution and tablet were not significantly different (P = .38). Dose-normalized CsA trough concentrations reached a plateau by day 30 for both tablet and solution formulations (Figure 3), suggesting that CsA pharmacokinetics was not affected by the sirolimus formulation. The interpatient/intrapatient %CVs for whole-blood CsA trough concentrations over 92 to 390 days with the sirolimus tablet and solution were 45%/35% and 39%/34%, respectively.



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  		Figure 3. Whole-blood CsA trough concentrations (Cmin,TN) at study visit times during 1 year after transplantation normalized to a 100-mg daily dose.

 
Based on a statistical comparison of whole-blood CsA pharmacokinetic parameters on days 1, 30, and 90 (Table V), significantly lower values of Cmax (P = .001) and AUC0-12 (P = .001) were observed on day 1 compared with days 30 and 90, while tmax was significantly greater (P = .008) on day 1 than on days 30 and 90. There were no significant differences between the sirolimus tablet and solution formulations in any of the CsA pharmacokinetic parameters. The intersubject variabilities were large, however, as reflected by CVs of the whole-blood CsA AUC0-12h values for the sirolimus tablet versus solution treatments on study days 30 (33.5% vs 40.0%) and 90 (59.9% vs 47.9%); the statistical power for detecting a 20% difference by formulation at an {alpha} level of .05 was <80% for all parameters.


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  		Table V Whole-Blood Cyclosporine Pharmacokinetic Parameters Among Study Days and Formulations

 

Pharmacodynamic Correlations
Linear mixed-effect regression analysis was conducted with a total of 8839 observations during the 2 to 6 months after transplantation among the following laboratory parameters: WBC (n = 928), platelets (n = 918), hemoglobin (n = 927), fasting cholesterol (n = 369), fasting triglycerides (n = 358), AST (n = 886), LDH (n = 737), potassium (n = 943), serum creatinine (n = 931), BUN (n = 918), and GFR (n = 924). Patients with missing values for independent variables were not included in the analysis. There were no statistically significant correlations between sirolimus formulations and any of the individual laboratory parameters (P values for final models ranged from 0.068 to 0.984). However, sirolimus and CsA drug concentrations showed significant correlations with laboratory parameters. Increasing sirolimus Cmin,TN values were correlated with increasing values for LDH (P = .001), AST (P = .007), and BUN (P = .005) and decreasing values for platelet count (P = .001) and GFR (P = .002). Similarly, increasing CsA Cmin,TN values were significantly correlated with increasing values for fasting triglycerides (P = .006), fasting cholesterol (P = .002), BUN (P = .037), and hemoglobin (P = .022). With respect to time effects during the 2 to 6 months after transplantation, mixed-effect regression analysis showed statistically significant increases for platelet counts (P = .003), AST (P = .001), and hemoglobin (P = .001), together with statistically significant decreases in LDH (P = .001), WBC (P = .014), fasting cholesterol (P = .001), and GFR (P = .001).

Logistic regression analysis was conducted to determine the effect of drug concentrations and other covariates on the probability of acute rejection. A preliminary statistical analysis of the data showed that sirolimus Cmin,TN was significantly higher (P = .009) by a t test comparison in nonrejectors (8.96 ± 4.51 ng/mL, n = 284) than in rejectors (7.24 ± 4.71 ng/mL, n = 59). Within formulation groups, sirolimus Cmin,TN were marginally significantly higher in nonrejectors compared with rejectors for both the solution (P = .055) and tablet (P = .065) formulations. Mean CsA trough concentrations also tended to be higher in nonrejectors (293 ± 93 ng/mL, n = 354) compared with rejectors (275 ± 179, n = 79), but these differences were not statistically significant (P = .382). The numbers of HLA mismatches were significantly lower (P = .005) in nonrejectors (3.44 ± 1.69, n = 362) compared with rejectors (3.94 ± 1.42, n = 87).

Univariate logistic regression analyses mirrored the results from the preliminary statistical analysis, and P values of .009, .197, and .012 were obtained for sirolimus Cmin,TN, CsA Cmin,TN, and HLA mismatch, respectively. There were no significant P values for demographic, immunologic (other than HLA mismatch), and formulation (P = .808) variables. The outcome suggests that only sirolimus Cmin,TN and HLA mismatch would be useful predictors of acute rejection. The lack of statistical significance for CsA Cmin,TN values does not diminish the importance of CsA in triple immunosuppressive therapy. The outcomes of logistic regression analyses can be affected by the size of the population and variabilities in the inter-/intrasubject trough concentrations. Since CsA increases sirolimus bioavailability17 and provides a synergistic immunosuppressive effect in combination with sirolimus,21 it was decided that the 2 drugs would be forced into the follow-up stepwise regression model.

