Bioequivalence of Two Microemulsive Preparations of Cyclosporine in Renal Transplant Recipients with Stable Graft Functions

K Sud, B Singh*, R Pandey, HS Kohli, V Jha, KL Gupta, V Sakhuja.

Departments of Nephrology and Nuclear Medicine*, Postgraduate Institute of Medical Education & Research, Chandigarh.

Abstract

Six months after renal transplantation, transplant recipients with stable graft function in the preceding 4 weeks and at feast were studied prospectively in an unblended, cross over study design to compare bioavailability of cyclosporine A (CyA) using a reference formulation* and a test formulation**. In period I, patients entered the study on a stable individualised dosage regimen of the reference formulation. In period II, patients were switched on to the test formulation in a 1:1 dosage conversion- The CyA area under the curve (AUC) was estimated at weekly intervals for 2 weeks before and after the 1:1 dosage switch. For calculation of AUC, whole blood CyA levels were taken at 0,1,2,4,6 and 8 hours after the morning dose and analysed using a radioimmunoassay which uses mouse monoclonal antibodies specific against the parent CyA molecule. Serum creatinine was checked twice a week for 2 weeks before and after the conversion. Eleven patients, all males, with a mean age of 34.65±10.9 years and receiving CyA in a dose of 3.83±0.50 mg/kg/day were included. There were no changes in Cmm (156.9±210.3 ng/mf vs 136.9±93.5, p=0.36), Cmax (658.8±337.0 ng/ml vs 617.7±407.5 ng/ml, p=0.30), Tmax (2.6±0.94 hours vs 3.05±1.49 hours, p=0.22), AUC (2618.1±1313.7 ng.hr/ml vs 2474.6±1500.1 ng.hr/ml, p=0.28) and CyA clearance (35.59±23.29 ml/min/kg vs 32.69±26.94 ml/min/kg, p=0.26) while on the reference formulation and the test formulation respectively. The relative bioavailability of the test formulation was 94.5%. There was no change in serum creatinine before (1.36±0.37 mg/dl) and after conversion to the test formulation (1.32±0,43 mg/dl, p=0.25). There were no episodes of graft dysfunction during and 4 weeks after conversion to the test formulation. We conclude that the two microemulsive formulations are bioequivalent and conversion to the cheaper test formulation 6 months after transplantation is not associated with any adverse short term graft outcomes.

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Introduction

Cyclosporine A (CyA) is a highly lipophilic cyclic polypeptide with high molecular weight, which results in its extremely poor absorption profile in humans. Structural, chemical, pharmacokinetic and pharmacodynamic properties impede achievement of predictable bioavailability, resulting in a high degree of inter and intra patient variability in pharmacokinetic parameters. Its initial formulations required presence of bile for its absorption from the small intestine^¹. Bile results in emulsification of the drug and fulfils the prerequisites for its absorption. This realisation lead to the development of a CyA formulation in a microemulsion form, which would immediately become available for absorption. Such a formulation has demonstrated higher oral bioavailability and consistent absorption during chronic oral dosing.^² Microemulsive CyA soon became the formulation of choice as it reduced intra individual variability in CyA bioavailability³ and improved graft acceptance in transplanted patients.^4,5

In recent years a number of cheaper brands of CyA in microemulsion form have become available6'9 Substitutions between these products for economic or other reasons without close therapeutic monitoring have the potential to induce undesirable toxic effects. There are also doubts amongst transplant physicians about the bioequivalence of these different formulations,

In the current study, bioequivalence and tolerability at steady state of two microemulsion preparations was examined in stable renal allograft recipients at least 6 months after transplantation. The test CyA, is an optically clear and thermodynamically stable formulation in which polar part of the drug is extended into a hydrophilic solvent and non-polar part is extended into lipophilic solvents.

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Materials And Methods

Patients and Study design

Eleven male renal transplant recipients with stable graft function defined as <20% change in the serum creatinine value in the preceding 4 study weeks and having completed at least 6 months after transplantation were studied prospectively in an un-blinded cross over design to compare bioavailability of CyA using two marketed preparations. Patients initially stabilised on the reference formulation* in two equally divided doses were enrolled. They received the reference formulation and the test formulation** following one another after 1:1 switch. Patients with acute or chronic liver disease, steatorrhoea, uncontrolled diabetes metlitus and lactating females were excluded. In addition, patients having received any drug known to potentiate the nephrotoxicity of CyA in the previous 2 weeks or patients with planned co-administration of any other drug known to interfere in CyA pharmacokinetics were excluded. Serum creatinine was checked twice a week for 2 weeks before and after the conversion. Patients exhibiting a rise in serum creatinine >20% of the baseline values were required to undergo a graft biopsy to look for evidence of rejection/CyA toxicity. Those patients with an episode of rejection or CyA toxicity and not responding to dosage reduction were to be excluded from the trial and CyA resumed in the same dosage and formulation which they were receiving before entry into the trial.

