MXPA97000929A - Use and useful compositions in the prophylaxis and therapy of conditions related to endotox - Google Patents

Abstract

The present invention relates to the treatment and prophylaxis of toxicity caused by endotoxin. This is achieved by administration to the subject of compositions containing phospholipid. The compositions are free of protein and peptide, and may contain triglycerides, polar or neutral lipids, bile acids, or salts of bile acids.

Description

METHODS AND USEFUL COMPOSITIONS IN PROPHYLAXIS AND THERAPY OF CONDITIONS RELATED TO ENDOTOXIN RELATED REQUESTS This request is a continuation in part of No. Series 08 / 288,568, filed on August 10, 1994, which is a continuation in part of the * PCT Application, PCT / US93 / 07453 filed on August 9, 1993. , which is a continuation in part of U.S. Patent Application Serial No. 07 / 928,930 filed August 12, 1992, now U.S. Patent No. 5,344,822. All these applications and patents are incorporated by reference.

FIELD OF THE INVENTION • and This invention relates to the treatment of endotoxemia related to endotoxin. More particularly, this relates to the treatment of such poisoning via the administration of various compositions which act to neutralize and / or eliminate the endotoxins of the organism, as well as the prophylaxis using these compositions.

REF: 23973 ANTECEDENTS AND PREVIOUS TECHNIQUE Normal serum contains a number of lipoprotein particles which are characterized according to their density, namely chylous, VLDL, LDL and HDL. These are composed of free and esterified cholesterol, triglycerides, phospholipids, various other minor lipid components, and protein. Very low density lipoprotein (VLDL) transports energy, in the form of triglycerides, to the cells of the body, for storage and 'Utilization. As triglycerides are distributed, VLDL is converted to low density lipoprotein (LDL). Low-density lipoprotein (LDL) transports cholesterol and other lipid-soluble materials to cells in the body, while high-density lipoprotein (HDL) transports lipid-soluble, excess or unusable materials to the liver to its elimination. Normally, these lipoproteins are in balance, ensuring proper distribution and elimination of lipid-soluble materials. The abnormally low HDL can cause a number of disease states as well as being a secondary complication in others. Under normal conditions, a natural HDL is a solid particle with its surface covered by a phospholipid monolayer that encloses a hydrophobic core. Apolipoproteins A-I and A-II are bound to the surface by the interaction of the hydrophobic face of their alpha helical domains. In its nascent or newly secreted form, the particle is in the form of a disc and accepts free cholesterol within its bilayer. Cholesterol is esterified by the action of lecithin: cholesterol acyltransferase (LCAT) and is moved towards the center of the disk. The movement of cholesterol ester to the center is the result of spatial and solubility limitations within the bilayer. The HDL particle "is inflated" to a spheroid particle as more and more cholesterol is esterified and moved toward the center. The cholesterol ester and other water-insoluble lipids which are collected in the "inflated core" of HDL are then cleared by the liver. Anantharamaiah, in Segrest et al., Meth. Enzymol. 128: 627-647 (1986) describe a series of peptides that form "helical wheels" as a result of the interaction of the amino acids in the peptide with one another. Such helical wheels have a non-polar face, and a polar face in their configuration. The reference shows, in general, that peptides can replace apoproteins in these particles. Joñas and collaborators, Meth. Enzym. 128A: 553-582 (1986) have produced a wide variety of reconstituted particles that resemble HDL. The technique involves the isolation and delipidation of 0 HDL by standard methods (Hatch et al., Adv. Lip. Res. 6: 1-68 (1968); Scanu et al., Anal. Biochem. 44: 576-588 (1971). to obtain apo-HDL proteins Apoproteins are fractionated and reconstituted with phospholipid and with or without cholesterol using detergent dialysis Matz et al., J. Biol. Chem. 257 (8): 4535-4540 (1982) describe a micelle of phosphatidylcholine, with apoliprotein A-I. Various proportions of the two components are described, and it is suggested that the described method can be used to make other micelles. It is also suggested to use the micelles as an enzyme substrate, or as a model for the HDL molecule. This document, however, does not discuss the application 5 of micelles to the elimination of cholesterol, nor does it give any suggestion for diagnostic or therapeutic use. Williams et al., Biochem. & Biophys. Acta 875: 183-194 (1986) teach the phospholipid liposomes introduced into the plasma, which collect the apoproteins and cholesterol. Liposomes are described, which collect apoprotein in vi, as well as cholesterol, and it is suggested that the uptake of cholesterol is increased in the phospholipid liposomes which have interacted with them, and collected the apoproteins. Williams et al., Persp. Biol. & Med. 27 (3): 417-431 (1984) discuss lecithin liposomes as cholesterol scavengers. The document summarizes the initial work that shows that liposomes containing apoproteins eliminate cholesterol from cells in vi tro more effectively than liposomes, which do not contain it. They do not discuss the use in vivo of liposomes or micelles that contain apoprotein, and the caution advised in any work in vivo with liposomes. It is important to note that there is a clear and significant difference between the particles of the present invention, and the liposomes and micelles described in the prior art. The latter involve a two-layer structure of molecules that contain lipid, surrounding an inner aqueous core space. The structure of the liposomes prevents the filling of the internal space with a lipid-soluble component, however, and any molecular uptake of the lipid-soluble components is limited to the space defined between the two lipid layers. As a result, there is much less volume available for the uptake and discharge of materials such as cholesterol and other lipid-soluble materials than there is for the particles of this invention, which expand in a manner similar to a balloon, with the interior space filled with the material of choice. Endotoxin shock is a condition, frequently fatal, caused by the release of lipopolysaccharide (LPS) from the outer membrane of most gram-negative bacteria (eg Escheri chia coli, Salmonella typhimuri um). The structure of bacterial LPS has been clearly elucidated, and a single molecule, designated as lipid A, which is linked to the acyl chains via the glucosamine backbone of the lipid A molecule. See Raetz, Ann. Rev.