The initial multivariate logistic regression analysis gave P values of .0344, .722, and .043 for sirolimus Cmin,TN, CsA Cmin,TN, and HLA mismatch, respectively. Testing for scale linearity showed significant P values by the Box-Tidwell transformation22 for both sirolimus (P = .031) and CsA (P = .027) Cmin,TN values, which indicated a nonlinearity in the scale and that the 2 drugs should be dichotomized in the final model. The upper limit of the first quartiles for sirolimus Cmin,TN (5.62 ng/mL) and CsA Cmin,TN (240.84) was used for dichotomization. No significant interactions between the variables were found in this multivariate analysis.

The final multivariate logistic regression model (339 patients; nonrejectors = 284, rejectors = 55) based on dichotomized drug concentrations gave P values of .053, .369, and .049 for sirolimus Cmin,TN, CsA Cmin,TN, and HLA mismatch, respectively. The corresponding odds ratios (95% CI) associated with sirolimus Cmin,TN, CsA Cmin,TN, and HLA mismatch were 1.867 (0.981-3.488), 1.345 (0.692-2.535), and 1.208 (1.005-1.467), respectively.

The effect of sirolimus as a categorized variable was only marginally significant (P = .053) after adjusting for HLA mismatch, indicating that patients having average sirolimus concentrations less than 5.63 ng/mL tended to be at increased risk of acute rejection. Dichotomized CsA average trough had no significant effect on the probability of acute rejection (P = .369). However, the effect of HLA mismatches on the frequency of acute rejections was significant (P = .049), indicating that the odds of an acute rejection were 1.2 times higher (6 to 5) for each increase in 1 HLA mismatch.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
APPENDIX
 ACKNOWLEDGEMENTS
 REFERENCES
 
The current study demonstrates the therapeutic equivalence of the tablet and oral solution formulations of sirolimus for prophylaxis of acute renal allograft rejection. Equivalence was demonstrated for the composite end point of efficacy failure. Furthermore, there were no significant differences in patient and graft survival between the treatment arms. There were no clinically significant differences between the 2 formulations with respect to the incidence of treatment-emergent adverse events, clinically important infections, malignancies, or death. In addition, there were no significant differences in average pharmacokinetic parameters by formulation except for tmax, in which sirolimus was more slowly absorbed from the tablet formulation than from the solution.

In 2 large, well-controlled, pivotal US and global trials, sirolimus oral solution (2 mg/d) significantly reduced the rate of efficacy failure when compared with placebo or azathioprine, respectively.10,11 At 6 months, the efficacy failure rate in the global study (30%) was similar to the rates in the current study (27.2% with the tablet, 26.1% with the solution), although these rates were considerably higher than that reported for the 2 mg/d group in the US study (18.7%). The lower rate in the US study was most likely due to a selection bias introduced by posttransplant randomization because the acute rejection rate in the azathioprine-treated patients was also lower than is typically reported with this agent.

Acute rejection has been associated with an increased risk of chronic rejection,4-6 and therefore, immunosuppressive agents such as sirolimus that provide enhanced prophylaxis against acute rejection compared with existing therapies might be expected to have a positive effect on long-term graft survival. Recent studies of CsA withdrawal in sirolimus-treated patients indicate a positive influence on graft survival.15 In addition, the negative effects of rejections tend to be minimized by the fact that they are predominantly mild in severity in sirolimus-treated patients,23,24 as in the present study.

The safety profiles for the tablet and oral solution formulations were similar in this study and paralleled those seen for sirolimus-treated patients in large pivotal trials.10,11 The most frequently reported treatment-emergent adverse events were anemia, reduced platelet counts, and elevations of fasting cholesterol and triglyceride levels. Although modest elevations of cholesterol and triglycerides have been associated with sirolimus treatment, these elevations are readily manageable with conventional statin and fibrate therapy and do not significantly increase cardiac risk in this patient population.23 Kidney function, as assessed by serum creatinine levels and GFR, was modestly impaired, as has been seen previously in patients treated with sirolimus, CsA, and steroids. This decreased function is attributable to an enhancement of CsA nephrotoxicity by sirolimus. Animal studies24-26 and clinical trials27-29 using sirolimus as base therapy have shown that sirolimus is not inherently nephrotoxic. In fact, a recent study of renal transplant patients who had CsA withdrawn 3 months after transplantation showed significantly better renal function through 36 months compared with those who remained on a sirolimus plus CsA treatment regimen.15

Analysis by linear mixed-effect regression and logistic regression showed that there were no statistically significant differences between the solution and tablet formulations with respect to effects on either clinical laboratory parameters (P = .068-.984) during months 2 through 6 after transplantation or on the probability of acute rejection (P = .808) during 105 days after transplantation.

