UA46739C2 - METHOD OF MANUFACTURE OF MEDICINE FOR PREVENTION OR TREATMENT OF HUMANS OR ANIMALS AFFECTED BY ENDOTOXEMIA, PHARMACEUTICAL COMPOZE COMPOSITION - Google Patents

Abstract

Treatment and prophylaxis of endotoxin caused toxicity is disclosed. This is accomplished by administering phospholipid containing compositions to the subject. The compositions are protein and peptide free, and may contain triglycerides, other polar or neutral lipids, bile acids, or bile acid salts.

Description

Description of the invention

This application is a partial continuation of the application Mo 08/288 568, filed on August 10, 1994, which is a partial continuation of the application PCT/О593/07453, filed on August 9, 1993, which is a partial continuation of the US application Mo 07/928930, filed on 12 August 1992, according to which the US patent No. 5,344,822 was issued. All these applications and the patent! usage is described in the following links.

The invention relates to the treatment of zondoxemia caused by zondotoxins, in particular, to the treatment of zondotoxin poisoning by introducing various compounds into the body that neutralize and/or remove 70 zondotoxins from the body, as well as for prevention using these compounds.

Normal serum contains a certain amount of lipoprotein particles that differ in density, namely, chylomicron, LDL (very low-density lipoprotein), LDL (low-density lipoprotein), and HDL (high-density lipoprotein). They contain free esterified cholesterol, triglycerides, phospholipids, some other secondary lipid components, and protein. 712 DONP transports energy, in the form of triglycerides, to body cells for storage and use. When triglycerides enter the cells, LDL is converted into LDL. LNIP transfers cholesterol and other lipid-soluble materials! into the cells of the body, and HDL transports excess or non-lipid-soluble material to the liver for elimination from the body. Usually, these lipoproteins are in equilibrium, ensuring proper delivery and removal of lipid-soluble materials. Sometimes a very low level of HDL can be the cause of many diseases, and can also lead to secondary complications in other diseases.

Under normal conditions, natural HDL is a solid particle whose surface is covered with a phospholipid monolayer surrounding a hydrophobic core. Apoproteins A-I and A-II are attached to the surface of HDL particles as a result of the interaction of the hydrophobic surface of their alpha helical domains. When c 75 is formed or eliminated, this particle has a disk-shaped shape and is able to accept free (3) cholesterol in its bilayer. Cholesterol is esterified by lecithin cholesterol acyltransferase (LCAT) and moves to the center of the disk. The movement of the complex cholesterol sphere to the center occurs due to spatial limitation and low the solubility coefficient inside the bilayer. As cholesterol is esterified and moves to the center, the HDL particle "swells", acquiring a spherical shape. Cholesterol and other water-insoluble lipids) that accumulate in the "swollen core" of HDL are then removed by the liver .

In the publication Apapipagataiyan, ip Zedgevi ei aiI, May. Epgutoi 128:627-647 (1986) describes a series of peptides that form "spiral wheels" as a result of the interaction of amino acids in the peptide. Such spiral wheels in their configuration have non-polar and polar surfaces. In general, it follows from this source that 30 peptides can replace apoproteins in these particles.

In the publication of Dopashe A!., Me(y. Epgut. 128A: 553-582 (1986) it is shown the preparation of a large variety of reconstructed particles similar to HDL. To obtain apo-HDL proteins, HDL is isolated and delipidated by standard methods (see the works of Naisya ai., Adm.jr.Kevz. 6:1-68 (1968); Zsapy ei ai., ApaI.Viospet.44a: "576-588 (1971)). Apoproteins are fractionated and reconstructed by phospholipid with or without cholesterol, З7З 70 using dialysis against a detergent. c In the publication of Mai; Eyai, 9U.VioY.Spet.257(8): 4535-4540 (1982), a micelle of phosphatidylcholine with apolipoprotein A-1 is described. Different ratios of the two components are shown and it is suggested that the described method can be used to obtain other micelles. It is also suggested that the micelle can be used as an enzyme-catalyzed substrate or as a model for molecules! 75 lVp. However, the use of micelles for cholesterol removal has not been considered, and no suggested to use them in diagnostics or therapy. (He) In the publication UMiShatve ei ai!., Viospet. 85 Viorpuz. Asia 875: 183-194 (1986), concern phospholipid liposomes introduced into plasma, which absorb apoproteins and cholesterol. Descriptions of liposomes that themselves absorb apoproteins and mimo, as well as cholesterol, and it is suggested that the absorption of cholesterol (4) 50 is enhanced in those phospholipid liposomes that have interacted and absorbed apoproteins.

In the work of UMiPattz ei a!., Regzr. Axes 5 Med. 27(3): 417-431 (1984) consider lecithin liposomes! what a means of removing cholesterol. The article summarizes previous work! and it is shown that liposomes containing apoproteins remove cholesterol from cells more efficiently than liposomes that do not contain them. The authors do not consider the use of IP without apobelk containing liposomes! or 59 micelles, and caution is advised when working with liposomes.

