RU2259824C2 - Means possessing lipid-correcting and immunomodulating activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: means has lipid fraction obtained from Berryteuthis Magister Comandor squid liver containing 10% of polyunsaturated fatty acids and 50% of alkyl-diacylglycerides showing marked lipid-correcting and immunomodulating properties.

EFFECT: enhanced effectiveness in treating lipid metabolism disorders and immunity system disorders.

4 tbl

Description

The invention relates to medicine, in particular to clinical pharmacology and rehabilitation therapy.

As you know, many lipid-dependent diseases are accompanied by disorders in the immune system. Thus, cardiovascular diseases are combined with damage to the system of non-specific resistance caused by significant antigenic load from accumulating lipoproteins. Therefore, it is extremely urgent to search for prophylactic drugs that possess, along with lipid-correcting effects and immunocorrecting properties.

The traditional treatment regimen for patients with dyslipidemia is based on diet therapy, which includes a reduced lipid content in the diet, a moderate carbohydrate content, which ensures the normal course of life processes and is necessary to maintain a high level of body functioning. In combination with a diet, patients are prescribed synthetic drugs of various spectrum of action that lower blood cholesterol and triglycerides. These methods of treatment do not always contribute to the restoration of the physiological state of the body, damaged as a result of the disease, since they do not provide for the correction of the state of immunity, which is largely damaged as a result of impaired lipid metabolism. In this regard, it is advisable to search for drugs and food products that possess, in addition to lipid-correcting properties, immunomodulating activity.

In recent years, fats of marine aquatic organisms containing ω3 polyunsaturated fatty acids (PUFAs) have attracted much attention. Their diverse biological effects have been proven, allowing them to be used as sources of biologically active substances for the prevention and treatment of a number of diseases. It is known that ω3 PUFAs are included in the cellular structures of the body and have a modifying effect on the structural and functional organization of cell membranes, the activity of membrane-bound enzymes and the biosynthesis of eicosanoids. Quite often, marine aquatic organisms are used as potential sources of ω3 PUFAs. At the same time, the composition of the total lipid fraction of fats of marine aquatic organisms may include other lipids. It is the diverse composition of lipids from various marine sources that determines the specificity of the action of the means used and does not allow us to approach their use on the basis of analogy, which requires a detailed study of the biological action mechanism, analysis of the effectiveness and safety of their use in clinical practice. For example, fats from the liver of marine mollusks, in particular squid, are rich in unsaponifiable substances (Hayashi K., Okawa Y., Kawasaki K. Liver lipids of gonatid squid Berryteuthis magister: a rich source of alkyi glyceryl ethers. 1985, Bull. Jap Soc. Sci. Fish., Vol. 51, No. 9, p. 1523-1526) having a high biological activity that differs from the properties of ω3 PUFAs. Unsystematic and uncontrolled use of various natural sources of biologically active substances, including marine hydrobionts, can cause serious complications and carry a huge risk for the country's gene pool.

Most natural fats and oils are triglycerides - glycerol and fatty acid esters:

Among the variety of fats, there are frequent cases of the presence of significant amounts of alkyl diacylglycerides (ADH) in them. In these compounds, in the 1st position of the molecule, there is an alkyl residue bound to glycerol via an ether bond:

If free fatty acids are removed from the molecule, then the alcohols of the general formula are obtained:

If R = 16: 0 (C ₁₆ H ₃₃ ) * (* - Note: 16: 0 - indicates the presence of 16 carbon atoms and 0 double bonds in the radical; the elemental composition is given in brackets.), Then chemical alcohol, if R = 18: 0 (C ₁₈ H ₃₇ ), then batilic, if R = 18: 1 (C ₁₈ H ₃₅ ), then selachilic. The sum of these three alcohols of their total amount in natural lipids is 90% or more (Hayashi K., Okawa Y., Kawasaki K. Liver lipids of gonatid squid Berryteuthis magister: a rich source of alkyi glyceryl ethers. 1985, Bull. Jap. Soc. Sci. Fish., Vol. 51, No. 9, p. 1523-1526). In the scientific literature, this total composition of alcohols is called alkyl glyceryl ethers (AGE).

Local application (Bodman J., Maisin J.H. The α-glyceryl ethers // Clinica Chimica Acta, 1958, v.3, p. 253-274) of alkyl glyceryl esters for wound healing in Wistar rats from radiation injury is known. Some rats were treated with AGE, others with talcum powder containing an antibiotic. The results showed an almost 2-fold acceleration of wound healing from the stage of necrosis to repeated hair growth.