Biochem. 59: 129-170 (1990) in this regard. The lipid A molecule serves as the membrane anchor of a lipopolysaccharide structure ("LPS") and it is the LPS that is involved in the development of the endotoxin shock. It should be noted that the LPS molecules are characterized by a lipid A type structure and a polysaccharide portion. This latter portion may vary in molecular details in different LPS molecules, but it will retain the general structural portions characteristic of endotoxins. It could be incorrect to say that the LPS molecule is the same from bacteria to bacteria (see Raetz, supra). It is common in the art to refer to the various molecules of 5 LPS as "endotoxins", and this term will be used hereinafter to refer to the y-molecules of LPS collectively. In US Pat. No. 5,128,318 the description of which is incorporated by reference 0 herein, it was shown that reconstituted particles containing an apolipoprotein associated with HDL and a lipid capable of binding to an endotoxin to inactivate it, could be used as effective materials to relieve the toxicity 5 caused by endotoxin.

In the parent and predecessor applications cited in the Related Requests section, incorporated by reference herein, it was described that other materials may be used to treat the toxicity caused by endotoxin. Specifically, it was found that apolipoproteins are not required in reconstituted particles, and that the reconstituted particle can contain a peptide and a lipid wherein the peptide is not an apolipoprotein. The inventors also found that the toxicity caused by endotoxin can be treated via the sequential administration of either an apolipoprotein or a peptide, followed by a lipid. After sequential administration, the components look like a reconstituted particle and then act to eliminate the endotoxin. It was also found that at least some individuals possess native levels of apolipoprotein which are higher than normal levels, such that effective endotoxemia therapy can be effected by the administration of reconstituted particles that do not contain apolipoprotein or peptide, but that contain the lipid of the 5 description.

