This study seeks to compare the in vitro multi-point dissolution profiles between 3 off-patent products azithromycin dihydrate 500 mg and the innovator product. The paddle-type dissolution apparatus was used. This was rotated at a speed of 50 rpm using 0.1-N HCl as media. Random samples were withdrawn after certain time points and assayed for azithromycin dihydrate. Comparision between test samples and the innovator product was done by computing their similarity (f 1 ) and disimilarity (f 2 ) factors and by fitting them to various kinetics of drug release during dissolution. Test samples 1 and 3 were comparable to the innovator product because they complied with f 1  and f 2  specifications. Test sample 2, however, gave higher f 2 values making it non-equivalent with the innovator product. The Higuchi and Korsmeyer-Peppa kinetics of drug release characterized most of the dissolution profiles. This study showed that test samples 1 and 3 are equivalent with the innovator products in terms of comparative in vitro dissolution profiles where extra-Fickian release behaviors was exhibited by all the preparations

Alejandro, Grecebio Jonathan D.

Dacanay, Aleth Therese L.

Erguiza, Raymund S.

Guzman, Gerard Q. de

Rosal, Rosalyn M.

English

International Journal of Advances in Pharmaceutics

  Research Article  Comparative in Vitro Dissolution Profile of Commercial Azithromycin Dihydrate 500 mg Tablet Preparations in the Philipppines  *Gerard Q. de Guzman1,5,6, Aleth Therese L. Dacanay1,2,4, Raymund S. Erguiza5, Rosalyn M. Rosal,5                                                  and Grecebio Jonathan D. Alejandro 1,3,4  1The Graduate School, 2Faculty of Pharmacy, 3College of Science and 4Research Center for the Natural and Applied Sciences, Universiy of Santo Tomas, Espana Blvd., Sampaloc, Manila 1015 Philippines 5College of Pharmacy, Virgen Milagrosa University Foundation, Martin P. Posadas Ave., San Carlos City, Pangasinan 2420 Philipppines 6Dr. Francisco T. Duque Medical Foundation, Lyceum Northwestern University, Tapuac Dist., Dagupan City, Pangasinan, Philippines 2400   *Correspondence Info: Gerard Q. de Guzman  30 de Guzman St., Poblacion, San Fabian, Pangasinan, Philippines 2433 Tel. No.:  +63 75 511 2333 Fax No.:  +63 75 513 2573 Mobile:  +63 933 364 4312 Email:  gerardqdeguzman@yahoo.com   Keywords:  Comparative dissolution,  Azithromycin dihydrate,  Tablets.   1. Introduction Among the macrolide antibiotics, azithromycin has the longest biological half-life because of its high volume of distribution, where tissue concentrations are much higher than blood levels.  High concentrations are taken up into white blood cells and, therefore, plasma concentrations are of little value as a guide to efficacy [1].  A 500 mg daily dose for 3 days is optimum for patient compliance in the eradication of mostly respiratory tract infections without the inconvenience of multiple daily dosing with other antibiotics.  Several off-patent azithromycin dihydrate 500 mg tablet formulations have proliferated the Philippines market.  Most of these products are sold at highly cheaper retail prices than the innovator product, making their qualities, safety and efficacy oblivious to scrutiny among physicians and pharmacists.  To ensure safety and efficacy of generic products, the conduct of expensive bio-equivalence studies using human subjects are preferred because actual pharmacokinetic data are compared against the innovator product.  A cheaper alternative is the conduct of in vitro comparative multipoint dissolution studies as they correlate well with in vivo bio-equivalence studies [2]. This study compares the multi-point dissolution profiles of 3 off-patent azithromycin dihydrate 500 mg tablets currently available in the Philippines against the innovator product.      2. Materials and Methods 2.1. Product Procurement  Samples of azithromycin dihydrate 500 mg tables were provided either as gifts or were purchased at wholesale Abstract This study seeks to compare the in vitro multi-point dissolution profiles between 3 off-patent products azithromycin dihydrate 500 mg and the innovator product.  The paddle-type dissolution apparatus was used.  This was rotated at a speed of 50 rpm using 0.1-N HCl as media.  Random samples were withdrawn after certain time points and assayed for azithromycin dihydrate. Comparision between test samples and the innovator product was done by computing their similarity (f1) and disimilarity (f2) factors and by fitting them to various kinetics of drug release during dissolution.  Test samples 1 and 3 were comparable to the innovator product because they complied with f1 and f2 specifications.  