WO2013141759A2

WO2013141759A2 - Use of l-carnosine for the preparation of a nanopreparation having an antihypoxic and antioxidant activity

Info

Publication number: WO2013141759A2
Application number: PCT/RU2013/000191
Authority: WO
Inventors: Зинаида Александровна СУСЛИНА; Сергей Николаевич ИЛЛАРИОШКИН; Сергей Львович СТВОЛИНСКИЙ; Александр Александрович БОЛДЫРЕВ; Владимир Васильевич КАПЦОВ; Константин Юрьевич КУЛЕБЯКИН
Priority date: 2012-03-20
Filing date: 2013-03-13
Publication date: 2013-09-26
Also published as: RU2482867C1; WO2013141759A3

Abstract

The invention relates to medicine, specifically to neurology and cardiology, namely to the production of a drug in the form of a biologically active nanopreparation having antihypoxic and antioxidant activity. The use of liposomal containers comprising L-carnosine for the preparation of a nanopreparation having antihypoxic and antioxidant activity increases the stability of the L-carnosine and the life thereof in an organism whilst increasing the efficiency of the action of L-carnosine in small doses, as well as extending the range of such drugs. The mentioned use of L-carnosine for the preparation of a nanopreparation having antihypoxic and antioxidant activity comprises a combination of L-carnosine with a combination of substances selected from the group consisting of phospholipids and nonpolar lipids with the following ratio of components, in % by mass: L-carnosine - 1.1 - 1.2; nonpolar lipids such as triglycerides, cholesterol, free fatty acids, DL-α-tocopherol - 1.2 - 2.5; phospholipids such as phosphatidylcholine, phosphatidyl ethanolamine, lysophosphatidylcholine, lysophosphatidyl ethanolamine, sphingomyelin - 95.3 - 96.3. In this application as well, the drug prepared is used to improve tolerance to brain ischemia, to improve recovery after acute hypoxia, and to increase the antioxidant status of brain tissues. Furthermore, the drug is produced in the form of liposomes containing L-carnosine.

Description

APPLICATION OF L-CARNOSINE FOR PREPARATION OF A NANO DRUG POSSESSING ANTIGYPOXIC AND ANTIOXIDANT ACTIVITY.

1. Field of technology.

The invention relates to medicine, specifically to neurology and cardiology, in particular to the production of a drug in the form of a biologically active nanopreparation with antihypoxic and antioxidant activity.

2. The prior art.

It is generally recognized that such common diseases accompanied by hypoxia as coronary heart disease (CHD), heart failure and cerebral stroke occupy a leading position among the causes of disability and mortality. Therefore, the development of new drugs for the treatment of the cardiovascular system is a very urgent problem. Recently, in the clinical practice in the treatment of such diseases as biologically active substances with a wide pharmacological spectrum of activity, carnosine compounds are increasingly used (Ivnitsky Yu.Yu., Golovko AI, Sofronov GA Amber acid in the system of metabolic correction functional state and resistance of the organism. - SPb, 1998. - 82 pp. - Bulletin of the exp. biol. and honey., 2000, T.129, 2, 149-151).

L-carnosine is a natural neuropeptide that exhibits diverse biological activity. It has been shown to be highly effective in protecting neurons as in vitro (individual damage reactions of macromolecules, suspensions of isolated neurons or brain sections under conditions of a free radical attack), and in vivo - on various models of experimental ischemia of the brain and heart, hypobaric hypoxia (Boldyrev A.A. Karnozin. Biological significance and possibilities of application in medicine - Moscow: Moscow State University, 1998 , 320 p.). . It has been established, in addition, that carnosine is an important natural factor in the antioxidant defense system of the brain under conditions of oxidative stress (A. Boldyrev Karnozin and tissue protection from oxidative stress - M: Dialog - Moscow State University, 1999, 362 p) .