AGE has been tested in some patients with serious external lesions:

a) non-healing purulent wound

Local application of AGE within one week initiated granulation of wounds.

b) non-healing wounds associated with surgical removal of malignant

tumors

A patient with removed breast carcinoma was intramuscularly injected daily with 100 mg of AGE in 2.5 ml of peanut butter. The injections were painless and did not cause any reactions. After 4 days, fresh granulation tissue was visible on the wound. After 1.5 months, the wound had a healthy appearance, and at the 3rd month the treatment was discontinued.

c) non-healing purulent wounds associated with the surgical treatment of fractures

The patient was given injections of AGE, as in the previous case, and at the same time, AGE powder was applied to the wound. Treatment was continued throughout the transplantation period. Other therapy due to good results was not even considered.

d) non-healing wounds associated with radiation damage

Local application of AGE with their intramuscular injection led to the immediate onset of healing of radiation burns.

e) healing of ordinary wounds, factory or accidental burns

No effect was observed in this case. AGE delayed healing and stopped their treatment.

There are known examples of the internal consumption of AGE in the treatment of radiation injuries.

A clinical study was conducted (Brohult A., Holmberg J. Alkoxyglycerols in the treatment of leucopenia caused by irradiation / Nature, 1954, v.174, No. 4441, p.1102-1103) on the effect of AHE isolated from animal bone marrow fat, for leukopenia in patients, mainly children, caused by radiation. AGE was administered orally for a long period (several months). 36 patients were studied, of which 25 prescribed therapy caused an immediate increase in the number of white blood cells in the blood: 1.5 times in a few days, and at the end of treatment more than 2 times.

A study was conducted (Brohult A. Alkoxyglycerols in irradiation treatment / Nature, 1962) on 300 women who received radiation therapy for cervical cancer. Patients of prophylactic groups received 1.2 g of AGE. The effectiveness was evaluated by the survival of patients after 1 year and 5 years of treatment. The data presented, especially for stage II cancer, showed significantly greater survival in the prophylactic group than in the control group.

A study is known of the effect of AHE on leukopenia from radiation therapy (Ghys R. Effets des alkoxyglycerols (Kaby 700) sur la leucopenie consecutive a le radiotherapie / Laval Medical, 1960, v.30, No. 3, p.331-342). Patients selected for examination, a total of 30 people, had different diagnoses: lung cancer, biceps sarcoma, breast cancer with bone metastases, cervical cancer with metastases to the peritoneum, stomach cancer, pancreatic cancer, rectal cancer, anal sarcoma, coronary astrocytoma , laryngeal cancer and another 4 specific tumors. Patients received standard radiotherapy and AHE. The drug was obtained from polar shark liver fat and had the brand name Kaby 700. On the first day, patients were prescribed 3.4 g of fat after fractionation (with saturated fatty acids), containing about 30% batil alcohol, and on the following days, 1.1 g fat. The author obtained conflicting results, showing, on the one hand, the effectiveness of exposure to AGE, and on the other, the lack of a therapeutic effect. Regarding the interpretability of the results, the main drawback should be stated - the small number of patients observed with such a significant set of pathologies.

The use of AGE for allergic diseases is known (European Patent 0333678, 02.01.89, MKI ⁵ A 61 K 31/08 / Brohult S., Brohult A. Use of glycerol ethers in the treatment of allergic diseases). The product was isolated from the liver shark liver fat, removing all fatty acids from it. The results of asthma treatment are given as an example. After 3-4 months, the discomfort (from the disease) disappeared. In some cases, its complete disappearance was observed after 6 months. As an example, a case is given when a patient suffered from severe asthma attacks for 15 years and was completely dependent on various medications (corticosteroids and others). After 3.5 months, the discomfort went away, and drug treatment was reduced. After another 3 months, he was completely abandoned. The brand name of the drug is Ecomer. Each capsule contains 50 mg of active substance - AGE. Dosage is not indicated in the description.

However, all researchers used AGE from a destroyed natural complex, where they were located along with polyunsaturated fatty acids, discarding an important biological component. Thus, the overall therapeutic effect was reduced, and in the multi-stage process of obtaining the drug, rigid chemical treatment was used and the cost of the final product accordingly increased. Polyunsaturated fatty acids were considered as ballast, which must be removed in order to "concentrate" the drug. And in the future, such researchers, for the convenience of using the drug, dissolved it in ordinary vegetable oils (Bodman J., Maisin J.H. The α-glyceryl ethers / Clinica Chimica Acta, 1958, v.3, p. 253-274).

