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Project titleDesign and research of new mechanochemical complexes and micro-/nanofibers of echinochrome A with polymer matrices and antioxidants for the development of new dosage forms based on it

AffiliationG.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences,

Research area 05 - FUNDAMENTAL RESEARCH IN MEDICINE, 05-501 - Pharmaceutical chemistry, pharmacology (including clinical pharmacology)

KeywordsEchinochrome A, histochrome, drug form, antioxidants, polymers, electrospinning, mechanochemical method, stability, solubility, bioavailability, active agent release

Annotation
Excessive activation of free radical processes entails a whole cascade of negative reactions and pathological processes underlying a number of diseases, so-called free radical pathologies, such as coronary heart disease (СHD), arterial hypertension (AH), cataracts, uveitis, glaucoma, diabetes mellitus, inflammatory diseases of the lungs, as well as age-related changes and many others. At the same time, the incidence, prevalence and disability of all age groups from such diseases tend to increase. An antioxidant drug Histochrome has been developed at the Pacific Institute of Bioorganic Chemistry, which is used in cardiology (state registration number P N002363/01) and in ophthalmology (state registration number P N002363/02). Histochrome has no analogues in the world, since it simultaneously blocks a number of links of free radical reactions: it neutralizes reactive oxygen species (ROS), nitric oxide and peroxide radicals, chelates metal ions, inhibits lipid peroxidation, and regulates the levels of antioxidant enzymes. The active substance of the drug histochrome, which is produced by the Pacific Institute of Bioorganic Chemistry, is the natural pigment of sea urchins echinochrome A (7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone), INN: pentahydroxyethylnaphthoquinone, state registration number P N002362/01. In the last decade, the study of the properties of echinochrome A has attracted the attention of scientists from many countries, as evidenced by the increase in the number of publications on the mechanisms of action of echinochrome A. Anti-inflammatory, antidiabetic, gastroprotective, neuroprotective properties of echinochrome A have been found, and the molecular mechanisms of its cardioprotective action have been studied in detail. Since the search for active compounds and the creation of new drugs is a long, expensive and risky process, it is very attractive to study the possibility of using already known drugs for a new use. Observing the variety of activities exhibited by echinochrome A with an established mechanism of action, the development of new dosage forms based on it seems to be an urgent task. Histochrome is a registered and successfully used drug without identified side effects, however, it is produced only in the form of a solution for injection in ampoules due to the low solubility of echinochrome A in water and the instability of its solutions. The creation of tablets, capsules, eye drops, burn and wound healing films, due to its rational use, will significantly expand the boundaries of the use of echinochrome A preparations for the treatment and complex therapy of a number of socially significant diseases, such as cardiovascular, ophthalmological and neurodegenerative diseases, diabetes 1 and 2 types, inflammatory diseases of the lungs, stomach and intestines, and other diseases associated with the development of oxidative stress in the body. Within the framework of the proposed project, such modern methods of obtaining biomedical materials as electrospinning and mechanochemical mixing will be used for the first time for the development of dosage forms based on echinochrome A. For the first time, data on the physicochemical and biological properties of the obtained complexes will be obtained. Analysis of modern literary sources on the problem shows the promise of using the selected materials, approaches and methods to achieve the project's task. Since a large number of studies of the pharmacological and therapeutic properties of echinochrome A have been carried out, confirming its effectiveness, it can be confidently expected that the resulting solutions and complexes will exhibit biological activity and may turn out to be multitarget. The complexes obtained within the framework of the project based on the domestic drug substance echinochrome A will be the basis for the creation of innovative drugs that do not have domestic and foreign analogues and contribute to an increase in the level of national security.

Expected results
Upon completion of the project, new complexes of the drug substance echinochrome A with polymer matrices and antioxidants approved for use in the pharmaceutical industry will be obtained, which will be stable, have comparable or higher biological activity than the histochrome preparation, as well as with the possibility of targeted delivery and prolonged action of the active component. As a result of the work performed on the project, new knowledge in the field of pharmaceutical chemistry will be obtained on the interaction of the drug substance echinochrome A with polymer carriers and antioxidant additives and the properties of the complexes obtained. The study will open up prospects for the practical application of the obtained solutions and complexes for the creation of new dosage forms, which, due to the rational use of the histochrome preparation, will significantly expand the boundaries of its use for the treatment and complex therapy of a number of socially significant diseases. The results obtained within the framework of the project will be reflected in the application for an invention and in two publications in journals indexed in the Scopus and WoS databases, presented in the form of reports at scientific Russian and international conferences.