It was also found that L-carnosine, as a naturally active metabolizing compound, has a limited lifetime in the body, being cleaved by a specific enzyme, carnosinase. 15 minutes after intraperitoneal administration to rats, its blood content reaches a maximum, after which it immediately begins to decline, returning to its initial low level 30 minutes after administration. The brain and liver are characterized by a similar kinetics of carnosine accumulation, although the time to reach the maximum is shifted to 30 minutes, and decreased to the initial level by 45-60 minutes (Gulyaeva N.V. Prospects for creating drugs based on carnosine (some new principles). Biochemistry, T. 57, issue 9, 1992, pp. 1398-1403).

Therapeutic measures with the help of L-carnosine and its esters are carried out in case of pathology accompanied by hypoxia (cardiovascular diseases, diseases of the brain and nervous system).

The use of methyl and ethyl esters of carnosine is known as an antioxidant and antihypoxic agent obtained by enzymatic hydrolysis (RU 2191592, 10.27.2002). We considered this source of information as the closest analogue. The product obtained by enzymatic hydrolysis increases oxidative stability of biological structures. It was found that at a concentration of 150 μM, L-carnosine and its esterified derivatives after preincubation with equimolar concentrations of copper and zinc almost completely inhibit the restoration of HCT, which indicates the presence of pronounced superoxide-intercepting activity (SPA) in the studied compounds. The half-inhibition constant (K _0g5 ) for carnosine is 30 μM, and for ethyl and methyl esters - 25 and 60 μM, respectively. Thus, the effectiveness of SPA for these complexes varies significantly in the concentration range of 20-60 μM. High SPA was observed in carnosine ethyl ester and lower in carnosine and its methyl ester. The effective dose in an experiment on animals, in particular the antihypoxic and antioxidant effects of carnosine and its ethyl ester in a stabilized form with sulfate anions, is 100 mg / kg.

The specified drug is currently successfully used in medical practice. However, its use in the form of a biologically active substance in pathology developing against the background of hypoxia (heart attack, stroke, hypertension, etc.) in optimal doses is difficult due to the limited time of its life in the body. The creation of a nanopreparation containing L-carnosine allows to increase its lifetime in the body and, therefore, to introduce it in therapeutic doses in a small volume.

3. The technical problem.

The technical result of the invention is that liposome containers containing L-carnosine increase the stability of L-carnosine and its life time in the body against increase the effectiveness of its action in small doses, and also expand the range of such funds.

The technical result is achieved by the fact that L-carnosine is used to prepare a nanopreparation having antihypoxic and antioxidant activity in combination with a combination of substances selected from the group of phospholipids and non-polar lipids in the following ratio of components, in mass%: L-carnosine - 1.1-1 , 2, non-polar lipids, such as triglycerides, cholesterol, free fatty acids, DL-a-Tocopherol - 1.2-2.5, phospholipids, such as phosphatidylcholine, phosphatidyl ethanolamine, lysophosphatidylcholine, lysophosphatidyl ethanolamine, sphingomyelin - 95.3 -96.3 for the preparation of a medicinal product with antihypoxic and antioxidant activity.

An application is also proposed in which the drug is used to improve the tolerance of cerebral ischemia, recovery from acute hypoxia, and also to increase the antioxidant status of brain tissue

Also provided is an application in which the drug is in the form of liposomes containing L-carnosine.

4. The invention is illustrated by the following examples.

Preparation of liposomes containing L-carnosine.

Example 1

5 g of a mixture of dry lipids containing 4.76 g of phospholipids, such as phosphatidylcholine, phosphatidyl ethanol, lysophosphatidylcholine, lysophosphatidyl ethanolamine, sphingomyelin and 0.24 g of non-polar lipids, such as triglycerides, cholesterol, free fatty acids, DL-a mixture of DL-a with 50 ml of distilled water to the obtained emulsions add 50 ml of an aqueous solution of L-carnosine with a concentration of 50 mm (amount of L-carnosine 0.317 g), and placed in a glass, for stirring on a magnetic stirrer. Stirring is carried out for 30 minutes until complete hydration with the formation of a homogeneous suspension. Then, using a homogenizer, this coarse suspension is subjected to closed loop homogenization in a continuous process under a pressure of 50 MPa at a temperature of 30-35 ° C. The number of homogenization cycles (passes) can be from 5 to 10. The suspension after homogenization is obtained in the form of a translucent liposomal solution. Liposomes containing L-carnosine according to this example have a size of 120 nm. Then the resulting liposomal composition is sterilized at a pressure of 1 excess atmospheres. for 45 minutes. The average diameter of liposomal particles increases to 150-160 nm. The resulting liposomes consist of 96.3% phospholipids, 1, 2% non-polar lipids and contain 1, 1% L-carnosine (by weight), the rest is up to 100% water.

