DEVELOPMENT OF PERORAL HYPOLIPIDEMIC FORMULATION BASED ON SULFATED ARABINOGALACTAN IN THE FORM OF POTASSIUM SALT

For citation: Ya.A. Kostyro, K.V. Alekseev. Development of peroral hypolipidemic formulation based on sulfated arabinogalactan in the form of potassium salt. Pharmacy & Pharmacology. 2021;9(6):441-453. DOI: 10.19163/2307-9266-2021-9-6-441-453 © Я.А. Костыро, К.В. Алексеев, 2021 Для цитирования: Я.А. Костыро, К.В. Алексеев. Разработка пероральной лекарственной формы гиполипидемического действия на основе сульфатированного арабиногалактана в виде калиевой соли. Фармация и фармакология. 2021;9(6):441-453. DOI: 10.19163/23079266-2021-9-6-441-453 DEVELOPMENT OF PERORAL HYPOLIPIDEMIC FORMULATION BASED ON SULFATED ARABINOGALACTAN IN THE FORM OF POTASSIUM SALT Ya.A. Kostyro1, K.V. Alekseev2 1 Federal State Budget Institution for Science “A.E. Favorsky Irkutsk Institute of Chemistry Siberian Branch of the Russian Academy of Sciences” 1, Favorsky St., Irkutsk, Russia, 664033 2 Federal State Budgetary Institution “Research Zakusov Institute of pharmacology” 8, Baltyskaya St., Moscow, Russia, 125315 E-mail: yanakos@irioch.irk.ru


INTRODUCTION
Atherosclerotic lesion of blood vessels is one of the most widespread and serious disorders of the homeostatic system. The use of drugs with a polyvalent mechanism of pharmacological action for the prevention and treatment of this pathology is justified, and the application of heparinoids for this purpose is very promising [1][2][3][4][5][6][7][8].
Foreign hypolipidemics based on natural and semi-synthetic heparinoids are limited [9][10][11][12][13] due to their physico-chemical properties (they are extremely easily destroyed in the digestive tract that impairs their absorption and transport to blood) 1 .
In the Russian Federation (RF), heparinoids are represented by sulodexide-derived drugs, which exert angioprotective, profibrinolytic and antithrombotic effects 2 . Sulodexide is an organopreparation, extracted from the mucous membrane of the small intestine of a pig. It is a natural mixture of glycosaminoglycans (80% of heparin-like fraction with a molecular weight of 8 kDa and 20% of dermatansulfate) [14][15]. The effective application of Sulodexide for the secondary prevention of coronary atherosclerosis and its complications is proven by clinical multicenter placebo-controlled trials [16][17][18]. Sulodexide-based medicines cause serious side effects in view of the peculiarities of the technology for obtaining 1  organopreparations. For instance, these drugs contain trace proteins and histamine-like substances that can lead to allergic reactions 3 .
Thus, the search for heparinoids devoid of similar disadvantages is an urgent problem in medical and pharmaceutical sciences.
A directed chemical modification of arabinogalactan, the main polysaccharide of Siberian larch, made it possible to develop the original pharmacologically active compound -sulfated arabinogalactan in the form of potassium salt (Agsular®), in A.E. Favorsky Irkutsk Institute of Chemistry Siberian Branch of the Russian Academy of Sciences 4 . This is a semi-synthetic heparinoid exhibiting anticoagulant and hypolipidemic activities, having a high relative bioavailability after the peroral administration (54.4%) [19]. The improved technology for the synthesis of potassium salt of sulfated arabinogalactan made it possible to obtain a pharmaceutical grade substance [20,21]. The safety and efficiency of the latter has been proven by the pre-clinical trials conducted together with the Research Institute of Toxicology (St. Petersburg) in 2009-2011 [22,23].
Thus, sulfated arabinogalactan in the form of potassium salt is a promising building block for the development of peroral medicines. Today, the most popular are such peroral finished dose forms (FDFs) as tablets and capsules, which have a wide range of possibilities and advantages [24][25]. Том 9, Выпуск 6, 2021 ОРИГИНАЛЬНАЯ СТАТЬЯ THE AIM was to develop solid peroral dose forms (capsules and film-coated tablets) for the prevention and treatment of atherosclerotic lesion of blood vessels, on the basis of potassium salt of sulfated arabinogalactan which would be suitable for further clinical trials of these forms.

