EFFECT OF SOLID DISPERSIONS ON THE SOLUBILITY OF METRONIDAZOLE

The aim of the work is to study the effect of solid dispersions using polyethylene glycols of various molecular weights on the solubility of metronidazole in water. Metronidazole is an antimicrobial and antiprotozoal drug. Its low solubility in water limits the use of metronidazole, causing technological difficulties and reducing its bioavailability. The solubility and release of the active substance from dosage forms can be increased using the solid dispersion methods. Solid dispersions are bi- or multicomponent systems consisting of an active substance and a carrier (a highly dispersed solid phase of the active substance or molecular-dispersed solid solutions) with a partial formation of complexes of variable compositions with the carrier material.Materials and methods. The substance of metronidazole used in the experiment, was manufactured by Hubei Hongyuan Pharmaceutical Technology Co., Ltd. (China). To obtain solid dispersions, polyethylene glycols of various molar masses – 1500, 2000 and 3000 g/mol – were used. The solid dispersions were prepared by “the solvent removal method”: metronidazole and the polymer were dissolved in a minimum volume of 96% ethyl alcohol (puriss. p.a./analytical grade) at 65±2°C, and then the solvent was evaporated under vacuum to the constant weight. A vacuum pump and a water bath were used at the temperature of 40±2°C. The dissolution of the samples was studied using a magnetic stirrer with heating, and a thermostatting device. The concentration of metronidazole was determined on a spectrophotometer using quartz cuvettes at the wavelength of 318±2 nm. To filter the solutions, syringe nozzles were used, the pores were 0.45 μm, the filter was nylon. Microcrystalloscopy was performed using a microscope with a digital camera. The optical properties of the solutions were investigated using a quartz cuvette and a mirror camera (the image exposure – 20 sec).Results. Obtaining solid dispersions increases the completeness and rate of the metronidazole dissolution. The solubility of metronidazole from solid dispersions increases by 14–17% in comparison with the original substance. The complex of physical-chemical methods of the analysis, including UV spectrophotometry, microcrystalloscopy and the study of the optical properties of the obtained solutions, makes it possible to suggest the following. The increase in the solubility of metronidazole from solid dispersions is explained by the loss of crystallinity and the formation of a solid solution of the active substance and the solubilizing effect of the polymer with the formation of colloidal solutions of metronidazole at subsequent dissolution of the solid dispersion in water.Conclusion. The preparation of solid dispersions with polyethylene glycols improves the dissolution of metronidazole in water. The results obtained are planned to be used in the development of rapidly dissolving solid dosage forms of metronidazole with an accelerated release and an increased bioavailability.


INTRODUCTION
This work continues the promising scientific area of "solid dispersions in medicine and pharmacy".
At the moment, the study of solid dispersions (SDs) is being carried out at I.M. Sechenov First Moscow State Medical University on the basis of the departments of the Institute of Pharmacy n. a. A.P. Nelyubin: "pharmaceutical technology" and "analytical, physical and colloi-dal chemistry". The work is carried out within the framework of research & development (R&D): "Increasing the bioavailability of drugs using solid dispersions." The expected social-and-economic effect of R&D is the production of innovative drugs with an increased bioavailability at minimal economic costs, as well as an active import substitution.
In dentistry, metronidazole is used for various localized infections caused by anaerobes in periodontal diseases and maxillofacial inflammations. A gel, which includes a combination of chlorhexidine and metronidazole, is used in dentistry. Its indications are: infectious and inflammatory diseases of the oral mucosa and parodontium -acute and chronic -gingivitis, periodontitis, necrotising ulcerative Vincent's gingivitis, postextraction alveolitis, aphthous stomatitis. Metronidazole is used in dentistry for systemic pharmacotherapy.
In dermatology, metronidazole is used to treat rosacea [11][12][13]. The widespread use of metronidazole in gastroenterology, dentistry, dermatology, gynecology, etc. was the cause of the emergence of various dosage forms. Therefore, on the Russian pharmaceutical market, metronidazole is presented in the form of tablets, solutions, creams; is included in gels and suppositories along with other active ingredients. The substance of metronidazole ( Fig. 1) is a crystalline powder of light yellow or white; it is slightly soluble in water, acetone and ethanol (1 : 100), which can limit its use in some cases, causes difficulties of a technological nature in the creation of new drugs, and reduces their bioavailability.
It is possible to increase the solubility and accelerate the release of substances from the dosage form by "the method of solid dispersions" (SDs) [1-10; 14-17]. SDs are either multicomponent systems that include an active substance and a carrier (a solid phase of a drug dispersed in a polymer), or solid solutions of a drug in a carrier. In some cases, the formation of complexes of various natures of the active substance with the carrier material can be observed [1,2,13]. Various polymeric substances are used in the role of the SD carrier [17][18][19].
THE AIM of the work is to study the effect of solid dispersions using polyethylene glycols of various molecular weights on the solubility of metronidazole in water.