GF) It is important to note that there is a clear and significant difference between the particles according to the present invention and the liposomes and micelles described earlier. The latter have a bilayer structure of lipid-containing molecules, which surrounds an internal aqueous central space. However, the structure of liposomes does not allow the filling of the internal space with lipid-soluble components, and any molecular absorption of lipid-soluble components is limited by the space between two lipid layers. As a result, the volume where absorption and excretion of such materials as cholesterol and other lipid-soluble materials is possible is much smaller than the volume for particles according to the invention, which increases like an air layer, filling with selected material.

Zndotoxin bacterial-toxic shock is a condition, often with a fatal outcome, because it is provoked by the release of lipopolysaccharide (LPS) from the outer membranes! most gram-negative bacteria (for example, EzsPegispia soii: ZaItopea yutrpitigisht). The structure of bacterial LPS is fairly well understood, and a single molecule, called lipid A, is linked to acyl chains through its glucosamine chain (see Kayig, App. Rheu. Viospet. 59: 129-170 (1990)).

The lipid A molecule serves as a membrane fixator of the structure of lipopolysaccharide (LPS), which is involved in the development of xenotoxin shock. It should be noted that LPS molecules are characterized by a structure of the type of lipid A and part of a polysaccharide. This part may differ in details in different LPS molecules, but it preserves the general structural basis of xendotoxins. Damn it! incorrectly says that the LIPS molecule is the same in all bacteria (see Kaei7, above). A casual specialist! name different molecules! LIS "xendotoxins", 7/0 M ztot term will be used further to denote LPS molecules.

In US patent No. 5,128,318, the description of which is used by reference, it is indicated that the reconstruction of particles containing both an apolipoprotein associated with HDL and a lipid capable of binding zondotoxin for ego inactivation can be used as an effective material! to reduce the toxicity caused by zondotoxins.

In previous applications, which are mentioned in the section "Related applications" and uses! in the way described in this article, it was indicated that other materials can be used to suppress the toxicity caused by zondotoxins! In particular, it was found that apolipoprotein! are not required in the reconstructed particles, and that the reconstructed particle can contain a peptide and a lipid, where the peptide is not an apolipoprotein.

The inventors also discovered that the toxicity caused by zondotoxins can be combated by the sequential introduction of an apolipoprotein or peptide, and then a lipid. Consecutive introduction of the components form a reconstructed particle and become active in the injected zondotoxins.

It was also found that at least some patients have natural levels of apolipoprotein, which are higher than normal and allow effective zondotoxemy therapy with the introduction of reconstructed particles that do not contain apolipoprotein or peptide, but contain descriptions of lipid.

In addition, the invention disclosed in these applications suggests the use of reconstructed particles and the mentioned components for the prevention of toxicity caused by xenotoxins by introducing a prophylactically effective amount of them to the subjects of prevention. Such subjects would include infected patients or those recovering from surgery. You are a patient! sometimes they have very low levels of plasma HDL! blood, decreasing to 20 95 normal levels. In those cases, early prevention with the use of HDL is desirable to compensate for those low levels. uh-

It was unexpectedly discovered that phospholipids can be used separately or in combination with additional materials, such as neutral lipids, cholates, etc., as an effective agent for weakening and/or preventing zondotoxemia. In particular, it is preferred to use phosphatidylcholine (hereinafter FH) both separately and in combination with other phospholipids, such as sphingolipids, in compositions that are essentially free of peptides and proteins, for example, apolipoproteins or peptides derived from them. Neutral lipids, such as mono-, di-, and triglycerides, can be mixed with phospholipids until the total amount of natural lipids is below a certain mass content in percent when using these compounds in the form of balls for 7) c intravenous administration. With other methods of administration, for example, intravenously by continuous infusion, the percentage by mass is not so important, but it is desirable. ;" Particularly preferred examples of the embodiment of the invention suggest the use of emulsions in which bile acid or its salt is used together with phospholipid or neutral lipid.

The effectiveness of bile acids and their salts - cholates in the treatment of zondotoxemia is shown. These bile acids can be used alone or in combination with one or more phospholipids and/or neutral lipids such as phosphatidylcholine and/or triglyceride.

Me. The invention is described in more detail below. -I Brief description of illustrations

Figure 1 shows how reconstructed particles containing apo A-1, phospholipid, and cholate are formed.

Ge) In fig. 2 shows how to reconstruct the particles! absorb molecules! LPS

Fig. 3 shows! experiment, in which the peptide according to the invention was used to study the reduction of toxicity caused by zondotoxins in a muscle model. 5B Fig. 4, related to the level of technology, shows the formation of spiral wheels by various peptides.

See Apapinagataiyan vishe.

F) Figures 5A and 5B show the results obtained when testing different compositions in a model where the neutralization of zondotoxin was determined by showing the release of TFN in a model of human whole blood. Fig. 5A shows the value of protein, and Fig. 5B shows the value of phospholipid. The test compositions included natural lipoproteins (VLDL, LNP, HDL), reconstructed HDL (P-HDL) and IMTRAIIRIO F composition, as well as smuls containing phospholipid and protein.

Figures 6bA and 6bB show the comparative value of triglyceride (neutral lipid) and phosphatidylcholine, a phospholipid in the same model.