Study of physico-chemical properties
The shape and size of the particles were determined by scanning electron microscopy using a SEM 525-M scanning electron microscope (Philips, the Netherlands) and by dynamic light scattering using a Zetasizer Nano ZS analyzer (Malvern Instrument, UK). The measurements were carried out at the angles of 13° and 173° in plastic cuvettes (1×1 cm). The average hydrodynamic diameter was calculated by the analysis of fluctuations in the intensity of light scattering of spherical particles. The results were processed using the Dispersion technology Zetasizer family software v7.01.
The mass-average molecular mass was determined by high performance size exclusion chromatography on an Agilent 1260 Infinity chromatograph (Agilent Technologies, Germany), a PL aquagel-OH 408 nm column, 300×7.5 mm, a refractometer detector, a sample concentration of 1 mg/ml, a sample volume of 20 μL. The elution was carried out with a 0.1 M aqueous solution of lithium nitrate (LiNO 3 ) at 30°C with the eluent flow rate of 1 ml/min. The column was calibrated using D-galactose and dextrans with molecular weights of 5, 12, and 25 kDa (Sigma, United States). The calculations were performed using the Agilent ChemStation software.
The elemental analysis was performed on a Flash2000 automatic elemental analyzer (Thermo Scientific, Italy).
The average density (ρ av , kg/m 3 ) was determined by the ratio of the sample mass (m≥0.3 kg) to the entire volume occupied by it (V) in natural conditions, including the cavities and pores in it (V pore ), and calculated by the formula: (1) where: ρ av is an average density, kg/m 3 ; m is the mass of the sample, kg; V is the sample volume, m 3 ; V s.ph. is a volume of a solid phase, m 3 ; V pore is a volume of cavities and pores, m 3 .
Specific surface area (S sp.s. , m 2 /kg) was determined as the sum of the surface of all particles, the total mass of which was 1 kg, and calculated by the formula: . .
where: S sp.s. is a specific surface area, m 2 /kg; k is a particle shape factor; d s.ph. is a particle diameter of the solid phase, m; ρ av is an average density, kg/m 3 .
The hygroscopicity (H, %) was determined under the extreme conditions (in a chamber with a relative humidity of 100% for 24 hours) [26]. The kinetics of the moisture absorption was calculated by the formula: (3), where: H is a hygroscopicity, %; m 0 is the mass of the powder before being kept in a chamber with a 100% air humidity, g; m is the mass of the powder after the exposure in the chamber with a 100% air humidity, g.
The flowability (g/s), the angle of repose (°) and the bulk density (g/ml) were determined according to the methods described in GPhM.1.4.2.0016.15 "The degree of flowability of powders" 8 on a device for determining the flowability TK-1 (Pharmatech, Ukraine) and on a device for vibration compaction of powders 545-P-AK-3 (Pharmatech, Ukraine).
The compressibility (breaking strength, N) was determined according to the following procedure: a weighed portion (0.3 g) of the powder was compressed in a matrix with a diameter of 9 mm on a PGR 400 manual hydraulic press (Infraspek company, Russia) at the pressure of 120 MPa. After pushing the tablet out of the matrix, the compressive strength was determined using a tablet and granule strength tester TT-03 (China) in kg of load, which was recalculated in N [26].
The Carr index (%) was calculated by the formula [28]: where: ρ bd0 is a bulk density before the compaction, kg/m 3 ; ρ bd1 is a bulk density after the compaction, kg/m 3 .

Technology for tablets production
Briquetting was carried out from a powder mixture of active ingredients and auxiliary substances obtained in a PSM "plough" mixer (Pharmag, Germany) at 50-250-450 rpm for 30 min, on a hydraulic hand press PGR 400 (matrix diameter -25 mm) (Infraspek company, Russia) under the pressure of 200 bar. The briquettes were grinded in a dry granulator DG (Pharmag, Germany).
The obtained granules were fractionized on an automatic diffuser (WEB MLW-Labortechnik, Germany); for tableting, granules with a size of at least 1 mm were used.
Tableting was performed using the punches to the tablet press CPR-6 (Dott. Bonapace & C s.r.l., Italy), allowing biconvex tablets with a diameter of 12 mm to be obtained.
The application of 30% aqueous dispersion of the finished film coating Aquacoat ECD was performed in a CP-9 coater (Pharmag, Germany) with a fixed operating angle of the device (45°). The coated tablets were dried using infrared light (150 W).

