A Comparative Study of the Pharmacokinetics of Conventional and Sustained-release Tablet Formulations of Aceclofenac in Healthy Male Subjects

Purpose: To examine the pharmacokinetics of a formulated aceclofenac sustained release tablet formulation and determine if it is bioequivalent to a commercial brand of aceclofenac immediate release tablet (Zerodol ® 100 mg). Methods: Each of two groups of twelve fasting volunteers received either the reference standard (Zerodol 100 mg tablets) or the test formulation (200 mg aceclofenac) orally once, using a cross-over design with a one week wash-out period. Their blood samples were obtained at regular time intervals over 24 h and analyzed by high performance liquid chromatography (HPLC). Using the noncompartmental approach, plasma levels of aceclofenac were employed to compute their individual disposition kinetics, including peak plasma concentration (Cmax), peak time (Tmax), area under the plasma level-time curve (AUC 0-t), elimination rate constant (Kel) and elimination half life ( t1/2). Results: The Cmax values of 11043 ± 3073 ng/ml and 12301 ± 3000 ng /ml were attained in 2.58 ± 1.22 h and 1.29 ± 0.75 h for the test and reference products, respectively, while AUCO-∞ was 45996 ± 10427 and 50253 ± 8283 ng.h/ml, respectively. At 90% confidence interval, the C max, AUC 0-t and AUC0-∞ values of the test preparation were 96.4 101.3, 100.2 -101.9 and 98.5 99.8%, respectively, of the values for the reference. The t1/2 values were found to be 4.50 ± 1.25 and 2.20 ± 2.59 h for the reference and test products. Conclusion: On the basis of the pharmacokinetic data, it can be said that the test aceclofenac sustained release formulation and the reference product were bioequivalent in some respects. However, the test formulation exhibited a longer elimination half-life (t1/2), thus demonstrating sustained release properties, unlike the reference.


INTRODUCTION
Aceclofenac is a relatively new phenylacetic acid derivative showing marked antiinflammatory, anti-arthritic, analgesic and anti-rheumatic activities [1].The drug demonstrates better gastric tolerance than other non steroid anti-inflammatory drugs (NSAIDs) such as indomethacin and diclofenac [2].Its recommended dose is 100 mg twice a day orally in the form of tablets.After oral administration, it is rapidly absorbed and bioavailability is almost 100% [3].Peak plasma concentrations are reached within 1.25 to 3 h following ingestion [3].It is highly protein-bound (> 99.7 %) and penetrates into the synovial fluid, where the concentration reaches up to approximately 60 % of that in plasma [3].The volume of distribution is approximately 30 L while mean plasma elimination half-life and clearance are 4.3 ± 0.3 h and 5 L/h, respectively [3].
Aceclofenac is probably metabolized via Cytochrome P450 2C9 (CYP2C9, a protein encoded by the CYP2C9 gene in humans) to the main metabolite 4-hydroxyacefenac with negligible contribution to clinical activity [3].Its adverse reactions, as with other NSAIDs taken by mouth, are indigestion, heartburn, nausea and diarrheoa [3].
The objective of this study was to examine the in vivo bioequivalence of a test 200 mg aceclofenac sustained release formulation in relation to a commercial brand of 100 mg aceclofenac in male volunteers.

Preparation of aceclofenac sustained release tablets
The tablets were prepared by a wet granulation technique.The composition of the tablet formulations are given in Table 1.Aceclofenac, Methocel K4M, lactose/maize starch, sodium propyl para benzoate and fumaric acid were screened through a 425 µm sieve and mixed manually in a bowl for 5 min.The blend was granulated with the aid of PVP K-30 and water.The mass was sieved through a 500 µm sieve and then dried in a hot air oven at 50 0 C. Magnesium stearate, talc and colloidal silicon dioxide were then added to the dried granules, mixed for about 5 min in a polythene bag and compressed into tablets using a 12-station tablet compression machine (CIP Machineries, Ahmadabad, India) equipped with a 11 mm biconcave-faced punches.To mask the bitter taste of the aceclofenac, the tablets were coated in a laboratory coater (Model GAC-250, Gansons Ltd, Mumbai, India) with HPMC 5cps dissolved in a mixture of isopropyl alcohol and methylene chloride; titanium dioxide and Ponceau 4 R supra (colouring agents) as well as polyethylene glycol (PEG) 6000 and castor oil (plasticizers) were also incorporated in the coating mixture prior to coating.

Physiochemical characterization of the tablets
Tablet weight variation was evaluated on ten randomly selected tablets with an electronic balance (Mettler Toledo, Mettler, Griefensee, Switzerland) while tablet hardness and friability were also determined for ten randomly selected tablets using a Monsanto tablet hardness tester (standard type) and a Campbell electronic friabilator for 4 min at 25 rpm, respectively.Tablet diameter was evaluated with a digital vernier caliper.