Example 2

5 g of a mixture of dry lipids containing 4.76 g of phospholipids such as phosphatidylcholine, phosphatidyl ethanolamine, lysophosphatidylcholine, lysophosphatidyl ethanolamine, sphingomyelin and 0.24 g of non-polar lipids such as triglycerides, cholesterol, free fatty acids, DL-a-mix 50 ml of distilled water. Then, L-carnosine is added to this mass of coarsely dispersed liposome suspension to a final concentration of 25 mM (amount of L-carnosine 0.317 g). The resulting liposome composition is subjected to closed loop homogenization in a continuous process using a high pressure homogenizer at a pressure of 60 MPa and a temperature of 30-35 ° C. The number of homogenization cycles (passes) can be from 5 to 15. After homogenization, a suspension is obtained in the form of a translucent liposomal solution. Liposomes containing L-carnosine according to this example have a size of 90-100 nm. Then the resulting liposomal composition is sterilized at a pressure of 1 excess atmospheres. for 45 minutes. The average diameter of liposomal particles increases to 120-130 nm. The resulting liposomes consist of 96.3% phospholipids, 1, 2% non-polar lipids and contain 1.1% L-carnosine (by weight), the rest is up to 100% water.

Example 3

5 g of a mixture of dry lipids containing 4.5 g of phospholipids, such as phosphatidylcholine, phosphatidyl ethanolamine, lysophosphatidylcholine, lysophosphatidyl ethanolamine, sphingomyelin and 0.5 g of non-polar lipids such as triglycerides, cholesterol, free fatty acids, DL-a-Tocophe with 50 ml of distilled water, 50 ml of an aqueous solution of L-carnosine with a concentration of 50 mM (amount of L-carnosine 0.331 g) is added to the emulsion obtained and placed in a glass for stirring on a magnetic stirrer. Stirring is carried out for 30 minutes until complete hydration with the formation of a homogeneous suspension. Then, using a homogenizer, this coarse suspension is subjected to closed loop homogenization in a continuous process under a pressure of 50 MPa at a temperature of 30-35 ° C. The number of homogenization cycles (passes) can be from 5 to 10. After homogenization, a suspension is obtained in the form of a translucent liposomal solution. Liposomes containing L-carnosine according to this example have a size of 120 nm. Then the resulting liposomal composition is sterilized at a pressure of 1 excess atmospheres. for 45 minutes. The average diameter of liposomal particles increases to 150-160 nm. The resulting liposomes consist of 95.3% phospholipids, 2.5% non-polar lipids and contain 1, 2% L-carnosine (by weight), the rest is up to 100% water.

Studies of the atigipoxic and antioxidant activity of phospholipid nanoliposomes loaded with L-carnosine (with a content of 1.1-1.2% relative to the mass of liposomes), their stability and lifetime in the body, and the effectiveness of their action in small doses were carried out on the following models. In vitro, a cellular model of oxidative stress induced in cerebellar granular cells was used. Cerebellar granular cells of 10-12 day old SAMR1 mice were used. To loosen the intercellular substance, a collagenase solution prepared using Hanks solution (Wako collagenase, 2 mg / ml, 1 ml / 1 animal) was used. The brain tissue fragments crushed with a scalpel were incubated at 32 ° C for 20 min without stirring. After incubation by decantation, the collagenase solution was removed, the precipitate was washed three times with Hanks solution; after washing, Hanks solution was added to the pellet at the rate of 1 ml per animal and the cells were dissociated by pipetting to an opalescent suspension; the resulting suspension was passed through a filter with a pore size of 60 μm, then cells were counted using a Goryaev camera. To work on a flow cytometer, the number of cells should not be less than 10 ⁶ / ml. Before the experiment, the cells were left for 30 minutes at 32 ° C, after which dichlorofluorescein diacetate (DCF-DA) with a final concentration of 10 μM was loaded with a fluorescent dye onto free radicals.