Figure 7 presents information on toxicity when various FH and FH/G compositions are introduced into a 65 model muscle using a model infected with LPS Es.soy. with a fatal outcome of 55905.

Fig. 8 shows this data, comparable to the data obtained above, when a model of whole human blood was tested, but when a phospholipid with unesterified cholesterol, sphingomyelin or a mixture of the latter was used instead of triglycerides.

In Fig. 9A and 9B, the results are shown, comparable to the results in Fig. 5A and 5B, and in this case, phospholipid, unsterified cholesterol and/or sphingomyelin are mixed with triglycerides or esterified cholesterol as a neutral lipid.

Figure 10 shows a comparison of the results obtained from the introduction of emulsions containing cholesterol and triglycerides in the muscle model.

Figure 11 shows a diagram of the theoretical amount of triglycerides released into the blood after the introduction of various TG-containing compounds, with toxicity thresholds. The abbreviation "OPP" means "general parenteral nutrition", and "SY" - composition! according to the invention.

A detailed description of examples of the embodiment of the invention

Example 1

To determine the level of viability of mice infected with zondotoxins 5 utrpitigiit, unrelated /5 mice of the Zm/iz5-UUerzieg line were injected with saline solution (20 mice), reconstruction of particles! HDL (40 mice), or reconstructed peptide 18A (20 mice) by injection into the tail vein. Let's enter the material! prepared as follows: a. HDL particles

Particles were prepared from apo-Ni-HDL (8595 A; 15956 A-P and apo C), reconstructed with egg phosphatidylcholine (2:11 by weight), purified by 95,956 dialysis against detergent according to the well-known method described in the publication of Maya? hey! a)/., U.VioI. Spent 257: 4535-4540 (1982) and in US Patent No. 5,128,318, the description of which is incorporated by reference. b. peptide particles

Peptide 18A has the following amino acid sequence: su

Avr-Ttr-I ets-IG uz-AIa-РНе-Tug-Azvr-I uz-МаИ-АИа-с1у-Г uv-I ets-I uz-С1ИШ-АИа-РНе.

Peptide samples were mixed and reconstituted with pure egg phosphatidylcholine (2:11 by weight) 9590, purified according to the above method (Mai?ei AI., 9U.VioYi. Spect. 257: 4535-4540 (1982) and US patent No. 5 128 318) , also using dialysis against detergent. Obtaining as a result of particles! identical to the descriptions in pat. USA Mo. 5 128 318, except that instead of apo-HDL (Maikh ei ai., 9U.Vioi. Spet. 257: 4535-4540. du (1982) and references to the patent literature) a peptide component was present.

Within 15 minutes. after the introduction of the reconstructed material, the LPS of ZaiYitopeg, i. intraperitoneally LOmg/kg body weight. The evaluation criterion was durability. In Fig. Z, the result is shown! of the experiment, where the indicators are almost 4 times higher than in the control group using saline solution. The synthetic peptide is almost as effective as the reconstruction of particles containing apo-HDL. "AND

Example 2

factor) that influence the stimulation of FN-9 by means of LPS while preserving the integrity of the interaction of plasma proteins) and cellular elements of the blood, can be the appropriate way of "research and migo on the whole blood system of a person. Such a system was used to determine which of the lipoprotein components are important in LPS neutralization. - the following materials were tested: reconstructed high-density lipoprotein (HDL), natural lipoprotein! (LONP, LNP, HDL) "» blood plasma, lipoprotein-deficient serum (SNLP) and triglyceride-laden emulsion 2090

IMTRAGIRIO F (a mixture of triglycerides and phospholipids).

Blood was collected in a heparinized test tube, diluted with a balanced salt solution of Hznka (hereinafter referred to as SSRH) or the test material dissolved in SSRH was poured. The resulting material was poured into Starstedt tubes (250 μl/tube). LPS was dissolved in pyrogenic salt solution containing 10

F MM HEPES buffer and added (2.5 μl) to a final concentration of 1 ng/ml. After incubation for 4 hours at 37°C, the test tubes were cooled to 4°C, then centrifuged at 10,000 x d for 5 minutes. The supernatant liquid was poured off and with the help of the well-known enzyme immunosorbent assay (EGIZA) the FNN-o, iChe was determined. Table 1 gives comparative data on the composition of the tested materials. Figures 5A and 5B show! the result Data are presented in the form of graphs of the dependence of the quantity produced

FNN-o from the concentration of added protein (Fig. 5A) and phospholipid (Fig. 5B). To show the wide range of concentrations used, a logarithmic scale was used, where 10" corresponds to 1 mg/ml. All incubated whole blood systems contained 1 ng/ml of E. soy 0111:84 LPS, supplemented with one of the compounds, as an illustration of Fig. 5A and 5B) The fact that the effectiveness of the materials is different when the protein content is plotted on the graph (Fig. 5A) and is similar when the phospholipid content is plotted (Fig. 5B) indicates that phospholipid is an important component. the fact that a lipid emulsion that does not contain protein is more effective than natural HDL, but less effective than P-HDL. The protein turns out to be not so important for neutralization. lova 30390 pi: oo NE NS NES NE NANU (oh intalimd! 77777771) wa| vo

Example C

In this example, protein-free lipid emulsions containing different amounts of neutral lipid were tested on human whole blood. At the same time, the same analysis was used as in the investigation of human whole blood in Example 2.