MATERIALS AND METHODS
The substance of metronidazole used in the experiment, was manufactured by Hubei Hongyuan Pharmaceutical Technology Co., Ltd. (China). It corresponds to the Product specification file (State Pharmacopoeia, Russia, XIV th ed., Pharmacopeial monograph.2.1.0136.18). To obtain SDs, PEGs of various molar masses -1500, 2000, and 3000 g/mol -were used as carriers (Merck, Germany).

Technology for preparing solid dispersions with PEG
The literature analysis and the accumulated actual experience make it possible to assert that, in case of PEG, the optimal technology for obtaining SDs is "solvent removal" [3, 5-7, 10, 20, 21-23]. The calculated amounts of metronidazole and polymer were dissolved in a minimum volume of 96% ethyl alcohol (analytical grade) by heating to 65±2°C, then the solvent was evaporated under vacuum to the constant weight. A UED-Lab 115 vacuum pump (China) and a UT-4301E water bath (Ulab, China) were used at the temperature 40±2°С [1,2,16,18,22].

Study of metronidazole dissolution
Carried out according to the technique described in the works of Krasnyuk I.I. et al. [1] and Beliatskaya A.V. et al. [2]. The main problem was the impossibility of using the methods according to General Pharmacopoeia Monograph 1.4.2.0014.15 "Dissolution for solid dosage forms". This is associated with the preparation of saturated solutions of metronidazole under study. The SDs obtained in the work are very sticky, thick white masses or powders of soft consistency, prone to sticking together. The conditions described in GPM 1.4.2.0014.15 for studying the dissolution of these objects, are not always acceptable. In this regard, a modified technique was used during the work. Preliminary studies [6][7][8] prove that the dissolution test on the "rotating basket" device presents results similar to those obtained by the modified methods.
Thus, the dissolution of the samples was studied using a heated magnetic stirrer equipped with an RCT BASIC thermostatting device (IKA, Germany). The samples for dissolution were selected in such a way that a saturated solution of metronidazole would be achieved. The temperature of the dissolution medium was 37±1°C. The samples were immersed in 150 ml of purified water; they were continuously stirred (200 rpm).
To study the dynamics of the metronidazole dissolution, the samples (5 ml) were thieved at the intervals of 5, 10, 15, 20, 30, 40, 50, 60 min. The medium was replenished up to 150 ml with purified water. The samples were filtered.

Measurement of metronidazole concentration
In the experiment, a UNICO2800 spectrophotometer (Unitedproducts & instruments, USA) and quartz cuvettes (the absorbing layer of 10 mm) were used. If necessary, the samples were diluted with purified water, the optical density of the resulting solution was measured at the wavelength of 318±2 nm (the maximum absorption of metronidazole). The results are presented in Table 1, Fig. 2, 3.

Filtration
The filtration was carried out using syringe nozzles with Minisart® filters (Sartorius, Germany) with a pore diameter of 0.45 μm, the filter was nylon.