Annotation of the results obtained in 2022
At the end of the second year of the project, using mechanochemical technology, complexes of echinnochrome A (Ech) with polymers polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), hydroxypropylmethylcellulose (HPMC), acetylcellulose (AC) and antioxidants ascorbic acid (Asc) and cysteine (C) were obtained . Using X-ray analysis, it was found that echinochrome A after mechanical action is present in several polymorphic modifications, which is important for crystalline medicinal substances, as this increases their solubility and, as a result, bioavailability and increased pharmacological effect. Optimum conditions for obtaining mechanochemical complexes of echinochrome A with polymers and antioxidants have been selected. The physicochemical characteristics of mechanochemical complexes of echinochrome A with polymers and antioxidants were obtained by X-ray analysis, scanning electron microscopy, and absorption spectroscopy; the content, stability, and solubility of echinochrome A in the obtained complexes were determined. When comparing the X-ray diffraction patterns of complexes containing cysteine, it was found that with the visual preservation of the phases of echinochrome A and cysteine, the shift of peaks to the region of smaller angles is observed in the cysteine phase, which corresponds to an increase in the parameters of the unit cell of cysteine, which may indicate the formation of cocrystals with echinochrome A. It has been established that in the complexes Ech + PVP and Ech + HPMC in the ratios of 2.5:97.5 and 5:95% and in all complexes with antioxidants, the content of echinochrome A is about 98% of the load. In the complexes Ech+PEG, Ech+AC in ratios of 2.5:97.5% and 5:95%, the content of echinochrome A averaged about 95% of the load. The solubility of echinochrome A in the obtained mechanochemical complexes in water, phosphate-salt (pH 7.3) and phosphate-citrate (pH 5.2) buffers was studied. In Ech+PEG complexes, the solubility of echinochrome A in water was 0.3 mg/mL, and in buffers it was up to 0.5 mg/mL, while it was previously shown that co-dissolution in all these solvents in the presence of PEG increased the solubility of echinochrome A. In the complexes Ech + PVP and Ech + HPMC, echinochrome A turned out to be soluble up to 0.4 mg / ml in phosphate-buffered saline, in water and phosphate-citrate buffer - up to 0.3 mg / ml, which is also higher than the solubility of echinochrome A in solutions together with PVP and HPMC. The presence of cysteine and ascorbic acid in the complexes of antioxidants did not affect the solubility of echinochrome A. It has been established that in the absorption spectra of aqueous solutions of the Ech+PVP, Ech+HPMC, Ech+PEG complexes, the maxima of echinochrome A at 260 and 337 nm are shifted to the long wavelength region by 5–7 nm, and at 470 nm it has a smoother peak shape with a less pronounced "shoulder" in the region of 525 nm, as well as a rise in the region of 570-700 nm, which indicates the interaction of the functional groups of echinochrome A with those of the polymer components. The presence of such interactions is also confirmed by the difference spectra of the Ech+PVP, Ech+HPMC, Ech+PEG complexes. Interestingly, the absorption and difference spectra of echinochrome A complexes with polymers and cysteine did not differ from the spectra of echinochrome A complexes with the corresponding polymers. Probably, during dissolution, the cocrystals of echinochrome A and cysteine detected by X-ray analysis are destroyed and do not manifest themselves in the absorption spectra. The absorption spectra of echinochrome A complexes with polymers and ascorbic acid also did not differ from the spectra of echinochrome A complexes with the corresponding polymers; however, the maximum at 420 nm disappeared in the difference spectra, which indicates the contribution of ascorbic acid to the interactions of all components in the complexes. The antiradical activity of mechanochemical complexes of echinochrome A with polymers and antioxidants was determined on the model of decolorization of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) cation. Echinochrome A decolorized the ABTS cation by 50% at a concentration of 2.12± 0.08 µg Ech + PVP, Ech + HPMC and Ech + PEG in ratios of 2.5:97.5 and 5:95% showed comparable activity - about 2.2 µg of echinochrome A equivalent was required. Ech + AC in both ratios showed lower activity - about 2.7 µg equivalent of echinochrome A. Complexes of echinochrome A with polymers and antioxidants showed higher activity than echinochrome A, with the exception of complexes with PEG, which showed a comparable effect. For the complexes Ech+Asc+PVP, Ech+C+PVP and Ech+C+HPMC, the IC50 was 1.7-1.8 μg echinochrome A equivalent, and the most active was the complex Ech+Asc+HPMC, IC50 was 1.54±0.11 µg echinochrome A equivalent. A study of the antioxidant properties of mechanochemical complexes of echinochrome A with polymers and antioxidants was carried out on the model of oxidation of a lipid substrate, linoleic acid. All mechanochemical complexes of echinochrome A with polymers and antioxidants showed in this model an average of 10% lower activity. Perhaps this method is not applicable to determine the activity of water-soluble polymers. Thus, new complexes of the drug substance echinochrome A with polymer matrices and antioxidants approved for use in the pharmaceutical industry were obtained, which are stable, have comparable or higher solubility and biological activity than the drug histochrome. As a result of the work performed under the project, new knowledge was obtained in the field of pharmaceutical chemistry about the interaction of the drug substance echinochrome A with polymer carriers and antioxidant additives and the properties of the resulting complexes. The conducted study opens up prospects for the practical application of the obtained complexes for the creation of new dosage forms, which, due to the rational use of the drug histochrome, will significantly expand the boundaries of its use for the treatment and complex therapy of a number of socially significant diseases.

