BIOEQUIVALENCE STUDIES OF MEDICINAL PRODUCTS OF TRANEXAMIC ACID

This article presents results of complex of research on biowaiver procedure to establish bioequivalence of medicinal products of tranexamic acid in form of tablets 500 mg. As a result of solubility and extent of permeation studies for active substance tranexamic acid is established I class according to BCS. It was established, that the objects of the study belong to highly soluble medicinal products in all recommended dissolution media. The similarity of dissolution profiles demonstrates the equivalence of test and reference medicinal products of tranexamic acid.


INTRODUCTION
Today around the world, including in Ukraine, significant part of the pharmaceutical market consists of generic medicinal products (MP) of different pharmacotherapeutic groups and dosage forms, that are much more cheaper on price constituent than innovative products. According to information of various authors for different time periods [1,7], number of registered generic MPs in Ukraine was within 55-90 %. But along with economic benefits for generic MPs can be outlined also negative aspects, such as side effects and inappropriate effectiveness compared to original drugs. The basis for the prevention of significant pharmacological differences in medical use of generic MPs and main condition for registration of generics according to international regulatory practice is proof of their bioequivalence to reference product, that should be original MP with proven efficacy, safety and quality.
One of methods to prove bioequivalence of generic and reference MPs for solid dosage forms of systemic exposure with immediate-release for oral use is simplifiedprocedure of registration for generic MP, that is called "biowaiver" and is based on biopharmaceutical classification system (BCS) of active substances (AS) and category of MP regarding dissolution in media of gastrointestinal tract (GIT). This approach allows to evaluate substitution of generic and reference drugs on the basis of their comparison in conditions in vitro. In this case it is necessary to consider quantitative and qualitative composition of products in terms of AS and excipients (EX), balance of risks in terms of health of population and individual patient, results of solubility and permeation of AS into gastrointestinal tract and the results of comparative in vitro studies using dissolution profiles of MPs in media, that reflect physiological composition of different media of gastrointestinal tract [6,11]. So, to be able to use "biowaiver" procedure for registration of solid dosage forms of systemic exposure for oral use with immediate-release in is necessary to conduct complex of studies.

Analysis of recent studies and publications
Basic requirements for studies regarding "biowaiver" procedure are approved by the majority of world leading regulatory agencies on assessment of medicinal products (FDA, 2000, WHO, 2006, EMA, 2010 [3,4,8]. In Ukraine, this approach is governed by guidelines ST-N of MOHU 42-7.1: 2014 "Medicinal products. Bioequivalence Study" [11], that is harmonized with relevant regulations of the European Union [4]. Foreign and domestic literature provides many study results of different ASs and their MPs regarding in vitro study and evaluation of bioequivalence of generic and reference drugs. These studies are devoted to main directions of "biowaiver" procedure, such as determination of solubility and degree of permeation of AS and determination on the basis of AS class, according to biopharmaceutical classification system (BCS) [9], determination of MP category and comparison of dissolution profiles of generic and reference MPs [5,9], methods development for study of dissolution kinetics of AS of their medicinal products [15].

RESULTS AND DISCUSSION Highlighting of not resolved before parts of general problem
During conducting literature review on matter of studies of bioequivalence of generic and reference MPs of tranexamic acid, we have not found any publications highlighting relevant issues, to which this article is devoted, that makes it impossible to assess the results of such studies and to use them in our work.