The closest technical solution to the claimed invention is a method for the production of semi-finished squid and its use in medical, diet and baby nutrition (patent RU 20046580 C1 from 10.27.1995).

The disadvantage of this invention is that the agent obtained by this method does not have pronounced lipid-correcting and immunomodulating properties, since the raw materials in this case are not the squid guts, in particular the liver, but the mantle - a known useful protein product containing an extremely small amount omega-3 series polyunsaturated fatty acids.

The problem solved by the invention is the expansion of the arsenal of natural products with pronounced lipid-correcting and immunomodulating properties based on the use of a natural complex of lipids isolated from squid liver, combining AGE and polyunsaturated fatty acids.

The essence of the invention lies in the fact that a biologically active lipid complex was found, which is a lipid fraction from the liver of Commander squid Berryteuthis magister, which contains 10% polyunsaturated fatty acids and 50% alkyl diacylglycerides and has lipid-correcting and immunomodulating properties.

The agent with the above biological properties is obtained as follows: raw fat from the liver of Commander squid Berryteuthis magister is isolated by heat treatment of the liver at t = 90 ° C followed by separation of the mixture and separation of fat, then it is refined to obtain a fraction containing 10% polyunsaturated fatty acids and 50% alkyl diacylglycerides.

Our use of the proposed agent obtained from the liver of Commander squid Berryteuthis magister for model cardiac pathology yielded an unexpected result in effectiveness, superior to the expected prognosis from its appointment and consisting in directed preventive action on the sick body as a lipid-correcting and immunomodulating agent due to the presence of alkyl-diacylglycerides ( ADH) is a complex of polyunsaturated fatty acids and alkyl glyceryl ethers (AGE).

EXAMPLE 1. The use of funds obtained from the liver of squid Berryteuthis magister, in experimental cardiovasopathy with severe impaired immunity

In an experiment on rats, the agent isolated from the liver of Commander squid Berryteuthis magister, containing 10% polyunsaturated fatty acids and 50% alkyl diacylglycerides (ADH) (based on total lipids), was studied.

Table 1 presents the composition of the total lipid fraction of the liver of commander squid after refining, used as a means.

Table 1 The composition of the main lipids of fat from the liver of commander squid Component Content,%, of the total mass Alkyl diacylglycerides (ADH) fifty Triglycerides 26 Polyunsaturated Fatty Acids 10 Other Rest

The object of the study was white Wistar male rats weighing from 235 to 350 grams, bred in the laboratory animal nursery RAMS "Stolbovaya" (Chekhov, Moscow Region), veterinary certificate No. 32-361061, 2002. The animals were kept in a vivarium in accordance with the sanitary rules for the design, equipment and maintenance of experimental biological clinics, in plastic cages of 4-5 heads each, on a litter of hardwood under natural light and a temperature of 19 ± 2 ° C. Rat quarantine was carried out for 14 days or more. The studies were carried out in accordance with the "Rules for the work using experimental animals" (1977). We used sexually mature individuals at the age of 6 months.

As an experimental model, accompanied by dyslipidemia with severe impaired immunity parameters, the study reproduced cardiovasopathy according to the previously developed technique (RF patent No. 2192048 Ru, IPC 7 G 09 B 23/28. Method for modeling infarctoid cardiovasopathy in rats / TPNovgorodtseva, O.G. Vostrikova, I.V. Dobrynchenko. - Publish. 10.27.2002, Bull. No. 30). For the study, 3 groups of rats were identified:

1st group - intact animals;

2nd group - rats with simulated cardiovasopathy;

Group 3 — rats with cardiovasopathy who received dietary supplements with food per 1 g / kg of animal weight.