All the description of particles here! were obtained according to the same procedure, which provides for the mixing of phospholipid, sphingomyelin or phosphatidylcholine, triolein, and/or unesterified cholesterol, dissolved in chloroform, and weighing them before placing them in a flask.

Vitamin E (0.0295 wt/vol) was added as an antioxidant. Then a dry lipid film was prepared by blowing gaseous nitrogen or argon over the sample. Then pyrogenic salt solution was added to the flask, and then the contents of the flask were mixed in a vortex mixer until the entire lipid was suspended. Then the solution was homogenized in a high-pressure homogenizer. Samples containing phosphatidylcholine (PH), with or without triolein, were cycled through the homogenizer 10 times at a pressure of 137,895 kPa (20,000 psi). Samples containing cholesterol esters with one or more other lipids were cycled 15-20 times at a pressure of 206,842 kPa (30,000 pounds per square inch). The homogenized solution was filtered using a syringe with a filter nozzle having a pore size of 0.45 μm, the filtrate was left at room temperature (for 3 days) until use. In fig. bA and 6B are the result! of those studies, where the data on the LIPS-dependent FNN-o produced depend on the concentration of the added triglyceride (fig.bA) or phospholipid (fig.bB). Composition), as shown in the reproduction, contained 795 triglycerides (TG), 4595 TG, 8995 TG, 9495 TG, P-HDL or phospholipid without TG and) (shown only in fig.b6B). The composition of 8995 TG contains 1095 IMTRAGIRIO F, and 94951 - 2095 IMTRAGIRIO. In all other experiments, egg phosphatidylcholine (FH) and triolein were used.

Here is the result! show that the protein-free compositions differ greatly in their triglyceride content (33%, but they are very similar! in studies taking into account the content of phospholipid (PH). This confirms the role of phospholipid, since only phospholipid is effective, although to a lesser extent than emulsions, and containing up to 45905 TG. M

Example 4

The experiment continues! continued! ip mimo on the mass model, which was selected as a reliable /-« of 5 systems for predicting the effectiveness of action on people. "

In these experiments, the mice were given, in the form of balls, a sufficient amount of the compounds described in example 3, as well as other compounds! (pure phosphatidylcholine, 7905 TG, 25905 TG, 4595 TG, 7190 TG, 8190 TG, 9495 TG) or a control saline solution, with doses of phospholipid (200 mg/kg or 40 Ω/kg), together with 25 mg/kg of E. soy 0111 :84 LPS. The control group was injected intravenously with a saline solution in a volume corresponding to the volume of the emulsion. Durability after 72 hours is shown in Fig.7. Of the 344 animals in the control group, 155 survived.

The use of only FH gave a moderate protective effect, statistically insignificant with a confidence level of 9595, while compositions containing 790, 4596 and 71906 TG significantly increased survival. Compositions containing 8095 and 8995 TG provided the minimum effect, and those containing 9495 TG

Serviceability decreased. When the dosage was increased to 400 mg/kg FH, the smulsified with 8995 and 9496 TG contents significantly shortened the lifespan, probably due to the toxic effect of TG, the explanation of which follows.

Example 5-I In examples 2-4, it was established that phospholipids are an active means of suppressing zondotoxemia.

The fact that nonpolar lipids, instead of triglycerides, can form a complex with phospholipids, but not with FH, suggests that other substances can also be used, for example, sphingomyelin (another

He phospholipid), unesterified cholesterol (polar neutral lipid) and their mixtures. They can also be used for terifying cholesterol (non-polar sulfur), squalene (hydrocarbon) and vitamin E (non-polar antioxidant). To test these white substances, a number of experiments were conducted, with the analysis of the model of whole human blood according to example 2 and the viability of muscles according to example 4.

Get ready! emulsified as described above, from pure phosphatidylcholine, phosphatidylcholine with 1090 (F) (in mass ratio) of unesterified cholesterol, 1095 (in mass ratio) of sphingomyelin, or a total of 1096 of a mixture of unesterified cholesterol and sphingomyelin. Emulsified was added to whole blood at a concentration of 10 Ωg/dL in the ratio of FH and 1 Ong/ml LPS. The mixture was incubated and the release was measured in FNN-o.

The results of experiments are shown in fig.8. The allocation of FNN-o was significantly reduced when only FH was used. Emulsions containing unesterified cholesterol, sphingomyelin or their mixture also inhibited the secretion of FNN-o.

Example 6 65 Whole blood analysis was also used to determine the effect of unesterified cholesterol and/or sphingomyelin on an emulsion containing a neutral lipid. In addition, they used the subjunctive

10 Omg/dL FH. Different composition (in terms of mass relations) of representations! in the following table; at

Fig. 9A and 9B are representations! the results of the experiment. Emulsified FH, preparation with neutral /5 lipid in the presence of polar lipids or without them showed inhibitory ability. In addition, the concentration of LIS was 1Ong/ml, i.e. clinically relevant concentration of zondotoxin. Emulsions containing zero cholesterol were less effective than emulsions containing TG, while emulsions containing unsterified cholesterol did not suppress PN, as well as emulsions that do not contain cholesterol. Addition of sphingomyelin to the osmulsified led to an increase in suppression of the production of FNN-o.