Microcrystalloscopy
A Levenhuk D50LNG microscope (PRC for Levenhuk, Inc., USA) with a digital camera was used. The study was carried out according to the methodology [1,2,6,7,9]. In case of the metronidazole substance, the powder was placed on a glass slide, mixed with a drop of vaseline oil, covered with a cover glass, and microscoped. In case of SD, a drop of the solution of metronidazole and PEG (in the proportions corresponding to SD) in 96% ethyl alcohol was applied to a glass slide, the solvent was completely removed and microscoped.
Separately, PEGs were studied in a similar way. A drop of the PEG solution was applied to a glass slide, the solvent was completely removed, and the PEG was solidified and microscoped.
The recrystallized substance of metronidazole was additionally investigated after the removal of alcohol. A drop of the metronidazole solution in 96% ethyl alcohol was placed on a glass slide, microscoped after a complete removal of the solvent. The micrographs of the studied samples with the microscopic condition are shown in Fig. 4.

Study of the optical properties of solutions
A quartz cuvette (the layer of 50.0 mm) was used. The cuvette was filled with a filtered solution of the studied sample. An opaque partition with a hole (1 mm in the diameter) was placed between the cell wall and the light source. A thin beam of white light was directed through the hole onto the cuvette. In the darkened room, the digital images of the Faraday-Tyndall phenomenon were filmed. A Canon 5D MarkII SLR camera (the image exposure of 20 sec) was used. The results are shown in Fig. 5.

