RU2191583C1 - Method for correcting hemorrhagic shock - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: one should intravenously inject the preparation of multilayer liposomes at the dosage of 1.0 mg/kg body weight taken at the concentration of 0.5 mg/ml distilled water. The method provides the restoration of hepatic lipids peroxidation, phospholipid composition of plasmatic membranes of hepatocytes, and membranous fractions of medulla oblongata mitochondria and frontal lobes of large cerebral hemispheres. EFFECT: higher efficiency of correction. 3 dwg, 5 ex, 2 tbl

Description

 The invention relates to medicine and relates to a method for the correction of hemorrhagic shock using liposomes.

 Massive blood loss, accompanied by hemorrhagic shock, is the cause of the mortality of a number of diseases as a result of profuse bleeding (peptic ulcer, stomach cancer, hemorrhagic erosive gastritis), postpartum hemorrhage, rupture of aneurysm of large arteries, and others. In trauma, hemorrhagic shock develops when Despite severe mechanical damage to soft tissues and bones, the factor of massive blood loss due to damage to large arterial arteries predominates in the development of shock. blood vessels (for example, with acute damage to long tubular bones; traumatic brain injury, accompanied by extracranial injuries, characterized by abundant acute blood loss, which occurs mainly in field wounds, etc.). Currently, mortality from hemorrhagic shock reaches 15-30%, and there is no significant tendency to decrease it. Of particular importance is the problem of massive loss of blood, accompanied by hemorrhagic shock, in military field surgery, given that most of the wounded come in the late period of shock, when conventional methods of treatment, including massive transfusion-infusion therapy, are often ineffective.

 A known method for the prevention of severe local and general post-ischemic disorders and related complications during the resumption of blood flow in long-term ischemic tissues after prolonged compression using voiced liposomes containing phosphatidylcholine (FC) and cholesterol as a lipid component, which is used at a dose of 25 mg / kg the body of the victim (Rudaev V.I. et al. A method for the prevention of local and general postischemic complications with prolonged compression trauma to the limb. - Patent of the Russian Federation N 2142271, l. A 61 K 9/127, published. 10.12.99). We take this method as a prototype.

 In contrast to the specified method, the claimed method for the correction of hemorrhagic shock is carried out using multilayer liposomes, the rate of excretion of which from the bloodstream significantly exceeds that of voiced liposomes, which provides an urgent effect on the body. The absence of cholesterol in liposomes contributes to an increase in the rate of removal of liposomes from the bloodstream. Thus, the claimed method allows for the correction of the consequences of circulatory disorders in a shorter time. The difference of the proposed method is also the use of low doses of PF (0.5 mg / kg body weight), which allows the use of liposomes in first aid.

 The choice of PF as a material for the preparation of liposomes is due to the fact that the PF of cell membranes, which makes up the bulk of their phospholipid bilayer, is depleted during the development of hemorrhagic shock in such vital organs as the brain and liver. The incorporation of PF liposomes into the cell membranes of target organs helps protect them from damage in hemorrhagic shock.

Installed:
1. Liposomes are an effective means of correcting hemorrhagic shock, acting at the subcellular, organ and systemic levels.

 2. The use of liposomes in the late period of hemorrhagic shock facilitates its course and significantly increases the life expectancy of animals.

 3. In contrast to unprotected liposomes, stable liposomes do not lose a pronounced anti-shock effect for a long storage time.

 The method is confirmed by the following examples.

Example 1. Correction of lipid peroxidation in the liver
Under the conditions of experimental hemorrhagic shock, the effectiveness of using liposomes for the correction of lipid peroxidation in the liver was evaluated. Hemorrhagic shock was modeled on cats weighing 2.5-3.5 kg for 1.5 hours (blood pressure was reduced to 40 mmHg for 30-40 minutes and maintained at this level for 1 hour, after which stopped bleeding). Blood loss in cats produced over 1.5 hours was 3.6 ± 0.1% of body weight. Liposomes at a dose of 1 ml / kg were administered intravenously 30 minutes after the onset of blood loss.

 Liposomes were prepared by the following method: soybean PF (Sigma, USA) at a concentration of 0.5 mg / ml was shaken in distilled water until a liposome suspension formed. Received freshly prepared "empty" phosphatidylcholine liposomes (PF liposomes).

 The liver peroxidation products were determined spectrophotometrically using 2-thiobarbituric acid (TBA).

 As can be seen from the results shown in Fig. 1, injection of PF-liposomes reduces the level of lipid peroxidation products in the liver in animals subjected to hemorrhagic shock by 1.8 times (p <0.01), bringing it closer to the control, which indicates suppression of damaging free radical processes in the tissue.

 Example 2. Correction of the phospholipid composition of the plasma membranes of hepatocytes.

 Simulation of hemorrhagic shock, preparation and administration of animal liposomes was carried out as described in Example 1. After isolation of the plasma membranes of liver cells by differential ultracentrifugation and extraction of total lipids from them using polar solvents, phospholipids were fractionated by thin-layer chromatography. The developed chromatograms were recorded on a Chromoscan-201 densitometer (England), and densitograms were analyzed on a Leitz-ASM semi-automatic analyzer (Germany).