Test sample 2, however, gave higher f2 values making it non-equivalent with the innovator product.  The Higuchi and Korsmeyer-Peppa kinetics of drug release characterized most of the dissolution profiles.  This study showed that test samples 1 and 3 are equivalent with the innovator products in terms of comparative in vitro dissolution profiles where extra-Fickian release behaviors were exhibited by all the preparations.             International Journal of Advances in Pharmaceutics ISSN: 2320–4923; DOI: 10.7439/ijap Volume 4 Issue 3 [2015] Journal home page: http://ssjournals.com/index.php/ijap   International Journal of Advances in Pharmaceutics 4 (3) 2015                                                                                   22   price from medical representatives on the condition that the samples have a remaining shelf-life of at least 18 months at the time they were donated or purchased.  These are as follows: a. Pfizer, Inc., Makati, Philippines (ZithromaxTM, designated as the innovator product with lot no. B141464102 and expiring on April 30, 2017); b. Farma Iberica Co., Pasig City, Philippines (OD MacTM, designated as test sample 1, with lot no. EMM401 and expiring on September 30, 2016); c. Stallion Lab., Gujarat, India (AnzalTM, designated as test sample 2 with lot no. PH240 and expiring on October 31, 2016); and d. Interphil Lab., Cabuyao, Laguna, Philippines (ZithramycinTM, designated as test sample 3 with lot no. 14ZAT-1 and expiring on August 2016).   2.2. Dissolution Testing Each of the 6 vessels of the USP dissolution type 2 apparatus was filled with 900 mL of 0.1-N HCl.  One tablet coming from any of the 3 test samples and the innovator product were randomly distributed into each vessel and the paddles were made to rotate at 75 rpm.  From each vessel, 2 mL aliquot portion was taken at the end of 5, 10, 20, 30, 40, 60, 80 and 120 minutes and then filtered (Millipore No. 1).  The filtrates were diluted to 100 ml with 0.1-N HCl and then assayed for azithromycin dihydrate content. Two mL of fresh medium was replaced after each aliquot withdrawal to maintain sink conditions.  More cycles of dissolution tests were repeated similarly so that at the end of the study, 6 units of each of the 3 test sample and the innovator product have been subjected to multi-point dissolution testing.     2.3. Assay for Azithromycin Dihydrate by High-performance Liquid Chromatography (HPLC) Azithromycin dihydrate reference standard was dissolved in 0.1-N HCl and then filtrered (Millipore no. 1) to produce a 0.1 mg/mL standard solution.  The standard solution and the finally diluted test sample solutions from 2.2 were injected at 20-µL quantities in a Merck Hitachi 24-1 (Tokyo, Japan) equipped with a reverse phase C-18 column (5 x 4.66 mm) that was heated at 500C and a UV detector at 210 nm and eluted with HPLC grade 0.03-M phosphate buffer – methanol (20:80 v/v, pH = 7.5; Merck Co.) at a flow rate of 2 mL/minute [3].   2.4. Quality Control of the Materials Procured The 3 test samples and innovator products were tested for assay for azithromycin dihydrate content by the spectrophotometric method  of Patil et al [4] (2011), friability (Roche ED-2), hardness, (Stokes-Monsanto),  disintegration (USP Electrolab) in 0.1-N HCl for 2 hours at 37 ± 20C and weight variation (Sartorius PG 03-S).    3. Results and Discussions 3.1. Comparative Multi-point Dissolution Profiles The area under the dissolution-time curves for the 3 test samples were compared against the innovator product by the similarity (f2) and difference (f1) factors, such that f2 = 50log [{1 + 1/n Σ (Rt – Tt )2}-0.5 x 100 and f1 = [{ Σ ǀ Rt – Tt ǀ / Σ Rt} x 100], where Rt and Tt  are the percentage drug dissolved at each time point for the innovator and test products, respectively, n is the number of dissolution sampling time and t are the time points for collecting dissolution samples.  Figure I compares the dissolution-time curve of the 3 test samples and the innovator product.   Figure 1:  Comparative Multi-point Dissolution between the Three Test Samples and the Innovator  International Journal of Advances in Pharmaceutics 4 (3) 2015                                                                                   23   The dissolution-time curve of test sample 3 was comparable to the innovator product, assuming almost mirro-image super imposable dimensions.  Test sample 2 failed to achieve this, with Q values being significantly lower than the innovator product and the 2 other test samples after 30 minutes.  Test sample 1 gave significantly higher Q values than the innovator starting at 40 minutes. All 4 products exhibited an initial phase of high release (i.e., burst effect) with a common asymptote after 40 minutes, followed by a second phase of moderate release.  