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This study was performed to investigate the bioequivalence of cyclosporine between a generic test product [Sigmasporin Microoral, 100 mg cyclosporine per 1 mL solution; Gulf Pharmaceutical Industries (Julphar), U.A.E.] and reference product (Sandimmun Neoral, 100 mg cyclosporine per 1 mL solution; Novartis Pharma, Switzerland). The clinical protocol called for 30 healthy volunteers, at least. The subjects received 1 mL of each product, Sigmasporin Microoral solution and Sandimmun Neoral solution (100 mg cyclosporine per 1 mL), in a randomized fashion with a washout period of seven days. Thirty healthy male volunteers completed the crossover..

The bioanalysis of clinical plasma samples was accomplished by the in-house LC-MS method, which was developed and validated in accordance with the international guidelines. The LLOQ for cyclosporine A was 5 ng/mL. Samples collection, handling, transfer, storage, and

Study title

IPRC study code Objective

Protocol/design

Phases of study

Study medications

Dosage regimen

Tolerance Drug bioanalysis Pharmacokinetics analysis were all conducted according to the international GLPs under the supervision of IPRC's QAU.

Pharmacokinetic parameters, determined by standard noncompartmental methods, and ANOVA statistics were calculated using Kinetica 2000 statistical software.

Regarding the pharmacokinetics, the extent of absorption was determined by AUC0 _, t and AUC0 _, m, while the rate of absorption was assessed from and imax. The adequacy of sampling time was judged from the ratio (AUCo-, t/AUCo -, <» )%. The elimination half-life was calculated (t(1/2)e), in addition to the elimination rate constant (Ke), which were invested for further characterization of the pharmacokinetics outcome of this study. ANOVA for both the untrans-formed pharmacokinetics t(1/2)e, Ke, Cmax/ imax, AUCo ->■ t, and AUCo ->■ », and the log-transformed Cmax, AUCo ->■ t, and AUCo ->■ » was executed. The variance model included sequence, subjects nested in sequence, period, and product as factors, employing 5% level of significance. The significance of a sequence effect was tested using the subjects nested in sequence as the error term.

The ANOVA analysis showed no statistically significant differences between the two products with respect to the calculated pharmacokinetic parameters t(1/2)e, Ke, Cmax, imax, AUCo->■ t, and AUCo->■ ». Results of ANOVA are summarized in Table C.10 of Appendix C. The geometric mean for the ratios expressed as a percentage (test product/reference product) are given in Tables C.7-C.9 (see Appendix C). Consistent with the two one-sided tests for bioequivalence, 90% confidence intervals for the ratios of means was calculated for the untransformed and the log-transformed Cmax, AUCo ->■ t, and AUCo ->■ ». The values obtained indicated that the 90% confidence limits for all geometric means are within the recommended criteria to conclude bioequivalence (80125% for Cmax, AUC0-1 and AUC0^ » ) (Figs A8-A10).

In conclusion, the study demonstrated that the test product, Sigmasporin Microoral Solution [Gulf Pharmaceutical Industries (Julphar), U.A.E.], 100 mg cyclosporine per 1 mL, is bioequivalent to the reference product, Sandimmun Neoral Solution (Novartis Pharma, Switzerland), 100 mg cyclosporine per 1 mL (Tables A.1 and A.2).

A.9.1. Introduction

A.9.1.1. Chemistry

Cyclosporine is an oral and parenteral immunosuppressive agent. Cyclosporine is a cyclic polypeptide consisting of 11 amino acids, and it is produced by the fungus Beauveria nivea. It has the following structural formula: Cyclosporine A

D-Ala-MeLeu-MeLeu-MeVal-N-C-CO-Abu-MeGly-MeLeu-Val Ala H MeLeu

A.9.1.2. Pharmacology

Cyclosporine induces immunosuppression by inhibiting the first phase of T-cell activation. The first phase of T-cell activation causes transcriptional activation of immediate and early gene products (e.g., interleukins—IL-2, IL-3, and IL-4, tumor necrosis factor alpha, and interferon gamma) that allow T cells to progress from the Go to Gi phases. Cyclosporine binds to an immunophilin termed cyclophilin. Immunophilins (e.g., cyclophilin and FK-binding proteins) are immunosuppressant-binding proteins that are distributed in all cellular compartments and play an important role in protein regulation. The cyclosporine-cyclophilin complex then binds to and inhibits the calcium-calmodulin-activated phosphatase calcineurin. The calcineurin enzyme catalyzes critical depho-sphorylation reactions necessary for early lymphokine gene transcription, and subsequent early activation of T cells. Calcineurin inhibition results in the blockade of signal transduction of the nuclear factor of activated T cells (NF-AT). The blockade of signal transduction results in failure to activate NF-AT-regulated genes. NF-AT-activated genes include those required for B-cell activation including IL-4 and CD40 ligand and those required for T-cell activation including IL-2 and interferon gamma. Cyclosporine does not affect suppressor T cells or T-cell-independent, antibody-mediated immunity.

A.9.1.3. Pharmacokinetics

Cyclosporine is administered orally or intravenously. Cyclosporine is extremely hydrophobic. Because of the unpredictability of oral absorption, it is difficult to convert between oral and parenteral doses. Most clinicians use a 3:1 ratio when converting between oral and parenteral routes (e.g., 30 mg IV is roughly equivalent to 90 mg orally). First-pass metabolism, mode of administration, formulation, and drug interactions all affect cyclosporine absorption.

