RU2724021C1 - Liposomal agent for treating chronic atrophic gastritis - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine and discloses use of agent, which is a liposomal form α-fetoprotein (AFP). Liposomal form of the AFP can be an aqueous suspension of liposomes or a lyophilizate. Aqueous suspension of liposomes contains AFP, lipoid C-100, cholesterol, lactose, rheopolyglucin, alpha-tocopherol (vitamin E), sodium chloride, disubstituted sodium phosphate in aqueous form, aqueous sodium monophosphate, water in the following weight ratio of components: AFP - 0.040–0.160; S-100 lipoid - 41.6–43.8; cholesterol - 4.0–5.3; alpha-tocopherol (vitamin E) - 0.48–0.8; lactose - 70–120; rheopolyglucin - 4.0–6.0; sodium chloride - 7.0–9.0; disubstituted di-sodium phosphate - 4.3–4.9; mono-substituted 2-aqueous sodium phosphate - 0.5–0.7; water - 800–1300. Lyophilizate contains the above components, except water, in the same weight ratio. Average diameter of liposomes is 0.18 mcm. AFP is recovered from human umbilical blood.EFFECT: invention allows to achieve the same results in recovery of the gastric mucosa within 2 weeks of treatment, which is achieved by the prototype (water-oil form of AFP) for 4 weeks.9 cl, 12 dwg, 2 tbl, 1 ex

Description

The invention relates to medicine.

Known funds that are used in the treatment of chronic atrophic gastritis (CAH), namely:

, Erten R.,

, Cengiz N., Yilmaz O. Hepatoprotective and anti-inflammatory activities of Plantago major L. Indian Journal of Pharmacology. 2009; 41(3): 120-124. doi: 10.4103/0253-7613.55211), спазмолитическим, обволакивающим действием, способствует регенерации тканей и активирует желудочную секрецию (Samuelsen А.В. The traditional uses, chemical constituents and biological activities of Plantago major. Journal of Ethnopharmacology. 2000;71(1-2):1-21. doi: 10.1016/s0378-8741(00)00212-9; Zubair M., Nybom H., Lindholm C, Rumpunen K. Major polyphenols in aerial organs of greater plantain (Plantago major L.), and effects of drying temperature on polyphenol contents in the leaves. Scientia Horticulturae. 2011; 128(4):523-529. doi:10.1016/ j. scienta. 2011.03.00).1. Plantaglucid is a herbal preparation obtained from large plantain. Has anti-inflammatory (Hussan F, Mansor AS, Hassan SN, Tengku Nor Effendy Kamaruddin TN, Budin SB, Othman F. Anti-Inflammatory Property of Plantago major Leaf Extract Reduces the Inflammatory Reaction in Experimental Acetaminophen-Induced Liver Injury // Evid Based Complement Alternat Med. 2015; 2015: 347861. Doi: 10.1155 / 2015/347861;

, Erten R.,

, Cengiz N., Yilmaz O. Hepatoprotective and anti-inflammatory activities of Plantago major L. Indian Journal of Pharmacology. 2009; 41 (3): 120-124. doi: 10.4103 / 0253-7613.55211), an antispasmodic, enveloping effect, promotes tissue regeneration and activates gastric secretion (Samuelsen A.V. The traditional uses, chemical constituents and biological activities of Plantago major. Journal of Ethnopharmacology. 2000; 71 (1- 2): 1-21. Doi: 10.1016 / s0378-8741 (00) 00212-9; Zubair M., Nybom H., Lindholm C, Rumpunen K. Major polyphenols in aerial organs of greater plantain (Plantago major L.), and effects of drying temperature on polyphenol contents in the leaves. Scientia Horticulturae. 2011; 128 (4): 523-529. doi: 10.1016 / j. scienta. 2011.03.00).

However, there is evidence of its insufficient anti-inflammatory effect, and, in addition, despite the improvement of the clinical symptoms of the disease, histologically, on the contrary, there is an increase in the inflammatory process (Khalifeh MS, Hananeh W, Al-Rukibat R, Okour O, Boumezrag A. Clinical and histopathological evaluation of MDP / collagen induced arthritis rat model (MCIA) after treatment with Urtica dioica, plantago major and Hypericum perforatum L herbal mixture // Exp Anim. 2008 Apr; 57 (2): 101-10).

2. Solcoseryl - a reporter of animal origin. It improves the transport of oxygen and glucose to cells under hypoxia, increases the synthesis of intracellular ATP and increases the dose of aerobic glycolysis and oxidative phosphorylation, activates reparative and regenerative processes in tissues, stimulates the proliferation of fibroblasts and collagen synthesis of blood vessels (https: // health .mail.ru / drug / solcoseryl_1 /).

However, the anti-inflammatory effect of solcoseryl is negligible (Lalabonova N, Daskalov N. Clinical assessment of the therapeutic effect of low-level laser therapy on chronic recurrent aphthous stomatitis // Biotechnol Biotechnol Equip. 2014 Sep 3; 28 (5): 929-933). This leads to the absence of a persistent therapeutic effect.