To validate this finding, comparison of f1 and f2 values were made and are presented in Table I.   Table I:  Comparative Similarity and Difference Factors Among the Three Test Products Formulations Similarity (f2) Difference (f1) Test Sample 1 51.16% 8.88% Test Sample 2 51.99% 28.90% Test Sample 3 53.00% 3.01% *p < 0.001 vs. innovator up to 120 mins. **p < 0.05 vs. innovator up to 120 mins.   All 3 test samples passed the Food and Drug Authority (FDA) specification for f2 which should be at least 50% to make it equivalent to the innovator product in terms of comparative in vivo multi-point dissolution.  However, test sample 2 exceeded the maximum f1 value of 15% set by the FDA and that does not make it equivalent to the innovator product as far as in vitro bio-equivalence is concerned. 3.2. Kinetics of Dissolution To analyze the in vitro dissolution release of azithromycin dihydrate, the following kinetic models were applied: a. Zero order, where cumulative concentration Q was plotted against time t;  b. First order, where log Q was plotted against t;  c. Higuchi  model where Q was plotted against square root of time t;   d. Hixson-Crowell, where Mo1/3 – Mt1/3 = KHCt, such that Mt is the amount of drug released in time t and Mo is the initial amount of the drug in the sample (~ 500 mg), and where Mo1/3 –                  Mt1/3  was plotted against t; and e. Korsmeyer-Peppa, where log Q, up to 60%, was plotted against log time t.    Information on the dissolution kinetics of the 3 test samples and the innovator product are provided in Table II.  The plots of azithromycin dihydrate with zero order, first order and Hixson-Crowell kinetics showed low linearity, were r < 0.9 for zero order and r < 0.8 for first order and Hixson-Crowell kinetics, to signify that diffusion is independent of concentrations.  The best linearity was found with Higuchi’s and Korsmeyer-Peppa's kinetics (r > 0.9).    Table II: Comparative Dissolution Release Kinetics Formulation Drug Release Kinetics (r = correlation coefficient) Zero Order First Order Higuchi Hixson-Crowell Korsmeyer – Peppa r r r r r n Test Sample 1 0.82 0.64 0.91 -0.77 0.99 1.06 Test Sample 2 0.88 0.77 0.96 -0.81 0.99 0.93 Test Sample 3 0.81 0.72 0.91 -0.63 0.98 1.1 Innovator 0.82 0.72 0.92 -0.76 0.98 1.1   The Higuchi plots, shown in Figure II, assumes one-dimensional drug diffusion where matrix swelling and dissolution are negligible within a perfect sink condition [2].  It is apparent that after 30 minutes, the Higuchi plots of test sample 2 was not comparable to the innovator product and test samples 1 and 3.  The Korsmeyer-Peppa model was designed to to confirm whether release mechanisms of drugs from cylindrical tables obey Ficks' law of diffusion.  The release exponent n, which is derived from the slope of he Korsmeyer-Peppa plots in Figure III, characterizes different release mechanisms described in Table III [2]. All 3 test samples and the innovator product gave n values higher than 0.89 which means that these products follow super case II transport diffusion due to chain entanglement as a result of swelling of hydrophobic and hydrophilic polymers which are present in the formulations to provide sustained-release properties [5].    International Journal of Advances in Pharmaceutics 4 (3) 2015                                                                                   24   Figure II: Comparative Higuchi Plots of the Various Samples Tested   Figure III: Comparative Korsmeyer-Peppa's Plots of the Various Samples Tested   Table III: Interpretation of Diffusional Release Mechanisms from Polymeric Films Release Exponent n Drug Transport Mechanism Rate as a Function of Time 0.5 Fickian diffusion t -0.5 0.45 < n = 0.89 Non-Fickian transport t n-1 0.89 Case II transport Zero order release Higher than 0.89 Super Case II transport t n-1  3.3. Quality Control of the Finish Products Table IV compares the 3 test samples against the innovator product based on standard quality control tests for tablets.  Results showed that there are no appreciable differences among the 3 test samples and the innovator product in terms of disintegraion and weight variation tests.  However, test sample 2 exceeded the friability limit of 1%, making it oblivious to abrasion during transport.  Test sample 2 is the hardest among the samples tested at almost 7 kg, a factor that reflects the high amount of binders in the formulation.  Too much abrasion and the additive effects of excessive amount of binders are factors to consider why test sample 2 was not equivalent with the innovator product, let alone test samples 1 and 3.  