Development of a combined solution of pyrimidine nucleotides with vitamin B₆

M. S. Almakaiev¹, N. V. Dvinskikh¹, L. G. Almakaieva², Olga V. Kryvanych²*

¹National University of Pharmacy, Kharkiv, Ukraine.

²State Higher Educational Institution “Uzhhorod National University”, Uzhhorod, Ukraine.

*Corresponding Author E-mail: olga.kryvanych@uzhnu.edu.ua

ABSTRACT:

Pyrimidine nucleotides, namely uridine monophosphate and cytidine monophosphate, play an important role in the cellular metabolism of nerve fibers. The combination of these nucleotides with pyridoxine hydrochloride (vitamin В₆) in one dosage form will allow us to fully implement the strategy of the complex neurotropic pharmacotherapy in neuropathies of various origins. To develop a stable solution, an important step at the stage of the composition development is to study the compatibility of active substances (active pharmaceutical ingredients - APIs) in solution. Samples of binary solutions and solutions containing all active substances were prepared and examined. The API interaction was determined by various parameters – changes in color, transparency, рН, the total impurity content, etc. Based on a comprehensive study of the processes of the API dissolution the optimal pH limits of the solution required for the stable existence of a combination of substances with different pH values of the medium have been substantiated and experimentally confirmed. As a result of the research, the optimal pH value of the solution recommended is 4.0-4.8. In the composition of substances the amount of water can be in an adsorbed or crystallized, or combined form. This fact should be taken into account in order to correctly calculate the actual amounts of initial ingredients when preparing the solution. The nature of the water component of APIs was clarified when studying the phase composition of samples on a powder diffractometer. The studies conducted have allowed us to determine the directions of further research for developing the composition of an injection drug. This research is in choosing the optimal buffer system and excipients-antioxidants.

KEYWORDS: Pyrimidine nucleotides, Uridine monophosphate, Cytidine monophosphate, Pyridoxine hydrochloride, Parenteral dosage form.

INTRODUCTION:

Progress in the clinical practice of pathologies of the peripheral nervous system (PNS) is impossible without solving the problem of effective and safe pharmacotherapy of diabetic and alcoholic neuropathies. The share of these neurological complications of diabetes and alcoholism is increasing and is accompanied by high indicators of temporary disability, disability and significant socio-economic losses¹^,².

Pharmacotherapy of nerve lesions in many cases should be comprehensive, affect different links of pathogenesis. To treat alcoholic polyneuropathy (APN) and diabetic polyneuropathy (DPN) nonsteroidal anti-inflammatory drugs, tricyclic antidepressants, anticonvulsants, analgesics, and others, which act on the symptoms of diseases, are used³^,⁴.

The range of medicines containing active components that act at the level of peripheral nerves and promote their therapeutic regeneration is currently not rather wide⁵. Among them drugs based on pyrimidine nucleotides – uridine and cytidine are known^6,7.

Nucleotides are endogenous substances and are generally considered harmless. Therefore, therapy with these drugs is very well tolerated; practically, it is not accompanied by undesirable side effects. Pyrimidine nucleotides – uridine monophosphate and cytidine monophosphate are neurotrophic medicinal substances that play a key role in molecular metabolic processes. Exogenous replenishment of these substances contributes to the restoration of impaired neuronal functions, normalization of metabolic shifts and acceleration of regeneration, and it clearly correlates with the regression of clinical symptoms⁶.

The treatment of diabetic and alcoholic complications of PNS is very often accompanied with administration of vitamin preparations, among them B vitamins or neurotropic vitamins take the main place⁸^,⁹. B vitamins are involved in the processes of axonal transport of neurons, transmission of nerve impulses along motor and sensory fibres, regulate the balance of the nociceptive and antinociceptive systems¹⁰.

Currently, there are some drugs based on pyrimidine nucleotides and vitamin B₆ preparations that are used as part of the complex therapy of DPN and APN. Among them, there are drugs for parenteral use. They are “Nucleo C.M.P. forte”¹¹^,1², lyophilized powder for the preparation of a solution for injection produced by “Ferrer International”, Spain, and “Keltican”, lyophilized powder for the preparation of a solution for injection produced by Nicomed. Pyridoxine hydrochloride (PHC) solutions for injection are also well known¹^3,14,15,16.