Blood Sampling and Assay

For calculation of area under the curve (AUC), whole blood CyA levels while on the reference formulation and after a 1:1 dosage shift over to he test formulation were taken at 0 hours (before next dose and 12 hours after the previous dose), 1,2,4,6 and 8 hours after the morning dose. All blood samples for CyA estimation were refrigerated at 2-8°C until such time that these were analysed. Whole blood CyA levels were analysed using Cyclo-Trac SP radioimmunoassay kit (© Incstar Corporation, USA) which uses mouse monoclonal antibodies specific against the parent CyA molecule. Each sample was tested twice and mean of two values was taken as the CyA level.

Pharmacokinetic and Statistical Analysis

CyA AUC was calculated from CyA values obtained at 0 to 8 hours using the linear trapezoidal rule on Microcal Origin computer software. Blood CyA concentration at 0 hours (trough) is the CyA concentration before the next dose and is assumed to be equal to the blood CyA concentration at 12 hours (Cmin). Cmax was the maximum blood concentration of CyA and Tmax was the time at which Cmax occurred. CyA clearance was calculated using the equation:

CyA clearance (ml/min/kg)

= CyA dose (ng) x 1440 (min)
   Cmin (ng/ml) x Ideal body weight (kg)

*All values expressed as means±2SD

Statistical Analysis

Data is expressed as mean+2SD. Bioavailability parameters of AUC, Cmax, Tmax, Cmin and CyA Clearance were compared for both formulations. Paired students t-test was used to compare differences in means and p values less than 0.05 were considered to be statistically significant. Blood concentration vs time profile was dose normalised to 300mg and presented graphically on linear to linear scale using mean collective values of all patients during periods I and II.

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Results

Eleven patients, all males, with a mean age of 34.65±10.9 years and receiving CyA in a dose of 240.91±30.15mg of CyA per day (3.83+0.50 mg/kg/day) were studied. Pharmacokinetic performance of the test and reference formulations during periods I and II are presented in Table 1. There were no statistically significant differences in Cmin (156.9+210.3 ng/ml vs 136.9±93.5, p=0.36), Cmax (658.8±337.0 ng/ml vs 617.7±407.5 ng/ml, p=0.30), Tmax (2.6±0.94 hours vs 3.05±1.49 hours, p=0.22), AUC (2618.1±1313.7 ng.hr/ml vs 2474.6±1500.1 ng.hr/ml, p=0.28) and CyA clearance (35.59±23.29 ml/min/kg vs 32.69±26.94 ml/min/kg, p= 0.26) while on the reference formulation and the test formulation respectively. Mean whole blood CyA concentration dose normalised to 300mg/day in the 11 patients, period wise distributed on both products, are shown in Figure 1. The relative bioavailability of the test formulation was 94.5%.

There was no change in serum creatinine values before (1.36+0.37 mg/dl) and after conversion to the test formulation (1.32+0.43 mg/dl, p=0.25). There were no episodes of graft dysfunction during and 4 weeks after the conversion to the test formulation. The conversion between the two formulations 6 months after transplantation was not associated with any adverse short-term graft outcomes.

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Discussion

In study period I, patients entered the study on a stable individualised dosage regimen of the reference formulation. In period II, patients were switched on to the test formulation to compare the rate and extent of absorption of CyA at steady state concentrations and patients were dosed at a dosage strength which was identical to the period when patients were taking the reference formulation. No dose titrations were performed in either phases of the study. After the product conversion there were no episodes of graft dysfunction and no adverse short-term graft outcomes were noticed. This indicates the good safely and efficacy profile of the test formulation as compared to the reference formulation. No statistically significant difference was detected in the serum creatinine values in either period at similar dosage strengths. Identical Cmax and Tmax values indicate that after oral consumption of the test and reference formulations, the drug is absorbed equally well from the gastrointestinal tract.

The Cmax and Cmin observed in both periods were similar. There was no clinical or biochemical evidence of CyA toxicity or acute rejection during the study period and for at least 4 weeks after conversion to the test formulation. The relative bioavailability of the test formulation was 94.5% and therefore, both products are bioequivalent in stable renal transplant patients. Our study has indicated that maintaining the same CyA dose when changing between these formulations yields comparable steady state concentrations. Concomitantly, none of the bioavailability parameters changed statistically which indicates that absorption related differences between the products do not exist.