,

Suarez AJ,

Fernandez FP. Ultrastructural study of the kidney subjected to warm ischemia and perfused with inosine // Arch Esp Urol. 2013 Dec; 66(10):945-55; Szabo G, Stumpf N, Radovits T, Sonnenberg K, Gero D, Hagl S, Szabo C, Bahrle S. Effects of inosine on reperfusion injury after heart transplantation. Eur J Cardiothorac Surg. 2006; 30:96-102; Veres G, Radovits T, Seres L, Horkay F, Karck M, Szabo G. Effects of inosine on reperfusion injury after cardiopulmonary bypass. J Cardiothorac Surg. 2010; 5:106), что способствует восстановлению слизистой оболочки желудка (Korepanov AM, Nazarov AM, Nikitin EN, Bazhenov EL Riboxine effectiveness in combined treatment of chronic atrophic gastritis // Klin Med (Mosk). 1999; 77(9):28-30).3. Riboxin (inosine) - refers to a group of drugs that regulate metabolic processes (https://health.mail.ru/drug/riboxin/). The drug is a precursor for the synthesis of purine nucleotides: adenosine triphosphate and guanosine triphosphate, which provide antihypoxic and cytoprotective effects (Gallo

,

Suarez AJ,

Fernandez FP. Ultrastructural study of the kidney finally to warm ischemia and perfused with inosine // Arch Esp Urol. 2013 Dec; 66 (10): 945-55; Szabo G, Stumpf N, Radovits T, Sonnenberg K, Gero D, Hagl S, Szabo C, Bahrle S. Effects of inosine on reperfusion injury after heart transplantation. Eur J Cardiothorac Surg. 2006; 30: 96-102; Veres G, Radovits T, Seres L, Horkay F, Karck M, Szabo G. Effects of inosine on reperfusion injury after cardiopulmonary bypass. J Cardiothorac Surg. 2010; 5: 106), which helps to restore the gastric mucosa (Korepanov AM, Nazarov AM, Nikitin EN, Bazhenov EL Riboxine effectiveness in combined treatment of chronic atrophic gastritis // Klin Med (Mosk). 1999; 77 (9): 28-30 )

, Kuhel DG, Nemeth ZH, Mabley JG, Stachlewitz RF,

, Lohinai Z, Southan GJ, Salzman AL,

. Inosine inhibits inflammatory cytokine production by a posttranscriptional mechanism and protects against endotoxin-induced shock // J Immunol. 2000 Jan 15; 164(2):1013-9;

, Sitkovsky MV,

Trends Pharmacol Sci. Immunomodulatory and neuroprotective effects of inosine // 2004 Mar; 25(3):152-157), этот препарат не обеспечивает достаточный противовоспалительный эффект (патент РФ №2662077 «Средство для лечения хронического атрофического гастрита»).However, despite the fact that riboxin weakens the production of inflammatory mediators (

, Kuhel DG, Nemeth ZH, Mabley JG, Stachlewitz RF,

, Lohinai Z, Southan GJ, Salzman AL,

. Inosine inhibits inflammatory cytokine production by a posttranscriptional mechanism and protects against endotoxin-induced shock // J Immunol. 2000 Jan 15; 164 (2): 1013-9;

, Sitkovsky MV,

Trends Pharmacol Sci. Immunomodulatory and neuroprotective effects of inosine // 2004 Mar; 25 (3): 152-157), this drug does not provide a sufficient anti-inflammatory effect (RF patent No. 2662077 "Means for the treatment of chronic atrophic gastritis").

4. A-fetoprotein (α-fetoprotein, AFP) was proposed to eliminate signs of CAH (RF patent No. 2662077 "Means for the treatment of chronic atrophic gastritis"), namely: a significant reduction in venous vascular dilatation, normalization of the state of microvasculature, elimination of aggregation phenomena erythrocytes in the vessels, elimination of edema and infiltration by the cellular elements of the plate of the gastric mucosa (mainly due to a decrease in the number of neutrophils and lymphocytes); restoration of the gastric mucosa (there is an increase in the size of the glands and a decrease in the distance between them, a decrease in the severity of signs of sclerotic changes, the acquisition by the epithelium of the glands of a more highly specialized structure, due to the restoration of the pool of cells that produce gastric juice and hydrochloric acid in the fundus, and the elimination of foci of intestinal metaplasia in the pyloric department) (prototype).

Despite the pronounced therapeutic effect of the use of AFP, the used form of the drug (water-in-oil emulsion) did not fully restore the gastric mucosa in the pyloric department, which is most susceptible to pathological changes, even after using the drug for 4 weeks.

In this regard, the search for the most optimal form of the drug, allowing to achieve new results in the treatment of chronic atrophic gastritis, is of undoubted relevance.

Known liposome containing recombinant human AFP, which may also contain recombinant human granulocyte colony stimulating factor (RF patent No. 2642957).

This liposome is intended for the treatment of local radiation lesions of the skin. The drug containing it may be used subcutaneously or transdermally.

Disclosure of invention

For the treatment of chronic atrophic gastritis (CAH), a liposomal (liposomal) form of α-fetoprotein (AFP) is intended for enteral use (administration).

The liposomal form of AFP is an aqueous suspension of liposomes or a lyophilisate.

The lyophilisate contains AFP, C-100 lipoid, cholesterol, lactose, reopoliglukin, alpha-tocopherol (vitamin E), sodium chloride, sodium phosphate disubstituted 12-water, sodium phosphate monosubstituted 2-water in the following weight ratio of components:

AFP 0.040-0.160;

C-100 lipoid 41.6-43.8;

cholesterol - 4.0-5.3;

alpha-tocopherol (vitamin E) - 0.48-0.8;

lactose - 70-120;

reopoliglyukin - 4.0-6.0;

sodium chloride - 7.0-9.0;

sodium phosphate disubstituted 12-water - 4.3-4.9;

monosubstituted sodium phosphate 2-water - 0.5-0.7.

An aqueous suspension of liposomes contains AFP, C-100 lipoid, cholesterol, lactose, reopoliglukin, alpha-tocopherol (vitamin E), sodium chloride, sodium phosphate disubstituted 12-aqueous, sodium phosphate monosubstituted 2-aqueous and water in the following weight ratio of components:

AFP 0.040-0.160;

C-100 lipoid - 41.6 - 43.8;

cholesterol - 4.0-5.3;

alpha-tocopherol (vitamin E) - 0.48-0.8;

lactose - 70-120;

reopoliglyukin - 4.0-6.0;

sodium chloride - 7.0-9.0;

sodium phosphate disubstituted 12-water - 4.3-4.9;

monosubstituted sodium phosphate 2-water - 0.5-0.7.

water - 800-1300.

The average diameter of liposomes is 0.18 microns.