Cyclosporine is a substrate and inhibitor of P-glycoprotein, which is an energy-dependent drug-efflux pump located in intestinal epithelium and the blood-brain barrier. There appears to be overlap between inhibitors and/or substrates of cytochrome (CYP) P450 3A4 and P-glycoprotein. The P-glycoprotein efflux of cyclosporine from intestinal cells back into the gut lumen allows for CYP3A4 metabolism prior to absorption, thus limiting cyclosporine availability. When cyclosporine is administered with inhibitors of both CYP3A4 and P-glycoprotein (e.g., diltiazem, erythromycin, or ketoconazole) increased cyclosporine bioavailability leads to increased cyclosporine concentrations.

Oral absorption of cyclosporine, USP (Modified): The physical properties of the cyclosporine (modified) formulation (i.e., microemulsion) make the absorption of cyclosporine less dependent on bile, food, and other factors that assist dispersion and subsequent absorption of lipophillic substances from the gastrointestinal (GI) tract. Although, agents which influence pre-systemic metabolism (e.g., grapefruit juice) may still influence cyclosporine (modified) absorption. The absolute bioavailability of cyclosporine (modified) has not been determined in adults. Following oral administration, the tmax for cyclosporine (modified) ranges from 1.5 to 2 hours. Food decreases the absorption of cyclosporine (modified). The AUC of cyclosporine (modified) is linear within the therapeutic dosage range. Intersubject variability of cyclosporine exposure (AUC) ranges from about 20% to 50% when administered as cyclosporine (modified). There is less intrasubject variation in AUC with cyclosporine (modified), despite random changes in food intake, bile secretion, or time of trough concentration measurement. Intrasubject variability of AUC in renal transplant patients is 9% to 21% for cyclosporine (modified). In these same studies, the intrasubject variation in trough concentrations was similar for the two formulations.

Cyclosporine is distributed widely throughout the body, crosses the placenta, and is found in breast milk. Preferential uptake of cyclosporine occurs in the liver, pancreas, and adipose tissue, while it penetrates the CNS poorly. In blood, the distribution of cyclosporine is concentration dependent; as the hemocrit rises, the cyclosporine concentration in plasma decreases. Approximately, 22% to 47% of cyclosporine is found in plasma, 4% to 9% in lymphocytes, 5% to 12% in granulocytes, and 41% to 58% in erythrocytes. At high drug concentrations, the binding to lymphocytes and erythrocytes becomes saturated. In plasma, cyclosporine is approximately 90% bound to lipoproteins. In addition, binding to erythrocytes and lipoproteins is temperature dependent. As the temperature increases, binding to lipoproteins increases; however, binding to erythrocytes increases as the temperature decreases. Other medications that may affect the binding of cyclosporine to lipoproteins may modify the clinical response to cyclosporine. Cyclosporine is metabolized extensively by the CYP3A enzyme system in the liver and to a lesser extent in the GI tract and kidney. Agents that affect the CYP3A system may significantly alter the metabolism of cyclosporine. At least 25 metabolites of cyclosporine have been identified, some of which are biologically active. Although most cyclosporine metabolites show only 10% to 20% of the immunosuppressive activity of the parent drug, they do contribute to toxicity. The major metabolites of cyclosporine are M1, M9, and M4N, resulting from oxidation at the 1-beta, 9-gamma, and 4-N-desmethylated positions. At steady state, concentrations and AUCs of cyclosporine metabolites may exceed that of cyclosporine. Mean AUCs for blood concentrations of these metabolites are 70%, 21%, and 7.5%, respectively, of blood cyclosporine concentrations. The elimination half-life of cyclosporine is highly variable. In patients with normal hepatic function, the average half-life ranges from 16 to 27 hours, and in other reports 5 to 18 hours.

Semi Logarithmic Mean Drug Time Curve
Time (hours)

Time (hours)

FIGURE A.8 Semi-logarithmic presentation (A) and linear presentation (B) for cyclosporine A means after single-dose administration of 100 mg cyclosporine per 1 mLfor both treatments Sigmasporin Microoral® Solution (test product) and Sandimmun Neoral® Solution (reference product).

Elimination of cyclosporine and its metabolites is principally through the bile and feces. Cyclosporine undergoes enterohepatic recycling. Only 6% of the cyclosporine dose is excreted renally, of which 0.1% is excreted as unchanged cyclosporine. Although cyclosporine blood levels are widely used to assist dosing, accurate interpretation is hampered by variation in absorption, variation in protein binding, sampling error, type of assay, cross-reactivity of metabolites, enterohepatic recycling of drug, and drug interactions.

A.9.1.4. Therapeutic Uses

Cyclosporine is indicated in the following cases (anon-FDA-approved indication):

■ Aplastic anemia3

Crohn's disease3

■ Graft-versus-host disease3

■ Heart transplant rejection prophylaxis

Time (hours)
Time (hours)

FIGURE A.9 Linear presentation for cyclosporine A individual plasma concentrations (ng/mL) versus time (hours), after single-dose administration of 100 mg cyclosporine per 1 mL for both treatments; Sigmasporin Microoral® Solution, the test product (A), and Sandimmun Neoral® Solution, the reference product (B).

■ Idiopathic thrombocytopenic purpura (ITP)a

Kidney transplant rejection prophylaxis

■ Liver transplant rejection prophylaxis

Lupus nephritis3

Myasthenia gravis3

Psoriatic arthritis3

■ Rheumatoid arthritis

■ Ulcerative colitis3

Xerophthalmia3

(Plot B)

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