Technology for capsules production
The encapsulation was carried out using the finished semi-closed hard-gelatin capsules Empty Hard Gelatin Capsules (Shaanxi Genex Bio-Tech Co., Ltd., China) with the sizes No. 00-1. The capsules were filled with the help of manual machines MC-1.2 (Multipharma, Italy) of the appropriate size.

FDF quality assessment
The quality of the obtained FDFs was evaluated according to the standard pharmacopoeial methods 9 : 1. GPhM.1.4.2.0009.15 "Uniformity of the mass of dosage forms", the determination of the uniformity of the mass of the capsules contents, was carried out using laboratory electronic scales LV 120-A (Sartogosm, Russia); 9 Ibid.
4. GPhM.1.4.2.0014.15 "Dissolution for solid dosage form", the determination of the amount of active substance released from the pharmaceutical dose forms for a certain period of time, was carried out in purified water using a device for determining the solubility 545 AK7-00-00 (Pharmatech, Ukraine) by spectrophotometric method on a UV/VIS spectrometer Lambda 35 (Perkin Elmer, USA).

Statistical processing of results
All obtained data were statistically processed (P = 95%) using the Student's test in accordance with the requirements of GPhM.1.1.0013.15 10 .

Physico-chemical and technological properties
The most economically beneficial and technological method for tableting of powders is their direct compression. However, despite the advantages of this method, it is applicable for a limited number of pharmaceutical substances and depends on their physico-chemical and technological properties [29].
Sulfated arabinogalactan in the form of potassium salt (Fig. 1) is a hydrophilic functionalized biopolymer, where sulfate groups are located at the C2 and C4 atoms of the main galactan chain and at the C6 atom of the terminal galactose residues of the main and side chains of the polysaccharide. According to the data of elemental analysis and characteristics of the molecular weight distribution, the degree of substitution of the macromolecule is 0.4, i.e. about one sulfate group is per one structural unit of the biopolymer [21].

Figure 1 -Fragment of sulfated arabinogalactan in the form of potassium salt
Visually, it is a white or white-creamy amorphous powder with a weak characteristic odor. According to the data of scanning electron microscopy ( Fig. 2A), its particles have a spherical shape and form large agglomerates with the size in the range of 200-600 nm (Fig. 2B).
To evaluate suitability of a sulfated arabinogalactan potassium salt for tableting by direct compression methods, its physico-chemical (Table 1) and technological properties ( Table 2) were studied.
The results of the carried out experiments showed that sulfated arabinogalactan in the form of potassium salt is finely dispersed amorphous powder with high hygroscopicity (20.07%). It is almost uniform in the particle size distribution, which does not meet the requirements to powders for direct pressing, since it has low technological properties.
Powders with a particle size of 0.5-1 mm are known to be the best for a direct compression [24]. This parameter for sulfated arabinogalactan in the form of potassium salt is only 8.6%, and the amount of the fine fraction is more than 79%. That significantly affects the material flowability, leading to its almost complete absence (1 g/s). In this case, not the entire sample spilled out by gravity from the device funnel, there was a partial freezing, dusting and static-charge accumulation of the powder. The Carr (24.91%) and the Hausner (1.33) indices confirm a very poor compressibility (24.5 N) of sulfated arabinogalactan in the form of potassium salt, accompanied by the complexity of filling the matrix and ejecting the finished tablet from it. All these can lead to a violation of the active substance dosage accuracy in the FFC.
In this regard, the next stage was to bring technological properties of sulfated arabinogalactan in the form of potassium salt into compliance with the properties of a technologically conditioned mass, suitable for obtaining tableted FFC by the direct compression method.