Example 7

Emulsions containing zero cholesterol were tested immediately on the model used in example 4 with a lethal dose of xenotoxins. Preparations of smulsified from FH, TG or FH and zero cholesterol (ZH) were administered with a single dose of 200 mg/kg FH in the form of a ball together with 25 mg/kg E. soya 0111: 84 LPS (lethal dose) in the tail vein. The control group was injected intravenously with saline in a volume corresponding to the volume of the emulsion. at

Fig. 10 shows the comparative results of the action of smuls containing FH and TG. Both methods were tested in at least two experiments using 16 or more animals.

As can be seen in the illustration, the slurry containing 795 or 45956 ХХ by mass significantly increased /(Fdzu з Вживаемост) Zndotoxins

Example 8 h-

A protein-free emulsion of phospholipid with triglyceride effectively blocks the production

FNN-o, in whole blood, activated with LPS. Theoretically, these rioters can fight more effectively! ise) also on living objects, if they can be safely administered in doses that provide protection of the concentration of phospholipid in plasma. Our previous experiences! with P-HDL allow us to assume that the minimum dose of phospholipid is approximately 200 Ω/kg. Using this dose and volume of plasma! 4.595 masses! body, it is possible to calculate the concentration of triglyceride expected in the plasma after the introduction of smulsy with successively increasing triglyceride content. The result is shown in Fig. 11 in the form of a smooth line that bends upwards with increased TG content (906 by mass). Concentrations of TG in plasma rarely exceed 100 Ωg/dL in healthy adults even after eating fatty food. According to clinical data, pancreatitis also occurs in patients with a TG concentration in the plasma of more than 2000 mg/dL (Ragteg, ei ai!., Ateg. U. Med. i"? 54: 161-164 (1973); Kgaizev, ei ai,, Ateg. 9. May 62: 144-149 (1977); SichesK, ei ai., 9. Iar. Siip. May. 123: 59-61). The concentration of TG in the plasma more than 400 Ωg/dL occurs extremely rarely and becomes the cause of serious concerns. In Fig. 10, the two last thresholds of the readings are horizontal lines. When 1095 or 2095 IMTRAGYRIO 2 is introduced to obtain a dose with a content of 200 mg/kg of phospholipid, a significant increase in TG concentrations in plasma (see two contour circles) beyond the safe limit is expected !. V

On the contrary, the introduction of smulses containing 7905, 45 95, 7195 or 7895 (solid squares - from left to right) will increase the concentration of TSH in plasma, respectively, to 136, 477, 1300 or 200 Ω/dL. It is expected that smulsified with a TG content of up to -50905 will not be toxic due to the presence of TG. o Example 9

Ge) Effectiveness of combinations of phospholipid and bile acids, i.e. sodium cholate, tested in an experiment similar to that described in the previous examples. However, the method used to prepare the composition administered for testing on animals was different. In this and the following examples, the composition was prepared using a high-pressure homogenizer "Misgoichidileg. This device facilitates scaling.

F, Liquid triolein or liquid soy triglyceride was dissolved in the corresponding mass amount of water or water with MM, 18 mM or ZbmM sodium cholate. Solid granulated phosphatidylcholine was weighed on paper, and then slowly added to the solution while stirring. At the same time, approximately 3-bmin is required. for lipid dispersion. After dispersion, the resulting material was poured into a high-pressure homogenizer. In this device, hydraulic pressure is used to turn on the pump, which, in turn, directs two opposite streams of samples. The pressure can reach 172.4 kPa (25,000 psi). When the jets collide under pressure, they pass into the cross-shaped hole, thus homogenizing the sample. 65 The sample was passed through the homogenizer several times, and "one pass" is the time required to pump the entire sample through the device. Samples were passed 20 times to achieve the required level of homogenization. Dextrose was added to the final 590-concentration.

Zndotoxin, purified from E. soy 0111:84 (40 mg/kg), and the emulsion described below (200 mg phosphatidylcholine/kg) were mixed at room temperature and immediately injected into the muscles of the C57VI line 6/) (mass 19 -

ZOg) by intravenous injection into the tail vein. Those mice that received only cholate were injected with a volumetric dose of sodium cholate, equal to the dose of the preparation cholate/EMI (excitable), at the same concentration of cholate.

Control mice were injected with the same dose of 5BUo-dextrose solution to ensure plasma osmolality.

The result! bring on the experiment! in the table below. In that case, an emulsion containing 7/0 phosphatidylcholine and 795 triglycerides, described in the previous examples, was used. Sodium cholate was added at the specified concentration to the starting materials to obtain an emulsion. tet lvmmhk roviv viv 81111118 er yav vi1111111111v|vi pt ET NO TE OO PO NU tv riv vviovv1111tv

The content of the Zmulsy was largely white as follows. When using 9MM cholate, the percentage content of sugar by mass in the slurry was: 79Uo of cholate, 6.196 of triglyceride and 86.9 of phosphatidylcholine; at 18 mM cholate: 13.196 cholate, 5.790 triglyceride and 81.295 phosphatidylcholine; with ZbmM cholate: 23.290 cholate, 590 (o) triglyceride and 71.8 phosphatidylcholine.