AFP isolated from human umbilical cord blood.

The technical results of the invention are as follows:

1) the proposed tool more fully restores the mucous membrane of the fundus of the stomach for 2 weeks of treatment than the prototype (water-oil emulsion AFP according to the patent of the Russian Federation No. 2662077) for 4 weeks of treatment;

2) after 2 weeks of treatment with the proposed tool, the manifestations of the chronic inflammatory process in the fundus of the stomach are eliminated, after 4 weeks of treatment - it completely lacks inflammatory infiltration;

3) during treatment with the proposed agent in the pyloric stomach, within 2 weeks of treatment, the same stage of CAH is achieved, which when prototype is treated in 4 weeks;

4) the proposed tool provides in the pyloric department for 2 weeks of treatment the achievement of the same degree of CAH, which is achieved during treatment with the prototype in 4 weeks of treatment: the inflammatory process after 2 weeks of treatment with the proposed tool in the pyloric department is less pronounced than with the prototype for 4 weeks

5) treatment with the proposed agent for 4 weeks allows you to restore the mucous membrane in the most susceptible to pathological changes pyloric department, which is not achieved at the same time using the prototype;

6) in comparison with analogues, the claimed agent has a pronounced anti-inflammatory effect;

7) the above properties of the proposed means to reduce the duration of treatment can avoid or reduce the likelihood of side effects of treatment that may occur due to the fact that with prolonged use of AFP increases the functional (proliferative and cytotoxic) activity of mononuclear leukocytes, which can manifest itself in an exacerbation of the body of chronic infectious and inflammatory diseases.

The presence in the liposome membrane of cholesterol and vitamin E in the stated ratios ensure the relative stability of liposomes in the gastrointestinal tract (until lipids are absorbed in the small intestine), and, accordingly, the biological activity of AFPs contained in them is preserved.

Vitamin E in the claimed ratio provides a sufficient antioxidant and stabilizing effect to maintain the biological activity of AFP in the process of liposome production.

Lactose and reopoliglyukin in the stated ratio ensure the stability of liposomes during lyophilization.

The buffer-salt base in the stated ratio ensures the stability of the AFP solution during the preparation of the liposomal preparation.

List of figures illustrative material

FIG. 1. The mucosa of the fundus of the stomach 8 weeks after the start of the administration of sodium salicylate against the background of keeping animals in an alternative post: the development of sclerotic changes. Okr. hematoxylin and eosin, uv. 400.

FIG. 2. The mucosa of the fundus of the stomach 8 weeks after the start of the administration of sodium salicylate against the background of keeping animals at an alternative post: infiltration of the gastric mucosa with both neutrophilic-leukocytic and macrophage elements. Okr. hematoxylin and eosin, uv. 400.

FIG. 3. The mucosa of the pyloric stomach 8 weeks after the start of the administration of sodium salicylate against the background of keeping animals in an alternative post: infiltration of the gastric mucosa with both neutrophilic-leukocytic and macrophage elements. Okr. hematoxylin and eosin, uv. 400.

FIG. 4. The mucosa of the pyloric stomach 8 weeks after the start of the administration of sodium salicylate against the background of keeping animals in an alternative post: the presence of foci of intestinal metaplasia with goblet cells. Okr. hematoxylin and eosin, uv. 400.

FIG. 5. The mucosa of the fundus of the stomach after 2 weeks of administration of a water-oil emulsion of α-fetoprotein in animals with reproduced CAH: a decrease in the infiltration of the lamina propria by neutrophilic-leukocyte and macrophage elements. Okr. hematoxylin and eosin, uv. 400.

FIG. 6. Mucosa of the pyloric stomach after 2 weeks of administration of a water-oil emulsion of α-fetoprotein in animals with reproduced CAH: reduced infiltration of the lamina propria by neutrophilic-leukocyte and macrophage elements, lack of signs of intestinal metaplasia, enlarged glands, reduced distance between them, acquisition of epithelium glands of a more highly specialized structure. Okr. hematoxylin and eosin, uv. 400.

FIG. 7. The mucosa of the fundus of the stomach after 4 weeks of administration of a water-oil emulsion of α-fetoprotein in animals with reproduced CAH: lack of infiltration of the lamina propria by neutrophilic-leukocyte and macrophage elements, restoration of the gland epithelium with its highly specialized structure. Okr. hematoxylin and eosin, uv. 400.

FIG. 8. The mucosa of the pyloric stomach after 4 weeks of administration of a water-oil emulsion of α-fetoprotein in animals with reproduced CAH: lack of infiltration of the lamina propria by neutrophilic-leukocyte and macrophage elements, elimination of signs of mucosal atrophy. Okr. hematoxylin and eosin, uv. 400.

FIG. 9. The mucosa of the fundus of the stomach after 2 weeks of administration of the liposomal form of α-fetoprotein in animals with reproduced CAH: lack of infiltration of the lamina propria by neutrophilic-leukocyte and macrophage elements, complete restoration of the glandular epithelium of its highly specialized structure. Okr. hematoxylin and eosin, uv. 400.

FIG. 10. The mucosa of the pyloric stomach after 2 weeks of administration of the liposomal form of α-fetoprotein in animals with reproduced CAH: the absence of infiltration of the lamina propria by neutrophilic-leukocyte and macrophage elements, a significant restoration of the glandular epithelium of its highly specialized structure. Okr. hematoxylin and eosin, uv. 400.

FIG. 11. The mucosa of the fundus of the stomach after 4 weeks of administration of the liposomal form of α-fetoprotein in animals with reproduced CAH: lack of infiltration of the lamina propria by neutrophilic-leukocyte and macrophage elements, complete restoration by the epithelium of the glands of its highly specialized structure. Okr. hematoxylin and eosin, uv. 400.