Subjects and ethical issues
This study was performed in accordance with United States Food and Drugs Administration (FDA) Good Clinical Practice guidelines.
Volunteers enrolled for this study were appraised in detail about all aspects of the study in easy to understand language and terminologies.Before admission to the study each subject was informed of the nature and the risks of the study and a written informed consent was obtained from the volunteers.

Study design
All the volunteers were housed at the study centre.They were randomly divided into 2 groups of 12 subjects each.After an overnight fast (10 h), each group received orally, with 250 ml of water, either a single dose of the test sustained-release formulation or the reference product in a randomized fashion, using a two-treatment, two-way crossover study design.A sevenday washout period was allowed between dosing schedules.

Preparation of standard solutions
A stock solutions (1 mg/mL) of aceclofenac was prepared with methanol as the solvent.
From the stock solution, standard solutions were prepared to contain 1, 2, 5, 10, 20, 30, 50 and 70 µg/mL of aceclofenac.A solution of ibuprofen (500 µg/mL) was prepared in a methanol/water mixture (80:20) and used as internal standard in the assay of aceclofenac.

Analysis of drug in plasma
To 100 µL of plasma, 25 µL of the internal standard and 200 µL of acetonitrile were added and mixed for a minute, and then made up to 1 ml with acetonitrile.The resulting solution was vortexed for 60 s and centrifuged at 10,000 rpm for 10 min.The supernatant layer was separated and analyzed for aceclofenac content using a sensitive high performance liquid chromatographic (HPLC) method with a Shimadzu Class VP series incorprating Class VP 6.12 version software, two pumps (LC-10AT VP), a variable wavelength programmable UV detector (SPD-10A VP), a system controller (SCL-10A VP) and an RP C-18 column (Hypersil BDS C18).The operating conditions and parameters include: mobile phase: methanol + 0.3% tri ethyl acetic acid (60:40 v/v, pH 7.0); flow rate: 1.0 mL/min; injection volume: 20 mL; temperature: 25 o C; run time: 25 min; detection wavelength: 275 nm; and internal standard -ibuprofen.The response factor was measured by the ratio of the peak area of drug to that of the internal standard for both standard and test samples.Blank plasma samples were analyzed prior to the analysis of the standard and test preparations.Preliminary studies had shown that the presence of plasma did not interfere with the analysis of the drugs.The peaks were well resolved and the retention times were 5 min (aceclofenac) and 10.5 min (ibuprofen).

Data analysis
Using the non-compartmental approach, plasma levels of aceclofenac were employed to compute their individual disposition kinetics, viz, C max (maximum plasma concentration), T max (time to reach maximum plasma concentration), AUC 0-t (area under plasma concentration-time curve), AUC 0-∞ (area under plasma concentration time curve, 0 to infinity), K el (elimination rate constant) and t 1/2 (elimination half-life).Pharmacokinetic parameters were calculated with PK Solutions 2.0 TM Noncompartmental pharmacokinetic data analysis software.
Student's t-test was employed to analyze the results using Graph Pad Instat Software, version 1.13.Differences below the probability level of 0.05 were considered statistically significant.The ratios of the mean bioavailability parameters (test : standard) were obtained and 90 % confidence interval was used to determine bioequivalence.Log (natural) transformation of C max and AUC 0-∞ were performed prior to the statistical analysis.The equivalence criteria range was 80 to 125 % based on FDA guidelines.Calculations also included area under curve (AUC) from time t to ∞ (infinity) calculated with poly-exponential and trapezoidal methods.

Pharmacokinetic profiles of aceclofenac tablets
Administration of the reference product as a single dose produced C max of 12301 ± 3000 ng/ml at a t max of 1.29 ± 0.75 h while C max for the test preparation was 11043 ± 3073 ng/ml at a t max of 2.58± 1.22 h (Table 2).The bioavailability of the test preparation relative to that of the standard was 91.1 %.C max , AUC 0-t and AUC 0-∞ values of the test preparation were 96.4 -101.3 %, 100.2 -101.9 % and 98.5 -99.8 % of that of the reference, as shown in Table 3.However, the AUC 0-∞ values for the two formulations were not significantly different.
There was also no difference in the extent of absorption for both the test and reference products as indicated by the less than 10 % difference in relative bioavailability (91.1 versus 100 %) of the drug.This suggests that the aceclofenac contained in the test product was completely absorbed.

CONCLUSION
The test product showed sustained release characteristics unlike the reference.Overall, the pharmacokinetic profile of the former was either equivalent or superior to that of the reference.Thus, the test aceclofenac preparation can be further developed to achieve a suitable sustained release product.

Table 1 :
Composition of sustained release tablet formulations

Table 2 :
Pharmacokinetic parameters for the test (A) and reference standard formulations (B)

Table 3 :
Bioavailability data for test aceclofenac formulation (200 mg) relative to the reference at 90% confidence interval The half-life of the reference preparation was low which indicates rapid removal of the drug from plasma.This was also supported by the high elimination rate constant value.On the other hand, the test formulation exhibited higher half-life and low elimination rate constant values indicating slower drug disposition and prolonged effect.