In vitro oxidative stress was created by incubating the primary culture with hydrogen peroxide. The intracellular level of free radicals and the number of dead cells in the preparations were determined using a FACSCalibur flow cytometer (BD Biosciences, USA), (Boldyrev A., Song R., Dyatlov V., Lawrence D., Carpenter D. Neuronal cell death and reactive oxygen species., Cell. Mol. Neurobiol.,. 2000, 20: 433-450).

The phospholipid nanoliposomes loaded with L-carnosine obtained in Example 1 were tested as possible protectors of neurons against oxidative stress.

The cerebellar granular cells used in the experiments are characterized by a certain initial level of DCF fluorescence corresponding to the stationary level of radical compounds. Incubation of cells with “empty” (unloaded L-carnosine) nanoliposomes somewhat reduces, although unreliably, the level of fluorescence. Under the same conditions, the use of carnosine-containing nanoliposomes significantly reduces the level of intracellular radicals, which increases the resistance of cells to oxidative stress.

Stationary level of free radicals in isolated granular cells of the cerebellum of mice under different conditions. A - intact freshly isolated granular cells, B - cells after incubation with "empty" nanoliposomes, C - the same with nanoliposomes containing L-carnosine is shown in Fig. 1. The average fluorescence in case A is 15.6 ± 0.7 rel. units, in case B - 14 ± 0.5, and in case C - 10 ± 0.4. (* - p <0.05 with respect to intact cells).

It was found that nanosomes not containing L-carnosine under stress caused by hydrogen peroxide do not interfere with the accumulation of intracellular radicals and cell death. At the same time, nanosomes containing L-carnosine effectively reduce the accumulation of oxygen radicals in neurons. Induction of oxidative stress by hydrogen peroxide (10 mM, 30 min) in the absence of (A) and in the presence of nanosomes loaded with carnosine (B) is shown in Fig. 2. Gray background - intact cells, black line - after incubation with Н2О2: along the ordinate axis - the number of cells, along the abscissa axis - relative units of fluorescence reactive oxygen species (ROS).

Moreover, the mortality rate of neuronal cells is significantly lower: in the population of cells contained in a medium with hydrogen peroxide, this value was 21.2 ± 3.0%, and in the presence of carnosine-containing nanoliposomes it did not exceed 15.4 ± 2.9% .

For in vivo studies, a hypobaric hypoxia model was developed that simulates an acute ischemic condition using adult SAMR1 mice. The animals were 8 months old. Oxidative stress was caused by exposure to acute hypobaric hypoxia. The study was carried out in a flow chamber. In the process of creating a vacuum corresponding to a rise to a height of 10,000 meters, the time elapsing until the posture was lost and the time before breathing stopped, characterizing resistance to hypoxic damage, as well as the time of rehabilitation after exposure to hypoxia. The effect of n-liposomes loaded with L-carnosine on the resistance of SAMR1 mice to the effects of acute hypobaric hypoxia is shown in Fig. 3. Nanoliposomes loaded with L-carnosine obtained in example 2 was administered 1 hour before hypoxia at a dose of 20-25 mg per kilogram of the animal’s body (free carnosine shows activity at a dose of 100 mg per kilogram of the animal’s body).

From fig. Figure 3 shows that the introduction of the drug nanoliposomes loaded with L-carnosine before hypoxic exposure leads to increased resistance of SAMR1 mice to hypoxic shock, increasing the posture preservation time and the time to stop breathing. Moreover, the duration of rehabilitation was reduced, which indicates an increase in the adaptive status of mice.

