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Investigation of the Combined Injection April 30, 2021 Archives• 2021 • vol.1 • 145-155 INVESTIGATION OF THE COMBINED INJECTION SOLUTION ‘NEURONUCLEOS’ ACTIVE COMPONENTS STABILITY AND COMPATIBILITY Maksym Almakaiev, Svitlana Gubar, Anna Materiienko National University of Pharmacy, 53 Pushkinska str., 61002, Kharkiv, Ukraine *[email protected] Abstract The article presents experimental data on the study of active components stability and compatibility in a combined injection solution. The described medicine contains three active ingredients: two nucleotides (uridine-5-monophosphate disodium salt (UMP), cytidine-5-monophosphate disodium salt (CMP) and vitamin B6 (pyridoxine hydrochloride) (PHC). It was proved that the medicine components do not interact with each other. The research also showed that thermal sterilization leads to the decomposition of API and the growth of impurities quantity. Therefore, solution sterilizing filtration through membranes with 0.2 μm pore size and subsequent filling of sterile ampoules under aseptic conditions was proposed. As a result of the experimental studies the pH of the combined injection solution medium in the range from 4.0 to 5.0 was selected. Additionally the impurities of the medicine active substances were established and normalized. Keywords: : uridine-5-monophosphate disodium salt, cytidine-5-monophosphate disodium salt, pyridoxine hydrochloride, compatibility, injection medicine, liquid chromatography, buffer solutions http://pharmacologyonline.silae.it ISSN: 1827-8620 PhOL Almakaiev, et al. 146 (pag 145-155) Introduction monophosphate disodium salt (UMP), cytidine-5- monophosphate disodium salt (CMP) and vitamin B Nucleotides and vitamins of group B play a key 6 (pyridoxine hydrochloride – PHC). Complex role in the regeneration of a damaged peripheral experimental research was carried out, as a result of nerve [1-3]. Previous preclinical trials showed that which the pH limits of the solution were determined pyrimidine nucleotides uridine and cytidine possess and substantiated, the composition and positive effects in the treatment of various technological parameters of an injection solution neuropathies and affect the acceleration of the obtaining were clarified. nerves and muscle fibers regeneration [4-5]. They also increase the nerve conduction speed and the Since this injection medicine is original, one of the phospholipids level (phosphatidylcholine and main issues at the stage of pharmaceutical phosphatidylethanolamine) of the neurons development (in addition to studying the membranes [6]. physicochemical properties of the dosage form components) was to determine the stability and The literature data describes the results of clinical compatibility of the solution components. Therefore trials for cytidine and uridine usage in combination the aim of our work at the stage of pharmaceutical with vitamins B in such conditions as pain in the development was to investigate the decomposition lower back and cervix, diabetic neuropathy, products profile while studying the stability of a new traumatic-compression lesions and acute non- combined injection medicine. traumatic pain [7-10]. Methods Medicines containing uridine and cytidine nucleotides have been used in the treatment of In order to develop a methodology and to study peripheral nervous system pathology for more than stability and compatibility the standards of 40 years already, but interest in this area is not substances and possible impurities of synthesis and decreasing, new combined medicines appear all the decomposition were used: time [11]. This can be explained by the important 1. For uridine-5-monophosphate disodium salt: role of nucleotides in the restoration of damaged uridine-5-monophosphate disodium salt (Sigma structures of the nerve fiber – axons and myelin Aldrich, Product # U6375, lot STBB2499V); uracil sheath [12]. Clinical efficacy and a favorable safety (Sigma Aldrich, Product # U0750, lot 080M0067V); profile of this combination have been noted in uridine (Sigma Aldrich, Product # U3750, radiculopathies, diabetic and alcoholic lot 071M1511V); uridine-5-monophosphate disodium polyneuropathy, neuropathy of the facial nerve. salt hydrate (Sigma Aldrich, Product # 94300, While taking nucleotides the regeneration of nerve lot BCBH2041V); uridine-5-triphosphate trisodium tissue intensified, the processes of nerve impulse salt hydrate (Sigma Aldrich, Product # U6750, conduction were normalized, the severity of the lot BCBD4670V) pain syndrome weakened [13]. The further advantages of nucleotides as medicines should also 2. For cytidine-5-monophosphate disodium salt: include their good tolerability [14-15]. cytidine-5-monophosphate disodium salt (Sigma Aldrich, Product # C1006, lot 129K2161V); cytosine According to the