FIG. 12. The mucosa of the pyloric stomach after 4 weeks of administration of the liposomal form of α-fetoprotein in animals with reproduced CAH: lack of infiltration of the lamina propria by neutrophilic-leukocyte and macrophage elements, complete restoration by the epithelium of the glands of its highly specialized structure. Okr. hematoxylin and eosin, uv. 400.

Liposomes of the claimed composition are obtained by the modified method of extrusion of multilamellar vesicles through polycarbonate membranes with pore sizes of 0.1 and 0.2 microns.

Brief outline:

Obtaining a lipid film (lipoid, cholesterol, vitamin E), emulsification in a buffer solution, extrusion using filters with pores of 0.2-0.1 microns, the formation of a liposomal-protein complex, lyophilization in vials from a volume of 1 ml.

An example of obtaining the claimed liposomal funds

8.48 g of C 100 lipoid are added to a round-bottom flask (V = 10 L) containing 200 ml of ethanol, dissolved with stirring, 0.94 g of cholesterol dissolved in 20 ml of a mixture of chloroform and ethanol in a volume ratio of 1: 2 is added. stirred, add 0.12 g of alpha-tocopherol and evaporated in vacuo to obtain a dry lipid film on the walls of the flask. After evaporation, the lipid film is purged with an inert gas to prevent lipid oxidation. A substance solution containing 16 mg of human AFP and containing the following components in addition to it is added to the flask:

Sodium chloride 1.6 g Sodium Phosphate Disubstituted 12-Aqueous 0.94 g Monosubstituted sodium phosphate 2-water 0.12 g Lactose 20.0 g Reopoliglyukin 1.0 g Distilled water to 200 ml

The contents of the flask are stirred until the lipid film is completely hydrated for 30 minutes at room temperature. The resulting liposomal suspension is subjected to inert gas (argon) pressure to form and sterilize the filter through polycarbonate membrane filters (Nuclepore company) - first through a filter with a pore diameter of 0.2 μm, then with a pore diameter of 0.1 μm. Under aseptic conditions, 1 ml of suspension is poured into vials, frozen at -40 ° C and lyophilized to dry the drug until a residual moisture content of (7 ± 1) wt. % Immediately after drying, the drug vials are sealed in an inert gas atmosphere under aseptic conditions.

For reconstitution, 1.0 ml of water is added to the lyophilized dried preparation, the vial is shaken until a homogeneous aqueous suspension is obtained.

The average diameter of liposomes obtained by extrusion of multilamellar vesicles through polycarbonate membrane filters and having the claimed composition is 0.18 μm.

Description and origin of the ingredients of the drug

Biologically active substance - human AFP from umbilical cord blood, electrophoretic purity of not less than 92%, obtained by the method, including immunoaffinity fractionation and gel chromatography (RF patent No. 2123009, FSP 42-0244098601).

Protein content in the preparation: 80 μg / ml, including not less than 70% in the composition of the lipid-protein complex.

Lipoid C 100 (Lipoid C100) - Lipoid GmbH (Germany, RU No. 15370/01).

Cholesterol - MP Biochemicals, Cat. No. 0210138280.

Vitamin E (alpha-tocopherol) - MP Biochemicals, Cat. No. 0210056280.

Lactose (Lactose Pharma 200/15) - company ALPAVIT (Germany).

Reopoliglyukin - Russia, reg. No .: LSR-005290/10.

Sodium chloride - Russia, "hch".

Sodium phosphate disubstituted 12-water - Russia, "hch".

Sodium phosphate monosubstituted 2-water - Russia, "hch".

Justification of the invention

A comparative study of the therapeutic effect of CAH of a water-in-oil emulsion (prototype) and liposomal form of AFP isolated from cord blood was conducted.

Material and research methods

The experiment was conducted on 36 sexually mature Wistar male rats with an initial body weight of 180-220 g. To obtain a CAH model, 30 experimental animals were injected intragastrically with 0.2 ml of a 2% sodium salicylate solution every day for 8 weeks. 30% alcohol solution, in addition, the rats were kept at an alternative post (2 days - feeding, 1 day - no feeding) with free access to water (hereinafter - animals with reproduced HAG) (Shao XN, Yang YP, Dai J, Wu JF, Bo AH. Effects of He-Ne laser irradiation on chronic atrophic gastritis in rats World J Gastroenterol. 2005 Jul 7; 11 (25): 3958-61).

36 animals were divided into 6 groups of 6 animals in each group.

Group 1 - intact animals ("Int").

Group 2 (“HAG 8”, where the number “8” indicates 8 weeks of intragastric administration of 0.2 ml of a 2% solution of sodium salicylate in a 30% solution of alcohol and content in an alternative post (2 days - feeding, 1 day - no feeding) ) with free access to water - animals with reproduced HAG.

Group 3 (“HAG 8 + vmAFP 2”, where “vmAFP 2” means 2 weeks of administration of a water-oil emulsion of AFP) - animals with reproduced HAG, which were administered enteral water-oil emulsion of α-fetoprotein daily with content of AFP at a dose of 15 mcg / kg. Animals were removed from the experiment 10 weeks after its start.

Group 4 (“CAH 8 + vmAFP 4”, where “vMAF 4” means 4 weeks of administration of a water-in-oil emulsion of AFP) - animals with reproduced CAH, which were administered enteral water-in-oil emulsion of α-fetoprotein daily for the next four weeks AFP at a dose of 15 mcg / kg. Animals were removed from the experiment 12 weeks after its start.

Group 5 ("HAG 8 + lAFP 2", where "lAPP 2" means 2 weeks of administration of the liposomal form of AFP) - animals with reproduced HAG, which for the next two weeks were injected daily with an aqueous suspension of lyophilisate of the liposomal form of a-fetoprotein containing AFP at a dose of 15 mcg / kg. Animals were removed from the experiment 10 weeks after its start.