It should be noted that the amount of LPS administered in these experiments (4Omg/kg) is significantly higher than the amount used for lethality studies in previous examples. The purpose of introducing these Fo

The reason for the higher doses was that they would suppress any protective effect of phosphatidylcholine and/or triglyceride. Thus, it follows from these experiments that salt bile acid!, i.e. sodium cholate, have a protective property.

There are no descriptions here! experiment! with other salts of bile acid! and taurine, containing salts of bile acids. Among the bile acids, it should be noted, for example: allodeoxycholic, lithocholic, « 5PModeoxycholic, hyocholic acid, o, p, 0s - formic acid, murodecoxycholic, « ursodeoxycholic, ursocholic acid and all their salts, such as sodium salts or taurine or their conjugates wisteria See Noitapp below.

Example 10

Further experiments were conducted, the first of which investigated the viability of mice, "70 used as research animals. 2 p. In addition, they were divided into 4 groups. The first group - the control group - was given a 595 dextrose solution. The second introduced an emulsion containing 9395 (by mass) of phosphatidylcholine and 795 (by mass) of triglyceride and ;" prepared as described above. This emulsion contained 595 dextrose and soy phospholipid! with approx

bomg/ml of lipid.

The third and fourth groups of animals were given an emulsion similar to the emulsion administered to the second group, with the addition of 18 mM sodium cholate or 18 mM sodium deoxycholate. In this experiment, the same procedure as in example 9 was used.

Me, Viability was determined 72 hours after infection; this is a summary! in the following table. -I o so py PI POZ PO PO Ye r vzhdvyuru 30000004 z PI ss NOSA POL NO NOYA 8 | 111111 Cholatnatiyaett 16177714) bos ooo o 4) Diskolcholatnatriyaye tt 8/5) ooo) ooooot mo ime) "M - unacceptable in We note that the statistical significance of comparisons of the survival of groups of animals was checked by the method

Wilcox using computer programs". Comparative data with control group 1 of rooms under the index "1", comparison with group 2, which animals were injected with 790-smulsion - "2", and comparison with group Z of animals, which were injected with sodium cholate emulsion - "3".

Both viability (90) and statistical analysis reflect a definite, unexpected advantage of compositions containing salts of bile acids. b5 Example 11

The second group of experiments was tested on a rabbit model. In this model, the allocation was determined

FNN (newly formed necrosis factor)-o.

The rabbits were divided into the Z group and administered a 59o dextrose solution, a phospholipid and triglyceride emulsion (9390/795), described above, or the same emulsion, but with 18 mM cholic acid!. All emulsions were corrected with 595 dextrose solution, as in example 10. Rabbits were pre-injected with a ball of emulsion and after 2 hours - 10 Ωkg. E. soii 0111: 84 LPS. In addition to the smulsified ball, a composition of 5O0 mg of lipid per kilogram of mass was continuously infused intravenously! bodies in time. Intravenous infusion was carried out within 3 hours after control infection. 70 Blood was taken from rabbits in the area of the line separating the vault and the base of the skull, 30 minutes after the preliminary introduction of the ball and every time within 5 hours after the introduction.

The following table shows the maximum values of the TN-co. They are registered! 2 hours after zondotoxin administration.

The statistical significance of indicators was determined using the well-known Student's test. Kak 75 As can be seen from the tables, the indicators of FNN-o significantly decreased after the introduction of 18 mM cholic acids. gnome m 0 vdeyutoduyuroym tyu 9 s

These examples reveal in detail the invention, which assumes that one of the goals is to weaken or prevent zondotoxemia by introducing an effective amount of phospholipid to the patient, which is associated with zondotoxins. Associations of phospholipids and zondotoxins are then removed from the patient's body as a result of common biological processes, well known to those who are familiar with the processes of removal of lipoprotein particles. Phospholipid suppresses zondotoxin in association with it. Ge")

From these examples it is clear that the introduction of any of cholanic acids or salts of cholanic acids, such as bile acid or its salt, can also be used instead of phospholipids to achieve the same effect, ie. weakening or prevention of znodotoxemia. Thus, the composition), which does not contain peptides and proteins, but contains only bile acid or a bile acid salt or both, as well as phospholipids, can be used for the treatment of zondotoxemia. The description of cholanic acids (see, for example, the article Neutapp, Nerayiodu 4 (5): 45 - 145 (1984)) is included in this text by reference, in particular, "Ж page 55, fig. 1 and 2, where indications The characteristic structure! cholanic acids.

The invention can be used preferably for the treatment of humans, but its use in veterinary medicine is not excluded. "

The term "weakening", used here, means treatment to reduce the severity of the manifestation of zondotoxemia, caused by any of the zondotoxins produced, for example, by gram-negative bacteria /-- with (5. lutrpitigisht, E. soii, eis.). Prevention can be carried out by introducing the appropriate agent at the moment when there is a danger of poisoning with xondotoxins. A classic example is a surgical operation. So, an active ingredient can be administered to a patient who is being prepared for a surgical operation.