Formulation of objectives of the article
The aim of this work was to conduct complex of in vitro bioequivalence studies of medicinal product Cyclocapron-Zdorovye film-coated tablets 500 mg, with active substance tranexamic acid, to determine possibility to use biowaiver procedure for state registration of generic MP.
All studies were performed according to standardized procedure, that was developed in accordance with recommendations [4,11] and approved by the company.
Dissolution study was performed using dissolution apparatus PTWS610 manufactured by company "Pharma Test" (Germany) under following conditions: apparatus with basket, volume of dissolution medium -500 ml, temperature of dissolution medium -(37 ± 1) °C, stirring speed -100 rpm, sampling points: 10, 15, 20 and 30 min, buffer solutions with pH 1.2, pH 4.5 and pH 6.8. Buffer solutions were prepared according to requirements of SPU [10] using reagents of Pharmacopoeial grade, such as: hydrochloric acid, sodium acetate, glacial acetic acid, potassium dihydrogen phosphate, sodium hydroxide and sodium chloride. For each study were used 12 units of MP for statistical evaluation. In accordance with the requirements [4,11] was conducted control of pH values of buffer solutions before and after dissolution of MP of tranexamic acid to confirm absence of impact of AS on pH of buffer solutions.
Assay of tranexamic acid in all studies was conducted according to developed and validated method by liquid chromatography. For this purpose was used reference standard of tranexamic acid USP RS, catalog number 1672745, batch number RO21KO.
In the studies following analytical equipment was used: electronic laboratory balance AG 204, manufacturer "Mettler Toledo" (Switzerland); liquid chromatograph Agilent 1100 3D LC System, manufacturer "Agilent Technologies" (USA), column Hypersil ODS, Agilent, dimensions 250 × 4.6 mm with a particle size of 5.0 microns; pH meter Seven Easy pH equipped with electrodes "Mettler Toledo" (China); measuring glassware class A.
To solve task set at the beginning of the study was analyzed performance of general conditions regarding possibility of use of "biowaiver" procedure to objects being studied. It was established, that studied and reference MPs had solid dosage forms (tablets) with systemic exposure, immediate-release for oral use, containing AS tranexamic acid in same amount of 500 mg and don't have narrow therapeutic index. EXs, that were used in composition of test MP were well-studied, met requirements [4,11] and performed functions that were similar to that of EXs of reference MP, namely were qualitatively similar (Tab. 1). So, objects of the study for all criteria met the requirements for use of "biowaiver" procedure.
The next stage of the studies was to establish the BCS class for tranexamic acid. One of the criteria for classification AS in accordance with BCS [4,11] is the solubility of the highest single dose in a volume ≤ 250 ml of each of three buffer solutions pH 1.2, pH 4.5 and pH 6.8 at (37 ± 1) °C. This volume is calculated as the ratio of the highest single dose to the equilibrium solubility of AS (HSD/S av. , ml).
According to world leading pharmacopoeias [2, 12], tranexamic acid is trans-4 (aminomethyl) cyclo-hexanecarboxylic acid, that belongs to freely soluble in water substances. Solubility and acid-base properties of AS are determined by its chemical structure (Fig. 1), that has groups of a dual nature: acidic part in form of an carboxyl group and main part in the form of amino group. It describes tranexamic acid as highly polar substance that is responsible for good solubility in water and evidences of its presence in solution as zwitter-ions depending on pH of medium. Based on these considerations it can be suggested, that this AS is soluble in recommended dissolution media within pH range 1.2-6.8. In order to establish BCS class and to confirm this assumption was carried out complex of studies on solubility of tranexamic acid according to the requirements of regulatory documents [11], that included study of profile of pH-dependent equilibrium solubility of tranexamic acid and calculation of volume, in which highest single dose dissolves in studied media. According to results of conducted studies [13] it was established, that tranexamic acid refers to substances with high solubility in all studied buffer solutions with pH 1.2; 4.5; 6.8.
Extent of permeation of AS tranexamic acid was established in SI "Institute of Pharmacology and Toxicology of NAMS of Ukraine" using method of measurement of permeation rate through monolayer of cell culture Caco-2. Average permeation rate for tranexamic acid was (1.24 ± 0.11) E-05 cm/s, that corresponds to the criterion of "high extend of permeation".
Thus, based on obtained results of conducted studies for the substance (solubility parameters and extend of permeation) it was found that tranexamic acid belongs to BCS class I, meaning that it shows high solubility and extend of permeation.
Next stage of complex studies is devoted to the study of release kinetics of tranexamic acid from test and reference MPs and comparison of their in vitro dissolution profiles. Average values of the AS release rate and relative standard deviation (RSD), obtained in the dissolution study of generic and reference products of tranexamic acid are given in Tab. 2. Dissolution profiles of the objects of the study at pH 1.2, pH 4.5 and pH 6.8 are shown in Fig. 2-4.
According to the results in Tab. 2 it is shown, that release of tranexamic acid from tablets of generic and reference products in all 3 recommended dissolution media having pH 1.2, pH 4.5 and pH 6.8 after 15 min is more than 85 % of labeled amount of active substance, indicating that the objects of the study belong to very quickly soluble MPs in all recommended dissolution media.