Group 6 (“CAH 8 + lAFP 4”, where “laPA 4” means 4 weeks of administration of the liposomal form of AFP) - animals with reproduced CAH, which were injected daily with an aqueous suspension of lyophilisate of the liposome form of α-fetoprotein containing AFP at a dose of 15 mcg / kg. Animals were removed from the experiment 12 weeks after its start.

A liposomal agent can be administered enterally, not only in the form of an aqueous suspension, but also in the form of a lyophilisate.

The stomach of animals was fixed in 12% formalin. From paraffin embedded objects, serial sections were made with a thickness of 7 μm. The morphology of the gastric mucosa was evaluated by panoramic microscopy after staining the sections with Erlich hematoxylin and eosin. Mucin was detected by mucicarmin, followed by dyeing of the nuclei with Erlich hematoxylin. Acidic mucins were detected with Alcian blue at pH 1.0, followed by dyeing of the nuclei with Mayer karmalum.

The main signs of CAH proposed by the Sydney classification system for chronic gastritis (Genta RM, Dixon MF. The Sydney system revised. Am J Gastroenterol 1995; 90: 1039-41; Filipenko P.S., Velikorodny G.G. Morphological classification of chronic gastritis (Adapted option) // Successes in modern natural sciences. - 2009. - No. 7. - P. 107-109), namely: the severity of atrophy of the gastric glands, which determines the stage of chronic gastritis; lymphoplasmacytic and neutrophilic infiltration, which determine the degree of chronic gastritis; as well as signs that are not included in the above classification, but noted with CAH: the content of mucin and acidic mucins (Mandal PK, Chakrabarti S, Ray A, Chattopadhyay B, Das S Mucin histochemistry of stomach in metaplasia and adenocarcinoma: An observation Indian J Med Paediatr Oncol. 2013 Oct; 34 (4): 229-33. Doi: 10.4103 / 0971-5851.125232).

The relative area occupied by the glands of the stomach, as well as the cellular elements of the infiltrates, was determined using a morphometric complex based on a Micros MS 300A microscope, a CX 13c digital camera (Вumer Electric GmbH, Germany) and ImageJ 1.42g software (National Institute of Health, USA). For each group, 48 images were evaluated.

Statistical processing of the results of the study was performed using the software package for statistical processing of SPSS v 17.0 for Windows. For comparison of independent groups, the Kruskal-Wallis test was used, followed by intergroup comparison using the Mann-Whitney test. Differences between the values of the compared parameters were regarded as statistically significant at p <0.05. The data obtained during the study are presented as the median (Me) and interquartile range (Q ₁ ; Q ₃ ).

Research results

HAG model (HAG 8 group)

In a morphological study of sections of the fundus and pyloric stomach after 8 weeks from the start of sodium salicylate administration against the background of keeping animals at an alternative post (group “CAH 8”), a number of signs characteristic of CAH were noted.

Fundal department of the stomach

It was observed:

1) thinning of the gastric mucosa due to atrophy of the glands with increasing distance between them, which was confirmed by a significant decrease in the relative area of the glands and the development of sclerotic changes (Fig. 1);

2) a significant decrease in the number of main, parietal and endocrine cells with an increase in the number of mucocytes secreting mucus, while the production of acidic mucins (sialomucin and sulfomucin) has significantly increased;

3) pronounced dilatation of veins and microcirculatory vessels with aggregation of red blood cells in them, the phenomena of edema were noted;

4) pronounced infiltration of the gastric mucosa with both neutrophilic-leukocyte and macrophage elements (Fig. 2), while the severity of the above infiltration significantly increased compared to intact animals by 6.7 times (table 1), the ratio of cellular elements (neutrophils, lymphocytes , monocytes / macrophages, plasma cells), forming infiltrates, amounted to 2.5: 1: 1: 0.

Pyloric stomach

In the pyloric stomach, it was noted:

1) thinning of the gastric mucosa due to atrophy of the glands with an increase in the distance between them, which was confirmed by a significant decrease in the relative area of the glands and the development of sclerotic changes;

2) a significant decrease in the number of endocrine and parietal cells with a pronounced increase in the number of mucocytes secreting mainly acidic mucins (sialomucin and sulfomucin);

3) pronounced dilatation of veins and microcirculatory vessels with aggregation of red blood cells in them, the phenomena of edema were noted;

4) pronounced infiltration of the gastric mucosa with both neutrophilic-leukocyte and macrophage elements (Fig. 3), while the severity of the above infiltration significantly increased compared to intact animals by 16.5 times (table 2), the ratio of cellular elements (neutrophils, lymphocytes monocytes / macrophages, plasma cells) forming infiltrates, amounted to 2.7: 1: 1.3: 0.3;

5) the presence of foci of intestinal metaplasia with goblet cells (Fig. 4).

Treatment of CAH with a water-oil emulsion of AFP for 2 weeks (group "CAH 8 + vmAFP 2")

Fundamental Department

In the fundus of the stomach were noted:

1) signs of restoration of the gastric mucosa, which was characterized by a 2-fold increase in the relative area of the glands in comparison with the "CAH 8" group and a decrease in the distance between them, as well as some decrease in sclerotic changes;

2) the glandular epithelium acquired a more highly specialized structure, which was due to some restoration of the pool of cells that produce gastric juice and hydrochloric acid, and a decrease in the number of cells secreting acidic mucins;

3) a decrease in the dilatation of veins and microcirculatory vessels, there was no aggregation of red blood cells in them, edema phenomena were significantly reduced;

4) the severity of infiltration compared with the group "HAG 8" decreased by 4 times (table 1, Fig. 5), mainly due to a decrease in the number of neutrophils and lymphocytes. The ratio of cellular elements (neutrophils, lymphocytes, monocytes / macrophages, plasma cells) forming infiltrates amounted to 1: 1: 2: 0, while the relative area of neutrophils and lymphocytes did not differ from the corresponding indices in intact animals (group "Int").