Technology for tableted FDF preparation
Previously, a tableted FDF based on sulfated arabinogalactan in the form of potassium salt with a dosage of the active component of 500 mg, had been designed. Anhydrous lactose, low-molecular polyvinylpyrrolidone and aerosil were used as adjuvants [34]. The shelf life of the tablets, obtained by direct compression with a monocomponent composition of adjuvants, was no more than two and a half years because of a high hygroscopicity of such sulfated arabinogalactan and the absence of protective coatings from the atmospheric moisture in the tablets.
Therefore, in order to increase stability and extend the shelf life of tablets derived from sulfated arabinogalactan, the composition and technology (the selection of modern combined adjuvants to improve flowability and compressibility; the selection of film moisture-protective coatings for tablets; the approbation of wide used granulation method for the directed enlargement of the particles), were experimentally optimized.    Granulometric composition: more than 60% should be fractions with a particle size of 0.5-2 mm; less than 20% -fractions with a particle size of up to 0.2 mm [30].  [26]. Bulk density before compaction, g/ml 0.633±0.032 > 2.0 g/ml -very heavy; from 1.1 to 2.0 g/ml -heavy; from 0.6 to 1.1 g/ml -medium; < 0. 6    The content of aerosil and calcium stearate in film-coated tablets should be from 0.054 g to 0.066 g (0.06 g±10%) and not more than 11% of the average tablet weight (1.082 g) -0.119 g.

Quantification
Spectrophotometric Content of the substance of sulfated arabinogalactan in the form of potassium salt in one film-coated tablet should be from 0.475 g to 0.525 g (0.5 g±5%).   When a drop of 95% ethyl alcohol is added, the solution becomes cloudy.

Reaction of metachromasia.
When a 0.005% solution of toluidine blue is added, the color of the dye solution changes from blue to lilac. The content of aerosil and calcium stearate in capsules No. 00 should be from 0.054 g to 0.066 g (0.06 g±10%) and not more than 11% of the average weight of the capsule contents (1.061 g) -0.117 g. The content of aerosil and calcium stearate in capsules No. 0 should be from 0.052 g to 0,.064 g (0.058 g±10%) and not more than 11% of the average weight of the capsule contents (0.808 g) -0.089 g. The content of aerosil and calcium stearate in capsules No. 1 should be from 0.051 g to 0.063 g (0.057 g±10%) and not more than 11% of the average weight of the capsule contents (0.682 g) -0.075 g. From a large assortment of modern combined adjuvants, Ludipress®, consisting of a complex of α-lactose monohydrate (93.4%), polyvinylpyrrolidone (Kollidon 30) (3.2%) and 3D polyvinylpyrrolidone (Kollidon CL) (3.4%) 14 , was chosen. Ludipress has the same qualitative composition of ingredients that had been used earlier to obtain tablets based on sulfated arabinogalactan in the form of potassium salt, but it is characterized by the optimal quantitative combination. That makes it possible to obtain sufficiently stable tablets under a low pressure of compression and to improve the technological properties of the active substance [35].
Since sulfated arabinogalactan in the form of potassium salt is a hygroscopic and thermolabile compound, the briquetting technique (a kind of dry granulation method) was employed to improve the flowability of the tableting mixture and prevention of its separation [36][37].
For the experiments, the model compositions of tablet mixtures were obtained, and their main technological properties were studied ( Table 3).
As Table 3 shows, only tablet mixture No. 1 satisfies the technological requirements of the tableting process, because it has an excellent flowability, an average breaking strength and a very good compressibility [31,38]. The results obtained confirm the right choice of its optimal composition and technology. Tablet mixtures No. 2 and No. 3 can be used to prepare FDFs for the peroral administration as capsules.
To protect sulfated arabinogalactan in the form of potassium salt from the effects of the atmospheric moisture, an effective film coating for tablets was selected. Hydrophobic polymers or hydrophobic polymers in combination with hydrophilic additives or hydrophilic polymers in combination with hydrophobic additives are most often used as moisture-proof coatings [39]. Such structure-forming moisture-protecting compounds as ethyl cellulose (insoluble in water, but permeable to aqueous solutions) [40] and cetyl alcohol, possessing emulsifying and stabilizing properties, were chosen as film-forming agents [41].
In modern pharmaceutical engineering, finished commercial film-forming compositions (FFCs) are widely used in the form of solutions consisting of a film-forming agent, plasticizer, colorant and solvent taken in the optimum ratio, as well as semi-products in the form of granules (powders). From the latter, a film-forming solution (dispersion) is prepared immediately prior to application using an appropriate solvent [42][43][44][45]. From all the nomenclature of FFCs containing ethyl cellulose and cetyl alcohol, Aquacoat ECD film coating was selected for the experimental studies on the protection of tablets from the atmospheric moisture; the tablets were based on sulfated arabinogalactan in the form of potassium salt. This FFC is used as a 30% aqueous dispersion obtained immediately prior to use. The advantage of the specified HPC is the possibility of its application not only for creating a barrier coating (the application in the amount of 1-2% of the tablet weight) but also for maintaining the pH-independent mode of the release (the application in the amount of 8-15% of the tablet weight 15 . In addition, Aquacoat ECD is compatible with most alcohol-and propyleneglycol-soluble dyes, as well as various types of plasticizers. It promotes the formation of high-resistant, stable, easily dissolvable (in the stomach) coating, giving the tablet a pleasant appearance and effectively protecting the active substance from the atmospheric moisture [46].
The technology for the preparation of a solid pharmaceutical for a peroral administration in the form of film-coated tablets based on sulfated arabinogalactan in the form of potassium salt according to the formulation of the tablet mixture No. 1, was elaborated using Pharmag equipment and a tablet press with one CPR-6 compression station (Fig. 3).
As a result, biconvex tablets with a diameter of 12 mm of high strength (44.5±2.2 N) were obtained.