RESULTS AND DISCUSSION
Polyethylene glycols (PEGs) are promising carriers of solid dispersions [10]. PEGs are tasteless and odorfree, readily soluble in water and alcohol, chemically stable, biologically harmless, resistant to high temperatures during sterilization. PEGs are insensitive to fluctuations in pH and the presence of electrolytes; they are resistant to the action of microorganisms due to the presence of primary hydroxyl groups in the molecule [3,21]. The PEG consistency depends on the molecular weight. Up to 400 g/mol, PEGs are viscous colorless liquids; with a mass of more than 400 to 1000 g/mol; they are substances with the consistency of soft wax; PEGs with a mass of 1500 g/mol and more are solid. In view of the fact that the obtained data are later planned to be used in the development of solid, rapidly dissolving dosage forms of metronidazole, PEGs of a solid consistency with weights of 1500, 2000, and 3000 g/mol were chosen as the actual objects of research. The selected polymers are often used as auxiliary substances in the production of tablets and granules [5].
The use of preparations based on SD and PEG is promising due to the bioadhesive qualities of PEGs (as high molecular weight compounds). Upon contact with mucous membranes or skin, PEG macromolecules are adsorbed and, as a rule, increase the permeability of cell membranes, promoting active transmucosal transfer of the active substance. The analysis of patent and scientific literature did not reveal information on the use of PEGs in the technology of solid dosage forms as carrier polymers for the preparation of SDs with metronidazole in order to increase its solubility in water.
Based on the analysis of the scientific literature and preliminary actual research, the range of optimal metronidazole:PEGs were determined: from 1:1 to no more than 1:5 (by weight) [1][2][3][4][5][6][7][8][9]. Taking into account the physicochemical properties of PEG (as an excipient) and its effect on the technological characteristics of solid dosage forms (for example, on their strength), this range of ratios is optimal for the future inclusion of metronidazole SD with PEG in the composition of solid dosage forms. Том 9, Выпуск 3, 2021 оригинальнаЯ статьЯ Table 1     To study the dissolution of weighed portions of the studied samples, the SD was taken in excess with respect to the solvent (purified water). The relative error for the average concentration values is ≈4.79%. The change in the solubility was calculated as the ratio of the concentration of a saturated solution of the studied sample to the concentration of a saturated solution of the metronidazole substance 60 min after the beginning of the dissolution process. By the end of the experiment, the solutions of all studied samples were cloudy and saturated. The original substance of metronidazole dissolves rather slowly (Fig. 2). After 5 minutes from the beginning of the experiment, the concentration of metronidazole reaches 7.767 mg/ml and then slightly increases, reaching an almost constant value of ≈9 mg/ml by 30 minutes.
Based on the data obtained (Table 1; Fig. 2, 3), it can be seen that in some cases metronidazole dissolves better from SD, and its solubility depends on the selected polymer and on the mass ratio of metronidazole:PEG in SD.
When using PEG-1500, the greatest increase in the solubility of metronidazole is observed in the case of SD metronidazole: PEG-1500, obtained in the mass ratios of 1:3 and 1:5 -by 14-17%. The concentration of metronidazole in the solutions of these SDs by the end of the experiment reaches 10.244 mg/ml and 10.534 mg/ ml, respectively. When using PEG-2000, the greatest effect on the dissolution of metronidazole is observed in the case of SD metronidazole: PEG-2000, obtained in the mass ratio of 1: 1. The concentration of metronidazole in the solution of this SD by the time of 60 minutes reaches 10.402 mg/ml, which is 16% higher than that of the substance solution at the same time. An increase in the content of PEG-2000 in SD does not provide any pronounced increase in the solubility of metronidazole.
Thus, for SD metronidazole PEG-2000 (1:5), the solubility of metronidazole is 9.686 mg/ml, exceeding the substance solubility by 7.6% (Fig. 3). The use of PEG-3000 to obtain SD both in the ratios of 1:1 and 1:5, similarly slightly increases the solubility of metronidazole -up to 9.639 and 9.751 mg/ml, respectively (by 7.1 and 8.3%). The use of SD does not increase the dissolution rate of metronidazole in all cases. In this case, the dissolution rate of metronidazole can both increase and decrease in the first 20-30 minutes.
For solutions of some SDs, the phenomenon of supersaturation is observed. Upon dissolution of SD metronidazole: PEG-1500 (1:1) and SD metronidazole: PEG-2000 (1:1), the concentration of metronidazole sharply increases to the maximum value during the first 15 minutes. Then, probably as a result of recrystallization, the concentration decreases, with the output of the values "on the plateau" (50-60 minutes). Thus, the greatest increase in the rate of metronidazole dissolution is observed from SD with PEG-1500 (1:1). At the moment of 15 minutes from the beginning of dissolution, the concentration of metronidazole in the solution of this SD reaches its highest value -12.885 mg/ml, which is 57% higher than the value of the concentration in the solution of the substance at the similar point of time. However, further on, by the 40 th min, the concentration level of metronidazole in the SD solution decreases to the value of ≈9.8 g/ml. In the authors' opinion, the above-described fluctuations in the metronidazole concentration of the SD solutions are associated with a number of mutually opposite processes. On the one hand, these are the processes of the metronidazole release and the PEG matrix upon dissolution of SD, and its transition into an aqueous medium in a molecular colloidal form. In this case, PEG plays the functions of a solubilizer (with a low content in SD) and/or colloidal protection, stabilizing the previously achieved high level of the metronidazole concentration. On the other hand, the processes of metronidazole recrystallization, coagulation of its colloidal particles occur, and the salting-out effect of PEG may affect it. This is especially noticeable in the case of SD with a high polymer content. The balance of these processes and their result in achieving a certain level of metronidazole concentration in the solution of its SD is difficult to describe and is a topic for the SD research.