 As can be seen from the results shown in Fig. 1, the injection of PF-liposomes restores the level of PF and phosphatidylinositol in the plasma membranes of liver cells to control values, which indicates a correction in the content of phospholipids, the metabolism of which is most vulnerable to chocogenic effects.

Example 3. Correction of the phospholipid composition of the membrane fractions of mitochondria of the medulla oblongata
Modeling of hemorrhagic shock, preparation and administration of animal liposomes was carried out as described in example 1. After obtaining mitochondrial membranes by differential ultracentrifugation, lipid extraction, fractionation of phospholipids and quantification of the composition of phospholipids in the membranes was carried out as in example 2.

 As can be seen from the results presented in figure 2, the injection of FK-liposomes significantly normalizes the phospholipid composition of the membranes of the mitochondria of the medulla oblongata. In the outer membranes of mitochondria, the level of FC increases compared with untreated animals by 43.7% (p <0.05). In the inner mitochondrial membranes in treated animals, a complete restoration of the level of PF was observed. In addition, the content of phosphatidic acid was normalized in the inner membranes of mitochondria. The control indicators also corresponded to the level of lysophospholipids.

Example 4. Correction of the phospholipid composition of membrane fractions of mitochondria of the frontal lobes of the cerebral hemispheres
The studies were carried out as described in Example 3. As can be seen from those presented in FIG. 3 results, injection of PF-liposomes in animals subjected to hemorrhagic shock, leads to the restoration of the content of phosphatidylinositol, phosphatidylethanolamine, as well as lysophospholipids in the membranes of mitochondria of the cerebral hemispheres.

Example 5. The impact on systemic blood pressure and life expectancy
Hemorrhagic shock was modeled for 2 hours, after which liposomes at a dose of 1 ml / kg were administered intravenously 2 hours after the onset of blood loss. Three types of liposomes were used: two types of freshly prepared liposomes [from purified soya lecithin (Sigma, USA) (PF-liposomes) and from the same lecithin, but oxidized after storage for one year at -18 ^o C (OFC-liposomes)] and stabilized liposomes (CT liposomes).

 PF and OFC liposomes were prepared as described in Example 1.

The method of preparation of ST-liposomes differs from that for freshly prepared liposomes in that the lecithin is shaken in distilled water containing lactose at a concentration of 0.5 mg / ml. The resulting solution is subjected to sterilization, bottling and freeze-drying, after which the bottles are sealed and sealed. Vials with lyophilized liposomes are stored for use for one year at -18 ^o C. Then the liposomes are dissolved in a concentration of 0.5 mg of lecithin per 1 ml of distilled water, left at room temperature for 1 h until ready.

 The research results are shown in Tables 1 and 2. As can be seen from the presented results, the injection of FK-liposomes in the late period of hemorrhagic shock facilitates its course and significantly increases the life expectancy of animals. When freshly prepared PF liposomes are injected 2 hours after the onset of blood loss, blood pressure rises over an average of 70 mm Hg over 20-30 minutes. Art. (tab. 1). Further, stabilization of blood pressure near a subnormal level takes place over 4 hours of observation. The life expectancy of such animals is an average of 8 hours. In untreated animals that also underwent 2-hour hypotension, a progressive decrease in blood pressure was observed, and they died on average after 70 minutes.

 The use of liposomes prepared from oxidized PF did not affect the course of hemorrhagic shock in cats. The dynamics of blood pressure in animals with hemorrhagic shock, which were injected with OFX liposomes, was similar to that of untreated animals, and the average life expectancy was 80 minutes.

 The nature of the effect of CT liposomes on the course of hemorrhagic shock in its late stage is similar to that of freshly prepared liposomes (Table 2). The dynamics of blood pressure in animals with hemorrhagic shock injected with CT liposomes was similar to that of untreated animals, and the average life expectancy was 8.5 hours on average.

 Thus, “empty” PF liposomes have a significant advantage over other known hemorrhagic shock correctors, which is manifested primarily in their membrane-stabilizing effect, combined with an increase in blood pressure to a subnormal level and its long-term stability. Stabilized liposomes have an efficiency equal to freshly prepared liposomes, but unlike unprotected liposomes, CT liposomes have a therapeutic effect over a long storage time, which helps to solve the difficulties of the practical use of liposomes.

 There is currently no remedy for the correction of hemorrhagic shock as highly effective and reliable as proposed. In the future, liposomes can be used for the prevention of hemorrhagic shock in case of massive blood loss, for first aid in the early and late stages of hemorrhagic shock, as well as in the complex treatment of hemorrhagic shock. Liposomes are prepared from cheap natural raw materials and their preparation can be stored for at least 12 months.

Claims (1)

 A method for correcting hemorrhagic shock, characterized in that a multilayer liposome preparation prepared from phosphatidylcholine taken at a concentration of 0.5 mg / ml distilled water is injected intravenously into the body at a dose of 1.0 ml / kg.

Images