The effective amount of phospholipid and bile acid needed to treat a patient can vary. In general, the total weight dose is approximately 200-800 mg of phospholipid per kilogram of weight! body is preferable, although the amount may be more or less depending on the severity of zondoxemia or

F degree of risk in prevention. The applied dose of cholanic acids and salts, for example, bile acids and their salts, contains approximately 10-300 mg per kilogram of body weight, more preferably approximately 275 mg per kilogram of body weight! bodies It is desirable for me to introduce bile acid/its salt and phospholipids with neutral lipids, but it is not (Che) mandatory, since the introduction of phospholipid emulsions that do not contain neutral lipids is also provided. The expediency of the combined introduction of phospholipids is due to the fact that neutral lipids and phospholipids are combined in particles that are similar! with lipoproteins, but differ from them in that they do not contain protein peptide components, which of course is always present in lipoproteins.

Such forms of treatment are especially desirable, where the phospholipid is represented by phosphatidylcholine, for example, egg yolk phosphatidylcholine, soy phosphatidylcholine or sphingolipid. As a name) bile acid/its salts are preferably cholic acid and/or its salts, such as sodium cholate, sodium deoxycholate and sodium chenodeoxycholate. As for neutral lipids, it is preferable to use cholesterol esters or triglycerides, but you can also use other neutral lipids, for example, squalene or other hydrocarbon oils, di- and mono-glycerides, and antioxidants, such as vitamin E.

Form! for introduction, they can be different, and spherical or others for intravenous administration are especially preferable. When using a spherical form, for example, with triglyceride, it is necessary to exercise caution with the prescribed dose. It is well known that triglyceride! toxicity if they are introduced in too large a quantity. But a specialist can easily choose such a composition to eliminate or reduce the risk of triglyceride poisoning. By the way, when the spherical shape is used! the mass fraction of the triglyceride or the second neutral lipid should be approximately no more than 8095, preferably no more than 7095. More preferably, the mass fraction of the neutral lipid in the ball should be approximately no more than 50965.

However, when non-spherical forms are used, for example, other forms! for intravenous administration, the risk of poisoning is reduced. However, the limit, description is higher, preferred! for intravenous or other methods of administration, although it is obvious that they are not mandatory. Doses of bile acids and their salts preferably range from 25mg/kg to 50mg/kg of body weight! bodies and more, preferably - approximately 7 from B5Omg/kg to 10Omg/kg of mass! bodies Preferred doses of phospholipids are preferably from 10 Ω/kg to 1000 mg/kg of mass! bodies Here are the general dosages, however, they will vary depending on the patient and the method of administration.

As indicated above, in compositions that do not contain proteins and peptides, at least one phospholipid or bile acid/its salt is necessary. Of the phospholipids, the presence of at least 7/5 of one neutral lipid, for example, triglyceride, diglyceride or mono-glyceride, is preferable. These compositions can include additional materials, for example, sterols (for example, cholesterol, I-sitosterol), esterified or unsterified lipids (for example, cholesterol ester /- or unsterified cholesterol), hydrocarbon oils, such as squalene, antioxidants!, such as vitamin Well, they are not mandatory. There is no doubt that any such formulation may include more than one phospholipid and/or more than one neutral lipid. When using a combination of a neutral lipid and a phospholipid, the neutral lipid should be present in an amount of approximately 395 to 5095 by mass relative to the total amount of lipid in the composition.

Bile acid or its salts can be used separately or together with a phospholipid, a neutral lipid. As for additional materials (for example, phospholipids or neutral peptides), their preferred types will be mentioned above.

Partially according to the invention, the proposals also include compositions for the treatment of zondotoxemia. One of i) examples of the embodiment of the invention is a composition containing at least one of bile acids/its salt, a phospholipid and a neutral lipid, which in general contains the active ingredient in an amount that weakens the manifestation of zondotoxemia. This composition mainly contains approximately 595 to 3090 Ge! zr by mass of bile acids/ee salts, approximately from 395 to 5095 by mass of neutral lipid and approximately from 1090 to 9595 by mass of phospholipid. Especially preferred! a composition containing approximately 10-1595 by weight of i, bile acid/ee salts, approximately 595 to 1095 by weight of neutral lipid, and the rest is h-phospholipid.

It should be noted that these mass fractions refer to compositions containing three components. In the case of a combined three-component system with, for example, a carrier, an adjuvant, optional ingredients, similar to the descriptions above, the indication of the mass fate relative to the entire composition will be below. It should be borne in mind that in all cases such therapeutic compositions do not contain proteins and peptides.

If the compositions, which do not contain proteins and peptides, also do not contain bile acid or its salts, then it is possible to introduce preferably at least about 3956 to about 5095 by mass of neutral 7-3) c lipid, the rest - at least one phospholipid. Neutral lipid is the main thing

And triglyceride, or your favorite additional neutral lipid, described above. In addition, phospholipid is primarily phosphatidylcholine. Another aspect! inventions are clear! there is no need for a specialist to re-cite them here.