Based on the results of the microscopy (Fig. 4), the initial substance of metronidazole is particles of the substance with a clearly pronounced crystalline structure. The fragments of crystals are colorless, transparent, oblong, layered, and in most cases, they are of the same type. Regular parallel faces in the form of a rectangular parallelepiped are traced. Presumably, the powder of the substance had not previously undergone intensive micronization. Recrystallized metronidazole differs from the initial substance and has the form of pronounced needle-like, transparent crystals. The edges are even, sometimes collected in stellate clusters. Polymer carrier (PEG) is a colorless, transparent mass located on the surface of a slide with a film without an internal structure. The surface is folded. With a high degree of probability, it can be argued that this is a non-crystalline, amorphous structure. SD with PEG are heterogeneous systems consisting of at least 3-4 phases. Some structures have a partially needle-like architecture-probably, a recrystallized substance. Very small (presumably amorphous), difficult to identify objects were notified. They represent either a stopped initial stage of recrystallization of the substance in a viscous polymer, or (possibly) its polymorphic modification, or a product of complexation with PEG. Considering the fact that the content of metronidazole in SD is from 30 to 50% by weight, the transparent background is most likely to be a solid solution of metronidazole in PEG.
Thus, SD metronidazole: PEG is a complex microcrystalline pattern that combines the features of the initial substance of metronidazole (crystalline and amorphous in nature), PEG, their solid solution, and, possibly, complexation products. When studying SD, thermo-methods are very common. With regard to SD, they are based on the fact that melting or the thermal destruction of the active substance molecule incorporated into the polymer matrix, occurs during or after the thermal destruction of the carrier polymer. The main criterion for the formation of the complex is the disappearance of the thermal effects typical of the active substance, as an individual phase. In this case, when examining SD of metronidazole, the use of, for example, differential scanning calorimetry may be of an auxiliary nature. However, the information obtained by microscopy, in the authors' opinion, is quite sufficient to make an assumption about the effect of the crystallinity of metronidazole in SD on the increase in the solubility of the active substance from the PEG polymer matrix.
In a number of works devoted to the preparation and study of SDs, the method of IR spectroscopy is often used. Based on the analysis of the characteristic bands shift of the active substance in SD, it suggests the formation of hydrogen bonds in the complex between the carrier and drug molecules. The preceding investigation of the authors' suggest that this research method is not always possible to use when studying such multicomponent systems as SD [1][2][3][4][5][6][7][8][9][10]. First of all, this is due to the pronounced shielding effect of polymers (in this case, PEG), due to which it is often impossible to obtain a true picture of the SD components interaction. The IR spectrum of SD with PEG is almost completely identical to the IR spectrum of the studied polymer and contains almost no characteristic bands of the active substance itself. However, in the absolute majority of cases, even if the interaction with PEG was observed, it was not of a covalent nature; it was a weak interaction at the level of the occurrence of hydrogen bonds. The absence of any interaction between the components of the studied SD is indirectly confirmed by UV spectroscopy of the studied samples.
The UV spectrum of metronidazole in SD with PEG is completely identical to the UV spectrum of the initial substance of metronidazole. Microcrystalloscopy makes it possible to conclude that one of the reasons for the increase in the solubility of metronidazole from SD with PEG, is the loss of its crystalline structure even before the SD dissolution in water. At the stage of the SD preparation, when the common solvent is removed under vacuum, metronidazole is partially dissolved in the SD matrix in the medium of the PEG carrier, to form a solid solution. Then, when dissolved in water, SD, as the polymer dissolves, releases the active ingredient in a molecular colloidal form. In this case, PEG, possibly, additionally has a solubilizing effect, stabilizing the concentration of metronidazole. In addition, according to a number of reference materials [17,19,20,[23][24][25], the formation of their colloidal solutions is an important factor contributing to an increase in the dissolution of active substances from SD.
In this regard, the optical properties of the solutions obtained in the work, were studied. For the filtered solutions of all the SDs studied in the work with PEG, the Faraday-Tyndall cone is observed -scattering of light of a bluish tint due to the colloidal-dispersed state of the dissolved metronidazole (Fig. 5).
Herewith, the solutions of the auxiliary substances (PEGs) and the saturated solutions of the metronidazole substance or its mixtures with the studied polymers, similarly prepared for the study, did not demonstrate the Faraday-Tyndall effect. The results obtained, underline the fundamental importance of obtaining SD by the method of "the solvent removal" described in this work, for increasing the solubility of metronidazole in water.

CONCLUSION
The analysis of the data obtained indicates that the improvement in the dissolution of metronidazole from SD, carried out by the method of "the solvent removal" with the use of 96% ethyl alcohol as a common solvent, is associated with a decrease in the crystallinity of metronidazole upon receipt of its SD and solubilization, as well as with the formation of metronidazole colloidal solutions stabilized with PEG when SDs are dissolved.
The optimal PEG for the SD production is PEG with a molecular weight of no more than 1500 g/mol, and the best ratio of SD components (metronidazole: PEG) is 1:1 by weight. The results obtained will be used in the development of the technology of "effervescent" tablets and granules of metronidazole containing its SD with PEG.

FUNDING
The work was carried out without attracting external sources of funding.