Modeling of cardio-vasopathy in rats was carried out for 15 days, after which the rats were transferred to the standard diet of the vivarium and for 30 days the experimental animals received the studied drug per os daily. Animal euthanasia was performed by decapitation under ether anesthesia. The integral indicators of the condition of animals, the lipid spectrum of blood serum, hematological and immunological parameters in the blood were evaluated. The lipid spectrum of blood serum was studied on an autoanalyzer, the level of total cholesterol (cholesterol), triglycerides (TG), and high density lipoprotein cholesterol (HDL cholesterol) were determined, the results were expressed in mmol / L. Low lipoprotein cholesterol (LDL) and very low density (VLDL) was determined by the calculation method according to the Friedwald formula. The atherogenicity index (IA) was calculated using the formula OXC-HDL-C / HDL-C. The number of red blood cells, platelets, leukocytes, lymphocytes was calculated in the Goryaev chamber; color indicator was calculated; hemoglobin determination was carried out using the hemoglobin cyanide method (reagent kit of LLC Agat-Med) using an SF-56 spectrophotometer (LOMO, Russia), blood coagulation time was determined. To assess the functioning of the immunological resistance system, level 3 tests were used: determination of the phagocytic activity of neutrophils (FAN) by assessing the absorption of latex particles (particle diameter 1.3 μm, Moscow) with neutrophilic peripheral blood granulocytes; phagocytic reserve (RF) in relation to the number of phagocytes stimulated by prodigiosan to unstimulated; absorption activity (PS) and its reserve (PSF), dynamics of the phagocytic process (total percentage of final stages), its completeness after incubation of cells in an thermostat at 37 ° C for 2 hours (Modeling and clinical characteristics of phagocytic reactions / Ed. Mayansky A .N. - Gorky: Publishing House Gork. Medical Institute, 1989. - 242 p.). Quantitative and qualitative parameters of oxidative metabolism (NST, NSTR, IAN, IANR) were determined by the method of Shmeleva E.V. (Shmelev E.V., Bumagina T.K., Miterov G.G. Modification of the Park method // Lab. Business. - 1979. - No. 9. - P.13-15). Cell counting was performed visually using an MBI-15 microscope from LOMO (Russia). The content of circulating immune complexes of small (C3) and large (C4) sizes, their ratio (K) was determined by the method of PV Struchkova (Struchkov P.V. Screening test for assessing the pathogenic properties of immune complexes // Lab. Business. - 1985. - No. 7. - S.410-413). The measurements were carried out on an SF-56 spectrophotometer (LOMO, Russia).

The results of the study are presented in tables 2-4.

table 2 The serum lipid content in rats under the influence of the course administration of the agent from the liver of the commander squid, M ± m Indicators, mmol / l Intact (group 1) n = 8 Model (group 2) n = 10 The introduction of dietary supplements, 1g / kg, (group 3) n = 10 XC 2.73 ± 0.19 ** 3.82 ± 0.14 * 2.18 ± 0.23 *** TG 1.25 ± 0.16 ** 1.99 ± 0.13 1.38 ± 0.14 ** HDL 1,50 ± 0,15 1.24 ± 0.10 1.38 ± 0.21 LDL 0.66 ± 0.12 *** 1.68 ± 0.07 0.81 ± 0.15 *** VLDL 0.57 ± 0.06 * 0.91 ± 0.10 0.63 ± 0.05 * IA 0.82 ± 0.06 ** 2.08 ± 0.28 ** 0.58 ± 0.02 *** Note: * on the left - the values are reliable relative to the intact group, * on the right - relative to the 2nd group (model); * - p <0.05; ** - p <0.01; *** - p <0.001. Table 3 The parameters of immunological resistance in rats under the influence of funds obtained from the liver of commander squid, M ± m Immunological parameters Intact (group 1) n = 8 Model (group 2) n = 10 The introduction of dietary supplements, 1g / kg, (group 3) n = 10 FAN,% 52.3 ± 1.9 *** 24.70 ± 1.77 * 61.0 ± 3.5 *** FR, cu 1.08 ± 0.08 0.85 ± 0.22 1.00 ± 0.05 Ff, cu 4.10 ± 0.29 ** 2.80 ± 0.32 * 4.30 ± 0.28 ** FFR, cu 1.64 ± 0.82 ** 1.20 ± 0.09 1.30 ± 0.08 Completion FAN 0.93 ± 0.12 ** 1.43 ± 0.14 0.89 ± 0.02 ** Total percent completing stages 38.0 ± 2.2 ** 17.70 ± 3.47 32.0 ± 1.3 * HCT,% 6.50 ± 2.01 ** 27.0 ± 1.5 ** 15.0 ± 0.9 NSTR.u.e 2.00 ± 0.08 ** 0.90 ± 0.04 2.10 ± 0.05 * IAN, cu 0.17 ± 0.01 * 0.41 ± 0.15 0.14 ± 0.01 * IANR, cu 2.00 ± 0.11 * 0.89 ± 0.01 2.20 ± 0.11 **