Publications

1. Kikionis S., Papakyriakopoulou P., Mavrogiorgis P., Vasileva E. A., Mishchenko N. P., Fedoreyev S. A., Valsami G., Ioannou E., Roussis V. Development of Novel Pharmaceutical Forms of the Marine Bioactive Pigment Echinochrome A Enabling Alternative Routes of Administration Marine Drugs, Vol. 21, N 4. – P. 250 [1–16] (year - 2023) https://doi.org/10.3390/md21040250

2. Vasileva E. A., Mishchenko N. P., Fedoreyev S. A. Investigation of Pacific sea urchins quinonoid pigments and design of new dosage forms based on them PNU BK21 Pharm 4.0 International Online Symposium on Pharmaceutical and Biomedical Sciences Abstract Book, P. 12 (year - 2023)

3. Vasileva E.A., Mishchenko N.P., Fedoreev S.A. Новая лекарственная форма эхинохрома А, способ ее получения и применение Бюл. № 20, Пат. 2800382 Российская Федерация, МПК А61К 31/122, А61К 9/70, В82В 3/00, А61К 35/616, Бюл. № 20 - 16 с. 2023. (year - 2023)

4. - Новая лекарственная форма эхинохрома А, способ ее получения и применение -, 2022131850 (year - )

Annotation of the results obtained in 2021
At the end of the first year of the project, new complexes of the drug substance echinochrome A with polymer matrices and antioxidants approved for use in the pharmaceutical industry were obtained, which have comparable or higher stability than the histochrome preparation, as well as the possibility of targeted delivery and prolonged action of the active component. During the first year of the project, micro-/nanofibers containing echinochrome A were obtained with pharmaceutically approved synthetic polymers cellulose acetate, methyloxypropyl cellulose, polyvinylpyrrolidone, polycaprolactone, polyethylene oxide, as well as from a mixture of polyvinylpyrrolidone and polycaprolactone in different ratios. It was found that echinochrome A remains stable in the process of electrospinning and fully embedded in micro-/nanofibers (more than 95% of the load), and also remains stable in all polymer matrices for 6 months. It has been shown that the most promising polymers for delivery and release of echinochrome A in the gastrointestinal tract are polyvinylpyrrolidone, polycaprolactone, and polyethylene oxide. Polyvinylpyrrolidone nanofibers instantly (within 10 minutes) release up to 100% echinochrome A at pH 1.2, which makes polyvinylpyrrolidone a promising carrier for the preparation of gastrosoluble capsules or tablets with instant release of echinochrome A for the treatment of inflammatory diseases of the stomach, such as gastritis, gastroduodenitis and other. Polycaprolactone and polyethylene oxide provide a prolonged release of echinochrome A at pH 6.8, which makes them promising materials for the preparation of enteric capsules or tablets for the treatment of inflammatory bowel diseases. The use of micro-/nanofibers obtained during the project with the inclusion of echinochrome A is not limited to the proposed production of tablets or capsules. The versatility of the materials obtained by electrospinning will also allow them to be used for the preparation of eye drops, films, and dressings for wounds and burns, providing a prolonged controlled release of echinochrome A, which, however, will require further research in this direction. The stability of echinochrome A in buffer solutions in the presence of antioxidants sulfite, dithionite and sodium thiosulfate, ascorbic acid, cysteine, and carnosine was studied. Ascorbic acid was found to be the strongest antioxidant. The solubility of echinochrome A in the presence of water-soluble polymers methyloxypropyl cellulose, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol in water, saline solution, PBS, phosphate-citrate and HEPES buffers. It was found that polyethylene glycol and polyethylene oxide did not increase the solubility and stability of echinochrome A, methyloxypropylcellulose increases the solubility of echinochrome A in buffer solutions, but only slightly increases its stability. In the presence of polyvinylpyrrolidone, the solubility of echinochrome A in buffer solutions increases 4 times and amounts to 0.4 mg/ml, which is two times higher than in ampoules of the preparation histochrome. Solutions of echinochrome A with polyvinylpyrrolidone in phosphate-citrate buffer remain stable for a month. Thus, it has been established that polyvinylpyrrolidone is a promising carrier for the preparation of eye drops containing echinochrome A.

Publications

1. - Красные водоросли выбирают для своих экспериментов молодые ученые ДВО РАН Вести Приморья, - (year - )