Pyloric Department

In the pyloric stomach:

1) signs of restoration of the gastric mucosa, which was characterized by an increase in the relative area of the glands by almost 4 times in comparison with the “CAH 8” group and a decrease in the distance between them, as well as some decrease in sclerotic changes;

2) the glandular epithelium acquired a more highly specialized structure, which was due to some restoration of the pool of parietal and endocrine cells and a decrease in the number of cells secreting acidic mucins;

3) a decrease in the dilatation of veins and microcirculatory vessels, there was no aggregation of red blood cells in them, edema phenomena were significantly reduced;

4) the severity of infiltration compared with the group "HAG 8" decreased by 2.8 times (table 2, Fig. 6), mainly due to a decrease in the number of neutrophils and lymphocytes, the ratio of cellular elements (neutrophils, lymphocytes, monocytes / macrophages, plasma cells ), forming infiltrates, amounted to 1: 1: 2.5: 1;

5) there were no foci of intestinal metaplasia.

Thus, the use of an aqueous-oil emulsion of AFP for 2 weeks led to a decrease in the signs of CAH both in the fundus and in the pyloric departments, as well as in the disappearance of intestinal metaplasia in the pyloric section of the stomach. However, neither the restoration of the mucous membrane, nor the elimination of the signs of the inflammatory component of CAH have completely occurred.

Treatment of CAH with a water-oil emulsion of AFP for 4 weeks (group "CAH 8 + vmAFP 4")

Morphological examination and preparations of the stomach 4 weeks after the start of the use of water-in-oil emulsion AFP (group "CAH 8 + vmAFP 4") showed a further decrease in the signs of CAH.

Fundamental department:

1) the mucous membrane was completely restored, as evidenced by the absence of significant differences in the relative area of the glands from the same indicator in intact animals;

2) the epithelium of the glands restored its highly specialized structure, which was confirmed by a significant restoration of the pool of cells that produce gastric juice and hydrochloric acid, and a decrease in the number of cells secreting acidic mucins;

3) there was no dilatation of veins and microcirculatory vessels, there were no phenomena of edema;

4) the relative area of immunocompetent cells (area of infiltrates) corresponds to that in the "Int" group. Moreover, the relative area occupied by neutrophils was statistically significantly lower than in the control group, and the relative area of monocytes / macrophages was significantly higher, while the ratio of cell elements (neutrophils, lymphocytes, monocytes / macrophages, plasma cells) in the fundal department was 1 , 1: 1: 1: 0 (Fig. 7; table 1).

Pyloric Department

Was noted:

1) further restoration of the mucous membrane, as evidenced by an increase in the relative area of the glands, a decrease in the distance between them, as well as a decrease in sclerotic changes. However, complete restoration to the level of intact animals did not occur;

2) the glandular epithelium acquired a more highly specialized structure, which was due to a moderate restoration of the pool of parietal and endocrine cells and a decrease in the number of cells secreting acidic mucins;

3) a decrease in the dilatation of veins and microcirculatory vessels, there was no aggregation of red blood cells in them, there were no phenomena of edema;

4) the severity of infiltration decreased by 2 times compared with the group "CAH 8 + vmAFP 2" (table 1), the ratio of cellular elements (neutrophils, lymphocytes, monocytes / macrophages, plasma cells) forming infiltrates amounted to 1: 1: 1: 1 (table 2, Fig. 8), while the relative area of neutrophils, monocytes / macrophages and plasma cells was significantly higher than in intact animals;

5) there were no foci of intestinal metaplasia.

Thus, the use of an aqueous-oil emulsion of AFP for 4 weeks in the fundus of the stomach led to a complete restoration of both the relative area of the glands and their cellular composition.

In the pyloric section, intestinal metaplasia was absent, but complete restoration of the mucosa did not occur.

The indicators reflecting the content of neutrophils and lymphocytes in the mucous membrane of the fundus and lymphocytes in the pyloric department came to normal, which indicated a decrease in the inflammatory component of CAH.

Treatment of CAH using the liposomal form of AFP for 2 weeks (group "CAH 8 + lAPP 2")

When applying the liposomal form of AFP for 2 weeks (group "CAH 8 + lAFP 2") as well as in the group "CAH 8 + vmAFP 2", a marked decrease in the signs of CAH was noted.

Fundamental department It was noted:

1) complete restoration of the gastric mucosa, as evidenced by the absence of significant differences in the relative area of the glands compared with intact animals (group "int");

2) the epithelium of the glands acquired a highly specialized structure, which was due to a large number of main, parietal and endocrine cells in this group compared to the “CAH8 + vmAFP4” group, which was combined with a reduction to the normal number of cells secreting acidic mucins (sialomucin and sulfomucin), observed in CAH;

3) the absence of dilatation of veins and microcirculatory vessels, there were no phenomena of edema;

4) the infiltration by the cellular elements of the gastric mucosa plate itself significantly decreased with respect to the CAH8 group (Fig. 9), which, unlike the CAH8 + vmAFP 2 group, occurred due to a decrease in the number of lymphocytes, macrophages / monocytes and plasma cells, which lets talk about the cessation of the chronic inflammatory process. Moreover, the relative area of lymphocytes, macrophages / monocytes and plasma cells did not differ from the corresponding indices in intact animals (group “Int”). The ratio of cellular elements (neutrophils, lymphocytes, monocytes / macrophages, plasma cells) in the fundus was 3: 1: 0: 0.

It is known that lymphocytes, macrophages and neutrophils secrete cytokines and growth factors that regulate the inflammatory process (The role of cytokines in the pathogenesis of malignant neoplasms / Sosnina A.V., Velikaya N.V., Autenshlyus A.I. - Novosibirsk: Vector-Best, 2013 . - 80 p.). It is also known that to maintain inflammation, it is necessary not so much the number of immunocompetent cells as their functional activity, expressed in the secretion of pro- and anti-inflammatory cytokines. The fact that the mucous membrane has completely recovered (the relative area of the glands and their cellular composition correspond to their level in intact animals), despite the significant increase in the number of neutrophils with a normal number of lymphocytes, plasma cells and macrophages / monocytes in the mucous membrane itself, indicates that these immunocompetent cells have already lost their pro-inflammatory potential, and their presence does not negatively affect the state of the mucous membrane.