Quality evaluation of the tableted FDF
The quality of the tablets obtained was evaluated according to the following parameters: description, authenticity, dosage uniformity, disintegration, dissolution, determination of auxiliaries (aerosil and calcium stearate) and quantitative determination (Table 4).
According to the studies, the developed solid dosage formulation of FDF for the oral administration in the form of film-coated tablets based on sulfated arabinogalactan in the form of potassium salt, withstands all tests and fully complies with the requirements of the GPhM.1.4.1.0015.15 "Tablets" 16 .
The data obtained form the basis for the development of regulatory documents and laboratory regulation for the production of "Agsular® film-coated tablets 500 mg".

Technology for preparation of encapsulated FDF
In order to prepare FDF based on sulfated arabinogalactan in the form of potassium salt in the form of capsules, the granulate, according to the recipes of tablet mixtures No. 1-3, was placed in hard gelatin capsules of size No. 00-1, respectively ( Table 5).
The elaboration of technology for the preparation of a solid pharmaceutical for the peroral administration in the form of capsules based on sulfated arabinogalactan in the form of potassium salt, was carried out using Том 9, Выпуск 6, 2021

ОРИГИНАЛЬНАЯ СТАТЬЯ
Pharmag equipment and sets of manual machines for filling hard gelatin capsules (MС-1.2) of the appropriate size. That was due to the forced feeding of the tablet mixture into the capsule and additional pre-pressing of the material, according to the proposed technological scheme (Fig. 4).

Quality evaluation of FDF
The finished capsules were controlled according to the developed specification ( Table 6). The following quality indicators were investigated: description, authenticity, mass uniformity, dosage uniformity, disintegration, dissolution, determination of auxiliaries (aerosil and calcium stearate), quantitative determination.
According to the experiments, the developed peroral solid dosage formulation of FDF in the form of cap-

FUNDING
The work was carried out according to the plans of the scientific research (state task) under the program V.48 "Fundamental physico-chemical studies of physiological processes mechanisms and the creation of pharmacological substances and dosage forms on their basis for the treatment and prevention of socially significant diseases" (2013-2020) according to Project V.48.1.2 "Search for new biologically active substances based on the biomass of coniferous species of Siberia and the Far East, screening of the biological activity of promising compounds. Development of technologies for obtaining natural substances and determination of their practical significance".

CONFLICTS OF INTEREST
Authors declare no conflicts of interest.

AUTHORS' CONTRIBUTION
Yana A. Kostyro -experimental work execution, research results processing, text writing; Konstantin V. Alekseevconcept and design of the study development, management of experimental work, text editing.
sules based on sulfated arabinogalactan in the form of potassium salt withstands all tests and fully complies with the requirements of the GPhM.1.4.1.0005.15 "Capsules" 17 . The data obtained form the basis for the development of regulatory documents and laboratory regulation for the production of "Agsular® capsules 500 mg".

CONCLUSION
In conclusion, the optimum compositions and technology for preparation of solid peroral dosage forms have been experimentally developed on the basis of potassium salt of sulfated arabinogalactan (capsules and coated tablets) for the prevention and treatment of blood vessels atherosclerosis. The data obtained make possible to plan a placebo-controlled clinical trial of the 1 st phase.