It is clear that description and example! serve as an illustration of the invention and do not limit the scope, and the specialist may offer other variants of the invention that do not exceed the limit! ego essence and volume. (22)

She

Claims (30)

The formula of the invention about 50 Ge 1. The method of production of a medicine for the prevention or treatment of people or animals suffering from zondotoxemia, which consists in preparing a pharmacological composition that contains cholanic acid or a salt of cholanic acid!, phospholipid and neutral lipid. 2. The method according to claim 1, characterized by the fact that the composition contains no more than approximately 8095 mass of neutral lipid. (F) 3. The method according to claim 1 or 2, characterized by the fact that the composition contains no more than 7095 mass of neutral GI lipid. 4. The method according to one of claims 1-3, characterized by the fact that the composition contains no more than approximately 5095 mass! neutral lipid. 5. The method according to one of claims 1-4, characterized by the fact that the composition contains up to approximately 5095 mass of neutral lipid relative to the total mass! lipid in the composition. 6. Pharmaceutical composition, devoid of proteins and peptides, which is suitable for intravenous use, while the composition contains cholanic acid or a salt of cholanic acid!, phospholipid and 65 neutral lipid, and the content of neutral lipid is approximately 3-5095 of the total mass of lipid. 7. Pharmaceutical composition according to p.b., characterized by the fact that cholanic acid or a salt of cholanic acid is a bile acid or a salt of bile acid. 8. Pharmaceutical composition according to one of items b6-7, characterized by the fact that phosphatidylcholine is a phospholipid. 9. Pharmaceutical composition according to any of claims 6-8, characterized in that the phospholipid is a sphingolipid. 10. Pharmaceutical composition according to any of claims 6-9, characterized by the fact that the neutral lipid is a triglyceride. 70 11. Pharmaceutical composition according to any of claims 6-9, characterized by the fact that the neutral lipid is a complex cholesterol ester. 12. Pharmaceutical composition according to any one of claims 7-11, characterized by the fact that the salt is bile acid! vibrate from sodium cholate, sodium deoxycholate and sodium chenodeoxycholate. 13. Pharmaceutical composition according to claim 12, characterized by the fact that salt is bile acid! is cholate /5 sodium. 14. Pharmaceutical composition according to any of claims 6-8, 10 and 13, characterized by the fact that it contains phosphatidylcholine, triglyceride and sodium cholate. 15. Pharmaceutical composition according to claim 14, characterized by the fact that the mass ratio of phosphatidylcholine:triglyceride is 93:7. 16. Pharmaceutical composition according to claim 14, characterized by the fact that the total mass ratio of sodium cholate, phosphatidylcholine and triglyceride is 13.1:81.2:5.7, respectively. 17. Pharmaceutical composition according to any of paragraphs b6-16, characterized by the fact that it is used in the production of a medicine for the prevention and treatment of people or animals suffering from zondotoxemia. 18. Pharmaceutical composition, devoid of protein and peptide, which is an emulsion containing bile acid/bile acid salt, phospholipid and neutral lipid, while the composition contains approximately 5-30 wt.9o bile acid salt bile acid!, approximately 3- 50 wt.9o neutral i) lipid and approximately 10-95 wt.9o phospholipid. 19. Pharmaceutical composition according to claim 18, characterized by the fact that it contains approximately 10-15 wt.9% of bile acid salts, approximately 5-10 wt.7% of neutral lipid, and the balance of the composition is created by phospholipid. 20. Pharmaceutical composition according to any one of claims 18 or 19, characterized by the fact that it is suitable for intravenous use. uh- 21. Pharmaceutical composition according to any of claims 18-20, characterized in that the phospholipid is phosphatidylcholine. ise) 22. Pharmaceutical composition according to any of claims 18-20, characterized by the fact that the phospholipid is "E sphingolipid. 23. Pharmaceutical composition according to any of claims 18-22, characterized in that the neutral lipid is triglyceride. 24. Pharmaceutical composition according to any of claims 18-22, characterized by the fact that the neutral lipid is a complex cholesterol ester. in the village 25. Pharmaceutical composition according to any of claims 18-24, characterized by the fact that the bile acid salt is selected from sodium cholate, sodium deoxycholate and sodium chenodeoxycholate. ;" 26. Pharmaceutical composition according to claim 25, characterized by the fact that it is a bile acid salt! is sodium cholate. 27. Pharmaceutical composition according to any of claims 18-21, 23 and 26, characterized by the fact that it contains 5" phosphatidylcholine, triglyceride and sodium cholate. 28. Pharmaceutical composition according to claim 27, characterized by the fact that the mass ratio of Me, phosphatidylcholine: triglyceride is 93:7. -AND 29. Pharmaceutical composition according to claim 27, characterized by the fact that the total mass ratio of sodium cholate, phosphatidylcholine and triglyceride is 13.1:81.2:5.7, respectively. at 30. A pharmaceutical composition according to any one of claims 18-29, characterized by the fact that it is used in the production of a medication for the prevention and treatment of people or animals suffering from zondotoxemia. F) name) 60 b5