Pyloric Department

The following were identified:

1) signs of restoration of the gastric mucosa, which was characterized by a significant increase in the relative area of the glands by 4.9 times in comparison with the “HAG8” group, as well as a pronounced decrease in sclerotic changes;

2) the glandular epithelium acquired a more highly specialized structure, which was due to the restoration of the pool of parietal and endocrine cells and a decrease in the number of cells secreting acidic mucins to normal;

3) normalization of veins and microcirculatory vessels, there was no aggregation of red blood cells in them, there were no phenomena of edema;

4) the severity of infiltration compared with the group "CAH 8" decreased in the pyloric department by 5.5 times (table 2, Fig. 10), mainly due to a decrease in the number of neutrophils and lymphocytes. The ratio of cellular elements (neutrophils, lymphocytes, monocytes / macrophages, plasma cells) forming infiltrates amounted to 1: 1: 1: 0.

It is noteworthy that the relative area of macrophages / monocytes and plasma cells was significantly lower than in the group "CAH8 + vmAFP4", with significantly identical relative area of neutrophils and lymphocytes, which indicates that the inflammatory process in the pyloric department has become less pronounced than in the group with the use of water-in-oil emulsion for 4 weeks. This is also confirmed by the fact that the relative area of the glands in the “HAG8 + vmAFP4” and “HAG8 + lAPP2” groups did not significantly differ, and the cellular composition in the “HAG8 + lAPP2” group recovered more fully;

5) there were no foci of intestinal metaplasia.

Thus, the use of the liposomal form of AFP for 2 weeks led to the complete restoration of both the relative area of the glands of the fundus of the stomach and their cellular composition. In the pyloric section, intestinal metaplasia was absent, but complete restoration of the mucosa did not occur.

Significantly decreased signs of the inflammatory component of CAH in the fundus.

Treatment of CAH using the liposomal form of AFP for 4 weeks (group "CAH 8 + lAPP 4")

In a morphological study of the preparations of the fundus of the stomach 4 weeks after the start of the use of the liposomal form of AFP (group "CAH 8 + lAPP 4"), a further decrease in the signs of CAH was noted.

Fund department

Was noted:

1) maintaining a state of complete restoration of the gastric mucosa, as evidenced by the absence of significant differences in the relative area of the glands compared with intact animals (group "int");

2) the epithelium of the glands had a highly specialized structure, which was due to the high number of main, parietal and endocrine cells compared with mucocytes, especially secreting acidic mucins (sialomucin and sulfomucin);

3) the absence of dilatation of veins and microcirculatory vessels remained, there were no edema phenomena;

4) there was completely no inflammatory infiltration (although this indicator was significantly different from the group of intact animals, but it was less) and, unlike the group “CAH 8 + vmAFP 4”, not only the number of lymphocytes, but also the number of monocytes / macrophages and plasmocytes in the gastric mucosa corresponded this indicator in an intact group of animals (table 2, Fig. 11). The ratio of cellular elements (neutrophils, lymphocytes, monocytes / macrophages, plasma cells) in the fundus of the stomach was 1: 1: 0: 0.

Pzhorichesky department

The following were identified:

1) there were completely no signs of mucosal atrophy, as evidenced by the fact that the relative area of the glands did not significantly differ from that in intact animals, there were no sclerotic changes;

2) a decrease in the secretion of sialomucin and sulfomucin with normalization of the number of mucocytes secreting neutral mucins, as well as the restoration of the pool of lining and endocrine cells, which characterized the restoration of the gastric mucosa and, above all, the functional activity of the glands of its pyloric department;

3) normalization of the diameters of veins and microcirculatory vessels, there was no aggregation of red blood cells in them, there were no phenomena of edema;

4) the severity of infiltration in the pyloric department decreased 5.5 times compared with the group "HAG 8" and remained at the same level as in the group "HAG 8 + vmAFP 4" (table 2, Fig. 12), however, the number of plasma cells was significantly less. The ratio of cellular elements (neutrophils, lymphocytes, monocytes / macrophages, plasma cells) in the pyloric department was 1: 1: 1: 0;

5) there were no foci of intestinal metaplasia.

Thus, the use of the liposomal form of AFP for 4 weeks led to the complete restoration of both the relative area of the glands and their cellular composition in both the fundus and the pyloric stomach. In the fundic department, the signs of the inflammatory component of CAH were completely eliminated. In this respect, statistically significant differences with intact animals remained in the pyloric section of the stomach that was most altered with CAH, but their severity did not differ from similar indicators in the CAH8 + vmAFP4 group.

Comparison of the results of treatment of CAH with the help of water-oil and liposomal forms of AFP shows, as can be seen from the above and from the tables, that the liposomal form has a more pronounced therapeutic effect.

The results of treatment of CAH with a water-oil form of AFP for 4 weeks and with a liposomal form for 2 weeks should be separately considered and compared.

Comparison of the results of the treatment of CAH with the help of a water-oil emulsion of AFP for 4 weeks and with the help of the liposomal form of AFP for 2 weeks shows the achievement of the same or similar results in these groups in the following.

Restoration of the gastric mucosa

A comparative morphometric analysis of the fundus of the stomach in the groups “HAG8 + vmAFP4” and “HAG8 + lAFP2” revealed that when using the liposomal form of the AFP, the state and structure of the glands restored more fully in 2 weeks than when using an aqueous-oil emulsion of AFP during 4 weeks (the relative area of the glands in the compared groups "HAG8 + vmAFP4" and "HAG8 + lAPP2" did not statistically significantly differ from the same indicator in intact animals, but the gland area in the group "CAG8 + lAPP2" was significantly higher than in the group "KHAG8 + vmAFP4").