Table 4 Hematological parameters in rats under the influence of funds obtained from the liver of the commander squid, M ± m Peripheral blood parameters Intact (group 1) n = 8 Model (group 2) n = 10 The introduction of dietary supplements, 1g / kg, (group 3) n = 10 Coagulation time blood sec 7.40 ± 1.03 *** 49.60 ± 1.94 5.2 ± 0.9 *** Hemoglobin, g / l 102.00 ± 7.69 *** 198.00 ± 10.82 *** 272.0 ± 12.2 ** Average content hemoglobin in erythrocyte, pg 1.80 ± 0.08 1.80 ± 0.03 * 3.80 ± 0.09 ** Red blood cells * 10 ¹² 6.13 ± 2.97 * 34.00 ± 10.27 *** 46 ± 1,1 Platelets * 10 ⁹ 359.30 ± 23.95 ** 202.50 ± 10.38 435.2 ± 13.1 *** ESR, mm / h 1.80 ± 0.14 1.30 ± 0.34 * 2.10 ± 0.12 * Color indicator 0.55 ± 0.15 0.60 ± 0.08 0.80 ± 0.06 White blood cells, g / l 12.77 ± 1.70 9.30 ± 1.31 8.0 ± 1.7 Lymphocytes,% 24.50 ± 2.25 ** 37.00 ± 1.82 ** 40.4 ± 3.4

As follows from the data presented, the development of the cardiovasopathy model was accompanied by changes in lipid metabolism (Table 2). In rats with cardiovasopathy, there was an increase in total cholesterol by 28.5%, LDL cholesterol by 60.7%, and VLDL cholesterol by 37.4%. HDL cholesterol index decreased, TG level increased. The atherogenic index in rats with cardiovasopathy was 2.5 times higher than in the control group of animals.

The development of the model was also accompanied by significant violations of peripheral blood parameters: an increase in its coagulation rate (by 82%) was associated with a high level of platelets (2 times higher than in intact animals); an increase in the number of red blood cells (by 58%) was accompanied by a decrease in the average hemoglobin content and color index (by 76%), which may be due to a decrease in hemoglobin synthesis; the total number of leukocyte cells increased (by 32%); the percentage of lymphocytes was reduced (by 45%). Violations of immunological resistance were characterized by a decrease in the phagocytic activity of neutrophils by 53%, phagocytic number - by 32%, and the total percentage of the final stages - by 54%. The number of cells that restore diformazan (HCT) increased sharply by 121%, the intensity of oxidative metabolism (IAN) increased by 65%, there was a decrease in the functional reserve of phagocytes - RF by 10%, PCR by 27%, NCTR by 29%, IANR by 55 % compared with intact animals. The killing process, which determines the completeness of phagocytosis, slowed down more than 2 times.

Course introduction of the claimed drug, which is a fraction of lipids from the liver of Commander squid Berryteuthis magister, containing 10% polyunsaturated fatty acids and 50% alkyl diacylglycerides, in addition to the expected lipid-correcting effect (a significant decrease in the level of total cholesterol and cholesterol of atherogenic classes of lipoproteins almost to the level of intact animals decrease in TG) had immunomodulating and homeostatic effects. The drug had the greatest effect on erythro- and lymphopoiesis. The content of red blood cells increased (3 times), the level of hemoglobin in peripheral blood increased (by 79%). By reducing the number of platelets (by 87%), the coagulation time increased by 2 times. Inflammatory reactions decreased: ESR decreased by 23%, white blood cell count - by 31%. Almost to normal, the level of lymphocytes was restored (Table 4).

The immunomodulatory activity of the drug was manifested in the normalization of the functional reserve of neutrophils, the absorption capacity of phagocytes, and oxidative metabolism. An increase in the speed and efficiency of the antigen processing process was observed (Table 3). The drug from the liver lipids of the commander squid increased the level of the main parameters of phagocytosis: the phagocytic reserve of neutrophilic granulocytes increased by 30%, the phagocytic number by 22%, and the phagocytic reserve by 14%. The process completeness index and the number of cells entering the final stages of the process changed by a third.

Thus, the proposed tool, obtained from the liver of the squad squid Berryteuthis magister, containing 10% polyunsaturated fatty acids and 50% alkyl diacylglycerides, has pronounced lipid-correcting and immunomodulating properties, which are manifested in the activation of the phagocytic activity of neutrophils and foreign agent, stimulating the development of an adequate response to the additional load. The agent obtained from the liver of the squid squid normalizes the oxidative metabolism of phagocytes during experimental cardiopathy, probably due to a decrease in the level of respiratory explosion in the cells and an increase in their functional reserve. For the first time, the immunomodulating properties of a lipid-correcting agent of natural origin have been identified that make it possible to use the agent obtained from squid liver as an effective tool for the prevention of diseases accompanied by disorders of both lipid metabolism and the immune system.

Claims (1)

A tool with lipid-correcting and immunomodulating properties, representing a fraction of lipids from the liver of Commander squid Berryteuthis magister, containing 10% polyunsaturated fatty acids and 50% alkyl-diacylglycerides.