The study is however on a small number of patients. A larger cohort may be able to bring out any minor differences that might exist in between the two formulations that might influence bioavailability parameters. Moreover, a longer period of observation on each formulation would be necessary to conclude that there is no change in the number of acute rejection episodes when using either of the formulations. Because the initial post transplant period is the period associated with the greatest risk of rejections and fluctuations in CyA blood concentrations, studies utilising the test formulation in the initial post transplant period are warranted to conclude equivalent clinical efficacy of the test formulation.^10,11 Johnston et al have suggested that simple bioequivalence criteria may not relevant to critical dose drugs such as CyA. They have suggested that after documenting bioequivalence in healthy volunteers (as has already been done for the test formulation), 6 therapeutic bioequivalence should be documented in a small number of intensely monitored stable patients (present study), followed by larger multicenter trials in stable renal transplant recipients and in de novo transplant patients who are associated with changing CyA kinetics and require higher CyA doses.¹⁰

We conclude that the 12 hourly dosage schedule of the reference formulation works adequately for the test formulation which has similar bioavailability in stable renal transplant recipients at least 6 months post transplant. Utilisation of the 1:1 conversion between the two formulations yields comparable steady state concentrations. Further studies on larger number of patients and on de novo transplant recipients will be required to establish therapeutic bioequivalence.

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References

1. Shaw LM, Bowers L, Demers L, et al. Critical issues in cyclosporine monitoring: report of the task force on cyclosporine monitoring. Clinical Chemistry 1987; 33:1269-1288.

2. Sud K, Maitra S, Singh B, Kohli HS, Jha V, Gupta KL, Pathak CM, Sakhuja V. Microemulsive preparation of Cyclosporine achieves higher Cyclosporine trough levels. Indian Journal of Nephrology 1996; 6: 146-149.

3. Sud K, Maitra S, Singh B, Kohli HS, Jha V, Gupta KL, Sakhuja V. Reduced Intra-individual Variability in Cyclosporine Levels in Renal Transplant Recipients Treated with Microemulsion Formulation. Indian Journal of Nephrology 1997; 7: 147-150.

4. Holt DW, Mueller EA, Kovarik JM, Van Bree JB, Kutz K. The pharmacokinetics of Sandimmun Neoral (Novartis): a new oral formulation of cyclosporine. Transplantation Proceedings 1994; 26: 2935-2939.

5. Holt DW, Mueller EA, Kovarik JM, Van Bree JB, Kutz K. Sandimmun Neoral (Novartis): pharmacokinetics: impact of a new oral formulation. Transplantation Proceedings 1994; 27: 17997-18001.

6. Gulati R, Sharma S, Gupta U. Pharmacokinetics of cyclosporine from conventional and new microemulsion formulations in healthy volunteers. Journal of the Association of Physicians of India 1998; 46: 860-863.

7. Lee YJ, Chung SJ, Shim CK. Bioequivalence of Neoplanta Capsules to Sandimmun Neoral, microemulsion formulation of cyclosporine A in human subjects. International Journal of Clinical Pharmacology and Therapeutics 1998; 36: 210-215.

8. Park MS, Yu NC, Lee HK, Kirn YS, Kirn KH. Bioequivalence study of CIPOL - N (Cyclosporine microemulsion preparation) in healthy subjects. Proceedings of the 5th congress of the Asian Society of Transplantation, December 4-7, 1997, Manila, Philippines.

9. First MR, Weiskittel D, Burton M, et al. Conversion of the stable renal transplant recipients from Sandimmun to Sang-35, a novel cyclosporine formulation, using a dose normalised equivalence method. Transplantation Proceedings 1998; 30:1701-1705.

10. Johnston A, Keown PA, Holt DW. Simple Bioequivalence Criteria: Are they relevant to critical dose drugs? Experience gained from cyclosporine. Ther Drug Monit 1997;19:375-381.

11. Mueller EA. Recommendation of bioequivalence standards for the development of new oral formulation of cyclosporine A to avoid adverse clinical implications. Eur J Clin Res 1995 ; 7 : 79-85.

Address for Correspondence

Dr. V. Sakhuja, Professor & Head, Department of Nephrology, Postgraduate Institute of Medical Education & Research, Chandigarh-160012

* Sandimmun Neoral © Novartis, Basel, Switzerland
** Fanimun Bioral © Panacea Biotec, India

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