Thus, the combination of such active pharmaceutical ingredients (APIs) as two pyrimidine nucleotides – uridine monophosphate and cytidine monophosphate with B vitamin – PHC in one drug will allow us to fully implement the strategy of the complex neurotropic pharmacotherapy in neuropathies of various origins, i.e. the effective prevention of the nervous tissue destruction and promotion in preserving the functional life. This is an urgent and socially significant task.

The aim of this work was to determine the compatibility and conditions of a stable existence of uridine monophosphate, cytidine monophosphate and PHC in solution in the necessary therapeutic concentrations in order to create a new combined drug for parenteral use based on pyrimidine nucleotides and vitamin В₆.

MATERIAL AND METHODS:

The study objects were such substances as uridine monophosphate of disodium salt (UMP), cytidine monophosphate of disodium salt (CMP) and PHC, solutions based on these pyrimidine nucleotides and vitamin В₆.

The substances of uridine monophosphate, disodium salt, batch 111020, cytidine monophosphate, disodium salt, batch 111018, manufactured by “Shanghai Oripharm Co. Ltd.”, China were used as active substances for the research. The quality of substances met the requirements regulated by the manufacturer in the Drug Master Files (DMF) and the manufacturer certificates. The quality of PHC substance, batch UQ 11113337, manufactured by “DSM Nutritional Products GmbH”, Germany met the requirements of the European Pharmacopoeia (Ph. Eur.) and the State Pharmacopoeia of Ukraine (SPhU)^17,18.

To achieve the goal, we developed a methodological approach that included the following stages:

· Determination of critical characteristics of APIs, which were the raw material and main drug substances;

· Analysis of their quality specifications, determination of the indicators that could affect the compatibility of APIs in solution;

· Study of the compatibility of active substances in solution under the action of influencing factors, determination of factors that could affect the critical quality characteristics of APIs in the product;

· Determination of the optimal pH range of the solution for the injectable dosage form.

During the research work the qualitative and quantitative control of experimental samples of solutions was carried out by the indicators characterizing stability: рН, the impurity content, transparency, color, particulate matter according to the methods given in the Ph. Eur., SPhU¹^7,18 and the product specification file.

The ranges of critical parameters and characteristics were determined based on the experimental data and the previous work experience.

RESEARCH RESULTS:

The qualitative and quantitative composition of the new combined drug was developed on the basis of studying the literature data and the experimental work. Therapeutic concentrations of active substances were proposed by pharmacologists based on the results of exploratory pharmacological studies of the neuropathic activity of mixtures containing different ratios of these ingredients in injection administration. The maximum neuropathic effect was exhibited by the composition containing all these ingredients in the following amounts:

• Uridine monophosphate, disodium salt, 2.0 mg/mL

• Cytidine monophosphate, disodium salt, 5.0 mg/mL

• Pyridoxine hydrochloride, 25.0 mg/mL

When creating a new drug, in addition to the rationality from a medical point of view, including the pharmacological compatibility of the active components of the drug combination, their physico-chemical compatibility in solution should be considered. This is especially important for parenteral drugs, which are mainly solutions¹⁹.

UMP and CMP are salts of weak organic acids and a strong base; in more detail – these are salts of nucleotides, which, in turn, consist of a nucleoside and a phosphoric acid residue. Nucleotides have strong acidic properties with a high-density negative charge. Nucleotides are also monoesterified derivatives of phosphoric acid. Due to the presence of phosphoric acid residues they are strong dibasic acids, which ionization is added to the ionization determined by the acidic properties of heterocyclic bases.

PHC is a pyridine derivative, and it also belongs to the category of salts. It is a salt of an organic base and an inorganic acid.

All main active ingredients of the combined drug developed are complex organic compounds that have reactive groups and can interact in solution.