above-mentioned information, (Sigma Aldrich, Product # C3506, lot 069K1542); the development of a new combined medicine, cytidine (Sigma Aldrich, Product # С122106, based on the uridine and cytidine nucleotides lot BCBG3699V); cytidine-5-diphosphate disodium together with vitamin B, especially in an injection salt hydrate (Sigma Aldrich, Product # С9755, dosage form, appears to be a topical issue of lot 099K5164); cytidine-5-triphosphate sodium salt modern medicine and pharmacy. (Sigma Aldrich, Product # C1506, lot BCBG0248V) Such original injection medicine under the name 3. For pyridoxine hydrochloride: pyridoxine ‘Neuronucleos’ has been developed in the hydrochloride (EP CRS, lot 2); pyridoxine laboratory of parenteral and oral liquid medicines of hydrochloride impurity А (EP CRS, lot 2); pyridoxine National University of Pharmacy. It was based on three active substances: two nucleotides (uridine-5- http://pharmacologyonline.silae.it ISSN: 1827-8620 PhOL Almakaiev, et al. 147 (pag 145-155) hydrochloride impurity В (Sigma Aldrich, Product method for analyzing the APIs decomposition # D0501, lot 071M1615). products all available information was thoroughly analyzed [18-21]. Injection medicine (laboratory series 1610121, 1610122, 1010123). Water for injection according to In order to determine the main impurities of the the requirements of the Ph.Eur. and SPhU [16-17]. APIs and active components of the finished dosage form (FDF) decomposition the method of ‘artificial HPLC grade water was obtained from a water aging’ was used. When API and FDF were kept purifying system (Millipore, Bedford, MA, USA). under stress conditions, for example, at elevated Other chemicals and solvents were of analytical temperatures, under the action of oxidizing and grade. All the solvents used for mobile phase were reducing agents, the main impurities arising under filtered through membrane (0.22 m pore size) and degassed before use. these conditions were determined [22]. The main impurities of the APIs decomposition HPLC that can be formed during the storage of the The analysis of decomposition products has been prepared medicine are presented in Table 1. carried out on a ProStar liquid chromatograph, In Fig. 1 and 2 chromatograms, which show the equipped with an autosampler 410 and photodiode complete separation of both APIs and their possible array detector PDA 330, made by «Varian decomposition impurities under these Chromatography System» (USA). A Hydrosphere C18 chromatographic conditions, are depicted. column (150 x 4.6 mm, particle size 3 µm) with a precolumn has also been used. The sodium The standard conditions for the accelerated phosphate dibasic solution, adjusted to pH 5.5, was degradation of API in solution, used in the research, used as the mobile phase. The separation has been are presented in Table 2. carried out in an isocratic mode. The flow rate of the The analysis of the interaction in binary mixtures mobile phase was 1 ml / min, the injection volume (CMP – UMP, UMP – PHC, CMP – PHC) was carried was 20 μl, the detection has been carried out at a out according to the parameter – the total content wavelength of 262 nm and a column temperature of impurities. The results for each solution of the was 35 °C. sample (each binary mixture) under the action of all рН determination the critical factors selected for the investigation were determined. The total content of impurities for The pH of the solution medium was measured by each solution in the sum of impurities for all the potentiometric method on the ‘Seven Easy’ pH solutions was calculated. meter, manufactured by ‘Mettler Toledo’ (USA), in accordance with the Pharmacopoeia requirements As a result of the research the contribution of (SPhU/ Ph.Eur., 2.2.3). each factor to the instability of binary mixtures was analyzed with the following conclusions: Results - there is a significant contribution of the heat We proposed to determine the content of treatment to the API decomposition; impurities in the medicine by the high-performance liquid chromatography method, as the most - there is no increase in the amount of impurities sensitive and selective instrumental analysis with a decrease in pH, however, to establish relative method, which makes it possible to control pH, it is necessary to consider each API separately. A concomitant impurities both in active relative decrease in impurities at pH 9.18 was pharmaceutical ingredients (API) and in the obtained, which is associated with the absence of prepared medicine. UMP and CMP decomposition in this pH range; The literature data describes the - the API stability is influenced by oxidizing agents spectrophotometric and HPLC methods for as well as by reducing agents. nucleotides and pyridoxine analysis in biological fluids and medicines.
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