Linear mixed-effect regression showed that increasing sirolimus Cmin,TN values were correlated with increasing values of LDH (P = .001), AST (P = .007), and BUN (P = .005) and decreasing values of platelet count (P = .001) and GFR (P = .002). Similarly, increasing CsA Cmin,TN values were significantly correlated with increasing values for fasting triglycerides (P = .006), fasting cholesterol (P = .002), BUN (P = .037), and hemoglobin (P = .022). These effects were not unexpected and have been observed previously.

Multivariate stepwise logistic regression showed that the probability of acute rejection was significantly correlated with HLA mismatch (P = .049), but there was only a marginal correlation for sirolimus time-normalized troughs (Cmin,TN; P = .053) and no correlation for CsA Cmin,TN (P = .369). The model predicted that patients with sirolimus Cmin,TN <5.6 ng/mL (odds ratio = 1.867) would be expected to have a higher probability of acute rejections and that the probability of an acute rejection would be 1.2 times higher for each additional HLA mismatch (odds ratio = 1.208).

In conclusion, this study demonstrated that the tablet and oral solution formulations of sirolimus are therapeutically equivalent. Efficacy failure rates and safety profiles for the 2 formulations were similar to those obtained in other phase III studies. Patient survival and graft survival were excellent. The tablet formulation provides the added benefits of stability at room temperature and better palatability for the patient.


   APPENDIX
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
ACKNOWLEDGEMENTS
 REFERENCES
 
Other Members of the Rapamune 309 Study Group
J. Andrew Bertolatus, University of Iowa Hospitals, Iowa City; Jeremy Chapman, Westmead Hospital, Sydney, Australia; David Conti, Albany Medical College, Albany, New York; Pierre Daloze, Notre Dame Hospital, Montreal, Quebec, Canada; Gabriel Danovitch, UCLA School of Medicine, Los Angeles, California; John Dunn, University of California at San Diego Medical Center; Josette Eris, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia; Ralph Fairchild, Tufts University School of Medicine, Boston, Massachusetts; Robert Fisher, Medical College of Virginia Hospital, Richmond, Virginia; Paul Gores, Carolinas Medical Center, Charlotte, North Carolina; Bruce Julian, University of Alabama at Birmingham; Richard Knight, Mount Sinai Medical Center, New York, New York; Marc Lorber, Yale University School of Medicine, New Haven, Connecticut; Robert Mendez, National Institute of Transplantation, Los Angeles, California; Laura Mulloy, Medical College of Georgia, Augusta; John Neylan, Emory University Hospital, Atlanta, Georgia; Mark Pescovitz, Indiana University Medical Center, Indianapolis; Bruce Pussell, Prince Henry Hospital, Little Bay, New South Wales, Australia; P. Rajagopalan, Medical University of South Carolina, Charleston; Russell Rigby, Princess Alexandra Hospital, Wooloongabba, Australia; John Scandling, Stanford University Medical Center, Palo Alto, California; Ahmed Shoker, Royal University Hospital, Saskatoon, Saskatchewan, Canada; Steven Steinberg, Sharp Memorial Hospital, San Diego, California; Rodney Taylor, University of Nebraska Medical Center, Omaha; Rowan Walker, Royal Melbourne Hospital, Parkville, Australia.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 ACKNOWLEDGEMENTS
REFERENCES
 
Supported by a grant from Wyeth Research, Collegeville, Pennsylvania. Presented in part at the annual meeting of the American Society for Transplantation, May 2000, Chicago, Illinois. The authors wish to acknowledge the assistance of Quintiles, Inc, North Carolina, in conducting the statistical analyses of pharmacodynamic data. The authors gratefully acknowledge Bernadette Maida and Joseph Scarola of Wyeth Research, Collegeville, Pennsylvania, for their contributions to the design, conduct, and analysis of the study, and Susan A. Nastasee, Wyeth Research, Collegeville, Pennsylvania, for technical assistance in preparation of the article. All authors have received research funding from Wyeth Research, Collegeville, Pennsylvania. Timothy H. Mathew, Charles Van Buren, Barry D. Kahan, Khalid Butt, and Sundaram Hariharan were investigators for this study. James J. Zimmerman is an employee of Wyeth Research, Collegeville, Pennsylvania. The members of the Rapamune 309 Study Group are listed in the appendix.

DOI: 10.1177/0091270005282628


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 ACKNOWLEDGEMENTS
 REFERENCES
 

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