We analyzed the quality characteristics of UMP, CMP and PHC, determined indicators and characteristics affecting the stability of these compounds in solution with their simultaneous presence. The information concerning the API properties and quality requirements is given in Table 1.

Table 1: Characteristics and quality indicators of substances of UMP, CMP and PHC

Name	UMP	CMP	PHC
Structural formula
Solubility	Readily soluble in water	Readily soluble in water	Readily soluble in water
Transparency	Transparent or does not exceed the standard I by turbidity (1 % solution)	Transparent or does not exceed the standard I by turbidity (1 % solution)	Transparent (5 % solution)
Color	Colorless or does not exceed the standard ВY₅ by the degree of color (1 % solution)	Colorless or does not exceed the standard ВY₅ by the degree of color (1 % solution)	The color of 5.0 % solution should not be more intense than the standard Y₇
рН	7.0-8.5 (1 % solution)	8.0-9.5 (1 % solution)	2.4-3.5 (5 % solution)
Related impurities	Impurity А – not more than 0.15%; impurity В – not more than 0.15%; any other impurity – not more than 0.10%; total impurities – not more than 1.5%	Impurity А – not more than 0.15 %; impurity В – not more than 0.15 %; impurity С – not more than 0.15 %; impurity D – not more than 0.15 %, impurity Е – not more than 0.15 %, impurity F – not more than 0.30 %, impurity G – not more than 0.30 %, impurity а H – not more than 0.15 %; any unidentified impurity – not more than; total impurities – not more than 1.5%	Impurity В – not more than 0.15%; unspecified impurities – not more than 0.10%; total impurities – not more than 0.2%. Peaks with the area less than 0.5 of the area of the main peak on the chromatogram of the reference solution (а) (0.05 %) are not considered
Water	Not more than 26.0 %	Not more than 25.0 %	Not more than 0.5 %
Assay	Not less than 98.0% and not more than 102.0% calculated with reference to the anhydrous substance	Not less than 98.0% and not more than 102.0% calculated with reference to the anhydrous substance	Not less than 99.0% and not more than 101.0% calculated with reference to the anhydrous substance

UMP and CMP have a similar chemical structure, similar physical and chemical properties, and a close pH range in the alkaline region; therefore, they are predictably compatible in the same solution.

PHC has a different structure, and its solutions have a pH in the acidic region; thus, there may be incompatibilities in the solution between this compound and nucleotides.

At different pH ranges different ionic forms of pyridoxine are present in the PHC solution (Fig. 1).

Fig. 1: Ionic forms of pyridoxine

The presence of different ionic forms can lead to differences in the physical and chemical properties of PHC at different pH ranges. In the acidic region, up to рН 4.31, there is mainly a protonated form, solutions are colorless. At рН from 4.31 to 8.37 a neutral form forms solutions of a slightly yellow color; in the region of alkaline pH values (above 8.37) there is a negatively charged form. The second and third forms are more labile and subject to oxidation processes.

The stability studies for PHC were performed in buffer solutions with a pH from 1.2 to 9.18. The analysis of pyridoxine solutions at pH levels corresponding to the existence of each of the three forms showed the following. In acidic solutions PHC was more stable. At рН 1.2 (Form 1) impurities were detected at the level of the unidentified limit and impurity B due to pyridoxine oxidation was insignificant (less than 0.03%). At рН 5.0 an increase in impurities was observed, but their amount did not exceed the level of the unidentified limit, impurity B was at the level of the unidentified limit (0.1%). At рН 9.18 impurities could be compared with impurities at рН 5.0, and the amount of impurity B at the level of the qualified limit was 0.4%. Solutions of pyridoxal as a product of PHC decomposition in solution had a yellow color at alkaline pH values.

Therefore, it is most acceptable to create a pH level for a new combined drug not higher than 4.8 in order to avoid the appearance of pyridoxine impurities, and not lower than 3.0 for pharmacological reasons.