Complete restoration of the gastric mucosa in the fundus (the relative area of the glands and their cellular composition corresponds to their level in intact animals) in the “CAH8 + lAFP2” group, normalization of the number of lymphocytes, plasma cells and macrophages / monocytes in the lamina propria mucosa in this group, despite a statistically significant difference in the number of neutrophils between this and the intact group, it indicates that the above immunocompetent cells have already lost their pro-inflammatory potential, and their presence does not negatively affect the condition of the mucous membrane of the fundus.

Thus, in the fundal department in the group "CAH8 + lAPP2" there was a more complete restoration of the gastric mucosa achieved in 2 weeks than in the group "CAH8 + vMAFP4" achieved in 4 weeks.

In the pyloric department, the relative area of the glands in the groups “HAG8 + vmAFP4” and “HAG8 + lAFP2” was significantly lower than in animals in the intact group, but these groups did not differ statistically significantly (Table 1 and 2), which can be regarded as the same stage of CAH (Filipenko P.S., Velikorodny G.G. Morphological classification of chronic gastritis (Adapted version) // Successes in modern natural sciences. - 2009. - No. 7. - P. 107-109).

The degree of severity of CAH in the pyloric department in the group "CAH8 + lAFP2" does not differ from that in the group "CAH8 + vmAFP4"

A comparative morphometric analysis of the relative area of the cellular elements of the gastric mucosa infiltrates in the “CAH8 + vmAFP4” and “CAH8 + lAFP2” groups showed that in the pyloric stomach section most altered with CAH, there were significant differences in the relative area of neutrophils and lymphocytes that determine the degree of chronic gastritis (Filipenko P .C, Velikorodny GG Morphological classification of chronic gastritis (Adapted version) // Successes in modern natural sciences. - 2009. - No. 7. - P. 107-109), was not determined (in relation to the intact group, both of these indicators were reliably higher).

It is known that alpha-fetoprotein increases the functional (proliferative and cytotoxic) activity of human mononuclear leukocytes (Obtaining activated lymphocytes from mononuclear leukocytes of human peripheral blood when exposed to alpha-fetoprotein / Chereshnev V.A., Lebedinskaya O.V., Akhmatova N.K. , Rodionov S.Yu., Lebedinskaya EA, Gavrilova TE, Karamzin AM, Kiselevsky MV // Siberian Oncology Journal. - 2005. - No. 1. - p. 40-46; Effect of the drug “ Profetal ”on the functional activity of human mononuclear leukocytes and dendritic cells / Chereshnev B.A., Lebedinskaya O.V., Rodionov S.Yu., Akhmatova NK, Shubina I.Zh., Lebedinskaya EA, Gavrilova T .E., Kiselevsky MV Medical immunology. - 2005. - No. 5-6. - pp. 525-534; The effect of the drug "Profetal" on the differentiation and functional activity of human mononuclear leukocytes / Lebedinskaya OV, Velizheva N .P., Donenko F.V., Chereshnev V.A., Rodionov S.Yu., Akhmatova N.K., Shubina I.Zh., Lebedinskaya E.A., Kiselev Sey M.V. Cell technology in biology and medicine. - 2006. - No. 2. - from. 108-116), which leads to the activation of the inflammatory process.

Since the use of AFP not only enhances the proliferative activity of mononuclear leukocytes, but also increases the cytotoxic ability of lymphocytes, NK and NKT cells, which are potential producers of pro-inflammatory cytokines, prolonged use of AFP can exacerbate the chronic infectious and inflammatory diseases present in the body ( The role of cytokines in the pathogenesis of malignant neoplasms / Sosnina A.V., Velikaya N.V., Autenshlus A.I. Novosibirsk: Vector-Best, 2013. - 80 p.).

Reducing the duration of CAH treatment avoids the above complications.

Claims (28)

1. The use of funds, which is a liposomal form of α-fetoprotein (AFP), for the treatment of chronic atrophic gastritis (CAH) by enteral administration of the specified funds. 2. The tool according to p. 1, characterized in that it is an aqueous suspension of liposomes. 3. The tool according to p. 1, characterized in that it is a lyophilisate. 4. The tool according to p. 3, characterized in that it contains AFP, C-100 lipoid, cholesterol, lactose, reopoliglyukin, alpha-tocopherol (vitamin E), sodium chloride, sodium phosphate disubstituted 12-aqueous, sodium phosphate monosubstituted 2- water. 5. The tool according to p. 4, characterized in that its components are in the following weight ratio: AFP - 0,040-0,160; C-100 lipoid 41.6-43.8; cholesterol - 4.0-5.3; alpha-tocopherol (vitamin E) - 0.48-0.8; lactose - 70-120; reopoliglyukin - 4.0-6.0; sodium chloride - 7.0-9.0; sodium phosphate disubstituted 12-water - 4.3-4.9; monosubstituted sodium phosphate 2-water - 0.5-0.7. 6. The tool according to p. 2, characterized in that it contains AFP, C-100 lipoid, cholesterol, lactose, reopoliglyukin, alpha-tocopherol (vitamin E), sodium chloride, sodium phosphate disubstituted 12-aqueous, sodium phosphate monosubstituted 2- water and water. 7. The tool according to p. 6, characterized in that its components are in the following weight ratio: AFP - 0,040-0,160; C-100 lipoid 41.6-43.8; cholesterol - 4.0-5.3; alpha-tocopherol (vitamin E) - 0.48-0.8; lactose - 70-120; reopoliglyukin - 4.0-6.0; sodium chloride - 7.0-9.0; sodium phosphate disubstituted 12-water - 4.3-4.9; monosubstituted sodium phosphate 2-aqueous - 0.5-0.7; water - 800-1300. 8. The tool according to p. 1, characterized in that the average diameter of the liposomes is 0.18 microns. 9. The tool according to p. 1, characterized in that the AFP is isolated from human umbilical cord blood.

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