At the same time, testing mice in the "Morris water maze", allowing to evaluate the characteristics of the cognitive functions of animals, shows a more effective effect of nanoliposomes containing L-carnosine on SAMR1 mice. These animals were less successful in the process of learning to find a platform in the pool on the first day of the experiment after hypoxic exposure, but they retained better acquired skills the day after training, see Fig. 4, which shows testing of SAMR1 mice in the “Morris water maze”: 5 training attempts a day after hypoxic exposure and 5 attempts the next day to control memorization (p <0.05 with respect to hypoxia). The light bar is the number of successful attempts in intact mice, the dark bar is without exposure to nanoliposomes loaded with L-carnosine, the gray bar is when exposed to nanoliposomes loaded with L-carnosine at a dose of 20 mg / kg of animal body weight, obtained according to example 3. Nanoliposomes, loaded with L-carnosine was administered 2 times - 1 hour before hypoxia and 1 hour after hypoxia.

To assess the effect of nanoliposomes loaded with L-carnosine on the total antioxidant activity in brain tissue, a testing system based on the stable radical diphenylpicrylhydrazyl (DPPH) was used. The total recovery of the radical in 30 minutes was measured when an extract from the brain tissue was added to the system. It was shown that the total antioxidant activity of DPPH restoration provided by extracts from the brain tissue of mice undergoing hypoxic effects on the third day of reperfusion did not significantly differ from this indicator for intact animals. The introduction of L-carnosine-containing nanoliposomes provides an increase in the total antioxidant activity, which is consistent with the higher rates of these animals in the Morris test (Fig. 4).

Results of the influence of L-carnosine-containing nanoliposomes on the total antioxidant activity of extracts from brain tissue in the DPPH test shown in table 1. Nanoliposomes loaded with L-carnosine were administered to animals twice - one hour before hypoxia and an additional 1 hour after hypoxia. The decapitation was carried out 3 hours after the second test in the “Morris water maze”.

Table 1

Table 2 shows the effect of the study drug on the duration of antioxidant activity in SAMR1 mice under conditions of hypoxia and the lifespan of L-carnosine in mice (nanoliposomes loaded with L-carnosine, 20 mg / kg), n = 10 in each group.

table 2

Duration

Lifetime L-

Learning Saves

Dose, mg / kg of carnosine in antioxidant preparations

body, (hours) activity, hours

L-carnosine

received

150 3 3

enzymatic

hydrolysis

nanoliposomes,

loaded with L- 20 72 72 carnosine

nanoliposomes,

loaded with L- 25 80 80 carnosine As follows from the data presented in table 2, the introduction of n-liposomes loaded with L-carnosine obtained in example 1-3 when introduced into the body of an experimental animal at a dose of 20-25 μ / kg increased the duration of the antioxidant and antihypoxic effect against acute hypoxia and provided improved tolerance of cerebral ischemia, recovery from acute hypoxia, as well as increased antioxidant status of brain tissue compared with the introduction of L-carnosine obtained by enzymatic hydrolysis at a dose of 15 0 mc / kg.

Thus, the use of nanoliposomes containing L-carnosine at a dose of 20-25 mg per kilogram of the animal’s body increases the total antioxidant activity of brain tissue. This activity persists for 3 days after the introduction of nanoliposomes obtained according to examples 1-3 (for free carnosine, its decomposition in the body 3 hours after administration is shown).

Consequently, the nanopreparation of L-carnosine obtained in Example 1–3 can be considered as a promising nanoconstruction with antihypoxic and antioxidant properties and increasing the stability and lifetime of L-carnosine in the body against the background of increasing its effectiveness in small doses. In addition, this will expand the range of such funds.

Claims

Claim

1. The use of L-carnosine for the preparation of a nanopreparation with antihypoxic and antioxidant activity in combination with a combination of substances selected from the group of phospholipids, non-polar lipids in the following ratio of components, in mass%: L-carnosine - 1, 1-1,2, non-polar lipids such as triglycerides, cholesterol, free fatty acids, DL-a-Tocopherol - 1.2-2.5, phospholipids such as phosphatidylcholine, phosphatidyl ethanolamine, lysophosphatidylcholine, lysophosphatidyl ethanolamine, sphingomyelin - 95.3-96.3 for cooking medicinal with COROLLARY having antihypoxic and antioxidant activity.

2. The use according to claim 1, in which the drug is used to improve tolerance of cerebral ischemia, recovery from acute hypoxia, and also to increase the antioxidant status of brain tissue.

3. The use according to claim 1 or 2, in which the drug is made in the form of liposomes containing L-carnosine.