Since the pH of UMP and CMP solutions of disodium salts were in the alkaline region (Table 1), their properties and resistance to destruction at neutral and acidic pH values were studied. Uridine monophosphate did not form various ionic forms, unlike cytidine monophosphate, which second ionic form was due to protonation of nitrogen. In the experiment, this form did not show significant differences in stability. Some slight difference in the composition of UMP and CMP impurities was found at рН 1.2: there was an insignificant formation of an impurity of uridine in the UMP solution (0.15%) and cytidine in the CMP solution (0.12 %), both impurities were at the level of the identified limit. Other impurities were up to 0.2 and 0.4% for UMP and up to 0.2% for CMP.

At рН 5.0 and 9.18 other impurities for UMP and CMP were below the identified limit. An impurity of cytidine with a slight increase (up to 0.15%) at рН 5.0 was detected in the CMP solution. Uridine was not formed in the UMP solution at this рН. At рН 9.18 the main impurity in the solutions of UMP and CMP was not formed.

The analysis of UMP and CMP solutions in buffer solutions from 3.5 to 5.3 under the temperature exposure (at 120°C for 15 minutes and at 40°C for 7 days) showed an increase in the impurity of uridine and cytidine, and it was due to the hydrolysis reaction by the ester bond. At pH of 4.2 or more, this process was somewhat slowed down and stabilized.

Based on these data the conclusion about the predicted compatibility of APIs in solution with a slightly acidic pH level was made.

To experimentally confirm the compatibility of APIs, the samples of binary solutions and solutions containing all APIs were prepared. All solutions were prepared in compliance with therapeutic concentrations of APIs.

When calculating the actual amounts of substances for the preparation of solutions in therapeutic concentrations it is necessary to take into account the amount of water, which can be adsorbed or crystallized, or of combined composition, in the substances. The nature of the water component of API substances was clarified in a powder X-ray diffraction study of the phase composition of samples using “Siemens D500” a powder diffractometer²⁰ (Bragg-Brentano geometry /2, radiation CuKα, = 1.54184Å, a monochromator or a filter on the secondary beam, step-scanning 2 = 0.02 °).

Each sample was ground in a mortar and compacted in a glass cell with a 20x10x0.5 mm cavity for the sample, the cell was then placed in a sample holder for imaging. The phase identification was performed using a PDF-1 file included in the diffractometer software and data from the Cambridge structural data bank. The quantitative calculation of X-ray diffraction patterns was performed using the Rietveld method²¹^,2² (FullProf program). The instrumental profile of the lines required for calculating the average crystallite size using this method was determined by a LaB₆ X-ray diffraction pattern obtained under similar conditions. The calculation results are given in Fig. 2.

Fig. 2: The results of refinement of the X-ray diffraction pattern for sample 1 (UMP) using the Rietveld method

Fig. 2 shows the experimental and calculated X-ray diffraction patterns that practically coincide (Mark 1), a number of vertical lines (Mark 2) indicates the Bragg position of the lines; the lower curve (Mark 3) demonstrates the difference between the experimental and calculated intensity at each point.

It has been found that sample 1 (UMP) is UMP in the form of a heptahydrate, and the Cambridge bank has data only for this form; moreover, these data are not entirely accurate – there is a disordering of sodium atoms in the structure, which makes it impossible to reliably separate sodium atoms and water molecules that are part of their coordination sphere.

The results of refinement of the X-ray powder diffraction pattern show a satisfactory correspondence between the diffraction pattern observed and the bank model of the structure, but in the X-ray pattern there are several weak lines at the background level, and they do not correspond to this model. It can be assumed that these lines appear as a result of the partial loss of crystallization water and the formation of one or more hydrates with the different water content, and there are no literature data for them²³.

To confirm the assumption of the water loss, three additional X-ray diffraction patterns were taken: the initial freshly ground sample (sample 2а); the same sample after exposure in a cell for 3 days in air (sample 2b); the same sample after exposure in a cell for 4 days in a sulfuric acid desiccator (sample 2с).

It can be seen from X-ray diffraction patterns given in Fig. 3 that all three samples differ significantly in their composition and, most likely, samples 2b and 2с are not single-phased. Thus, it can be concluded that sample 1 is a heptahydrate of UMP, which, being an unstable substance, slowly loses crystallization water during trituration and maturation.