Furthermore, it is possible that the overage of 3.2% observed with test sample 1 after assay for azithromycin dihydrate content contributed to the high Q values during dissolution as reflected in Figures 1 and 2.  This overage, however, is still deemed acceptable as the upper monograph limit is set at 110% by the United States Pharmacopoeia 34.   International Journal of Advances in Pharmaceutics 4 (3) 2015                                                                                   25   Table IV:  Quality Control Tests for the Various Samples Tested Formulation (N = 20) Mean ± S.E.M. (Specification) Assay  Friability Hardness Disintegraton Weight Variation Test Sample 1 103.20 ± 0.32% 0.23 ± 0.02% 4.7 ± 0.21 kg 6.2 ± 0.23 mins. 777.2 ± 4.5 mg Test Sample 2 98.80 ± 0.17% 1.11 ± 0.03% 6.8 ± 0.11 kg 6.1 ± 0.19 mins. 713.5 ± 5.6 mg Test Sample 3 99.30 ± 0.21% 0.46 ± 0.01% 4.2 ± 0.02 kg 5.7 ± 0.13 mins. 792.3 ± 4.8 mg Innovator 100.30 ± 0.13% 0.25 ± 0.03% 4.4 ± 0.12 kg 5.9 ± 0.14 mins. 788.3 ± 5.8 mg  4. Discussions Cost analysis and survey of the azithromycin dihydrate 500 mg tablets tested in this study showed the following retail prices:  test sample 2 (AnzalTM) at 60 pesos per 500 mg tablet; test sample 3 (ZithramycinTM) at 40 pesos per tablet; test sample 1 (OD MACTM) at 140 pesos per tablet; and the innovator (ZithromaxTM) at 151.25 pesos per tablet.  This study showed that generic branding at relatively cheaper prices than the innovator is possible by formulating preparations that are bio-equivalent through comparative in vivo multi-point dissolution testing.  This study revealed that test samples 1 and 3 are comparable with the innovator product in terms of similarity and difference factors and exhibited similar extra-Fickian diffusion behavior as both fitted to Higuchi and Korsmeyer-Peppa release kinetics.  These findings are important since azithromycin dihydrate has a relatively long shelf-life and high volume of distribution as it is primarily stored in tissues so that it is important that their tablet preparations provide sustained-release mechanisms to maintain patient compliance, particularly when these tablets are given once daily for 3 days [6]. It was found out that test sample 2 was not equivalent to the innovator in terms of multi-point comparative in vitro dissolution testing but these findings do not translate to poor quality in terms of bioavailability, even if it also failed friability and hardness tests.  It has passed assay limits and, therefore, it should release azithromycin into the blood streams and underlying tissues at sufficient bactericidal concentrations [7].   5. Conclusions This study showed that 2 of the 3 off-patent generic azithromycin dihydrate 500 mg tablets are equivalent to the innovator product in terms of multi-point in vitro dissolution profile.  All 3 preparations tested exhibited extra-Fickian release mechanisms that are characteristics of drugs released from matrices that constains hydrolloidal polymers that are intended to provide sustained-release effects.   References [1] Wilms, E.B., Touw, D.J. and Heijerman, H.G.M. Pharmacokinetics and sputum penetration of azithromycin during once weekly dosing in cystic fibrosis patients. J. Cystick Fibrosis, 2008; 7 (1): 79-84.  [2] Dash, S., Murthy, P.N., Nath, L. and Chowdhury.  Kinetic modeling on drug release from  controlled drug delivery systems.  P.  Acta Pol. Phar. Drug Res., 2010; 67 (3): 217-223 [3] Al-Rhimawi, F. and Khaoaf, M.  Analysis of azithromycin and its related compounds by RP- HPLC with UV Detection.  J. Chrom. Sci., 2010; 48 (2): 86-90. [4] Patil, A., Payghan, S and Disouza J.  Formulation and evaluation of enteric-coated tablets of  azithromycin dihydrate. Int. J. ChemTech Res., 2011; 3 (3): 1479-1484.  [5] Althaf, A.S., Seshadri,T., Sivakranth, M. et al. Design and study of lamivudine oral sustained release tablets.  Pelagia Res. Lib. 2010; 1 (2): 61-76. [6] Sun, L., Zhang, W., Liu, X. et al. Preparation and evaluation of sustained-release azithromycin tablets in vitro and in vivo.  Asian J. Phar. Sci., 2014; 9 (3):  155-161. [7] Christopher, J.D.and Barradel, L.B. Azithromycin:  a review of its pharmacological properties and use as a 3-day therapy in respiratory tract infections.  Drugs, 1996; 51:  483-505.  

Comparative In Vitro Dissolution Profile of Commercial Azithromycin Dihydrate 500 mg Tablet Preparations in the Philipppines

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Comparative In Vitro Dissolution Profile of Commercial Azithromycin Dihydrate 500 mg Tablet Preparations in the Philipppines

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