Calculation of the X-ray diffraction pattern obtained for sample 2 (CMP) has allowed us to find out that it is CMP in the form of a thirteen-water crystal hydrate with a small admixture of another hydrate (Fig. 3). Both crystallohydrates are in a nanocrystalline state; thus, it has been assumed that efflorescence processes can also occur in this salt as in sample 1. It should be noted that the bank data from the structure of 13-hydrogen crystallohydrate is similar to that of sample 1 (disordered sodium and unreliable separation of sodium and oxygen peaks), while the structure of the second crystallohydrate contains a lot of crystallization water, which is not part of the coordination sphere of sodium atoms. Therefore, the phase content of both substances in the sample may differ markedly from that obtained as a result of the X-ray diffraction pattern calculation. The results of the X-ray diffraction pattern refinement of sample 2 (CMP) are presented in Fig. 4 (marks 1-3 indicate lines and curves as in Fig. 1).

Fig. 4: The results of refinement of the X-ray diffraction pattern for sample 2 (CMP) using the Rietveld method

The phase composition of the PHC substance sample was also studied. It was found that sample 3 (PHC) was in the form of pyridoxinium chloride, which corresponded to the literature data and confirmed by calculation. No impurity lines were observed on the X-ray diffraction pattern.

The results of the study of the phase composition of APIs substances determined the identity of substances, the presence of impurities and the type of water present in the substance (adsorbed or crystallized). These data were taken into account when choosing a method for determining the actual water content in substances (for UMP and CMP – according to Fischer, for PHC – by the loss on drying) and for factorizing their quantities when preparing binary solutions and solutions of all APIs in compliance with therapeutic concentrations to study compatibility.

The API interaction was determined by various parameters – changes in color, transparency, рН, the total impurity content, etc. In these studies, if binary mixtures showed an increase in impurities associated only with the influencing factors and the absence of impurities that would characterize the API interaction, it was considered that the ingredients were compatible²⁴.

The solutions were exposed to various factors (heating, рН, the action of an oxidizer, a reducing agent).

No impurities characterizing the API interaction were detected in binary solutions of UMP and CMP. In binary solutions of UMP with PHC and CMP with PHC there was an increase in the formation of impurity А of pyridoxine when heating on a boiling bath for one hour compared to the fresh solution. At room temperature no increase of impurities in the solutions was observed. Thus, an increase in impurities associated only with the influencing factor was detected. There were no impurities that would characterize the API interaction.

The study of binary model samples and solutions containing all APIs at рН 4.0-4.8, which is acceptable for PHC, has shown that the stable existence of disodium salts of UMP and CMP is also possible in this range. Therefore, it can be assumed that a new combined drug may include these APIs provided that the pH is maintained at approximately 4.0-4.8.

The next potential factor of instability of the APIs studied is the effect of oxidizing agents. The behavior of APIs in binary model samples and in solutions containing all APIs under the action of an oxidizer was also studied. The contribution of this influencing factor to the instability of model solutions was determined by the parameter “total impurity content”. Based on the analysis of the results the absolute value of the increase of impurities relative to the freshly prepared solution (in %) by the factor “action of oxidizing agents (hydrogen peroxide)” was calculated. This value was 11 %.

Therefore, based on the analysis of the effect of factors affecting the stability of API solutions of the drugs developed the ability to hydrolytic and oxidative processes was determined due to the pH level, the action of the elevated temperature and oxidizing agents.

CONCLUSIONS:

Thus, as a result of the research, the compatibility of APIs that have an alkaline and acidic nature has been studied, and the optimal pH level for a combined solution based on them has been determined. The studies conducted have allowed us to determine the directions of further research for developing the composition of a parenteral dosage form. This research is in choosing the optimal buffer system, which is very important for maintaining the stability^25,26 of active substances with different pH levels and their simultaneous presence. To achieve the chemical stability of the solution, it is necessary to choose a system of excipients that prevent possible oxidation processes.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 23.12.2020 Modified on 20.04.2021

Research J. Pharm. and Tech 2021; 14(12):6228-6234.

DOI: 10.52711/0974-360X.2021.01078