In addition, the invention described in these applications involved the use of the reconstituted particles and the components discussed therein for prophylaxis against toxicity caused by endotoxin, by administering prophylactically effective amounts to subjects in need of such prophylaxis. Such subjects include patients suffering from infections or recovering from surgery. These patients sometimes have very low plasma HDL levels, sometimes as little as 20% of normal levels. It is highly desirable, in these cases, for early prophylaxis with HDL, to compensate for these decreases. It has now been found, quite surprisingly, that phospholipids can be used alone, or in combination with additional materials, such as neutral lipids, cholates, etc., as effective agents for alleviating and / or preventing endotoxemia. It is especially preferred to use phosphatidylcholines (hereinafter "PC"), either alone or in combination with other phospholipids, such as sphingolipids, in compositions which are essentially free of peptides and proteins, such as apolipoproteins or peptides derived therefrom. . Neutral lipids such as mono-, di-, and triglycerides can be combined with the phospholipids, as long as the total amount of the neutral lipids is below certain percentages by weight, when the compositions are used in the form of a intravenous bolus. When used in other administration forms, such as intravenously, for example, by continuous infusion, the percentages by weight are not 'r critics, but they are desirable. The particularly preferred embodiments of the invention include emulsions where a bile acid, or a salt of bile acid in conjunction with a phospholipid and a neutral lipid is used. The efficacy of bile acids and salts of bile acids, which are cholates, in the treatment of endotoxemia, is shown in These bile acids can be used alone, or in combination with one or more phospholipids, and / or neutral lipids, such as a phosphatidylcholine, 0 and / or a triglyceride The invention is described in greater detail in the following description.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows how the reconstituted particles containing Apo-A-I, phospholipid and cholate are formed.

Figure 2 shows the reception of the LPS molecules by the reconstituted particles.

Figure 3 shows experiments in which a peptide according to the invention is used to study the reduction of toxicity caused by endotoxin, in a mouse model.

Figure 4 marked prior art, shows the formation of helical wheels by various peptides. See Anantharamaiah, supra.

Figures 5A and 5B show the results obtained when various compositions were tested in a model, which determined the neutralization of the endotoxin via the determination of the release of TNF in a human whole blood model. Figure 5A shows the role of the protein, and 5B that of the phospholipid. The compositions tested included natural lipoproteins (VLDL, LDL, HDL), reconstituted HDL ("R-HDL"), and INTRALIPID * compositions, as well as emulsions containing phospholipid and protein.

Figures 6A and 6B compare the role of the triglyceride (a neutral lipid), and of the phosphatidylcholine, a phospholipid, in the same model.

Figure 7 presents the information regarding the toxicity associated with the administration of various PC and PC / TG compositions in a mouse model, using a 55% lethality model where E LPS is administered. col i.

Figure 8 shows data comparable to those insured for the whole human blood test, supra, but using phospholipid with unesterified cholesterol, sphingomyelin, or mixtures of both, instead of triglycerides.

Figures 9A and 9B show results comparable to those shown in Figures 5A and 5B, except that in these new figures, the phospholipid, the unesterified cholesterol and / or the sphingomyelin are mixed with triglycerides or with cholesterol esterified as a neutral lipid.

Figure 10 compares the results obtained from the emulsions containing cholesterol ester and triglyceride, in the mouse model in vi.

Figure 11 graphically shows the theoretical amounts of triglycerides released into the blood after administration of various compositions containing TG, with the toxicity thresholds. "TPN" means "total parenteral nutrition" while "RI" means the compositions according to the invention.

J * "DETAILED DESCRIPTION OF THE MODALITIES Example 1 0 Studies were carried out to determine the survival rate of mice challenged with S_ typhimurium endotoxin. Multiple crossbred male Swis Webster mice received either saline solution (20 mice), reconstituted HDL particles (40 mice), or reconstituted 18A peptide (20 mice), via tail vein injection. The details of the injection materials are as follows: to. HDL particles The particles were prepared from apo-Hu-HDL (85% AI, 15% A-II and apo C), reconstituted with 95% pure egg phosphatidylcholine (2: 1 w / w), using dialysis with detergent , according to Matz et al., J. Biol. Chem. 257: 4535-4540 (1982), and US Patent No. 5,128,318, the disclosure of which is incorporated by reference herein. b. Peptide particles Peptide 18A has the amino acid sequence: Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Gly-Lys-Leu-Lys-Glu-Ala-Phe Peptide samples were also mixed and were reconstituted with 95% pure egg phosphatidylcholine, as per Matz et al., supra (2: 1 w / w), and US Patent No. 5,128,318 also using detergent dialysis. The resulting particles are identical to those described in US Pat. No. 5,128,318 except that a peptide component was present, rather than apo-HDL of the Matz and patent references. Within fifteen minutes of administration of the reconstituted material, the mice were administered, intraperitoneally, with 10 mg / kg of 0 body weight of Salmonella LPS. The criterion for the evaluation was survival. Figure 3 presents these results, and indicates a superiority of almost 4 times over the saline control. The synthetic peptide is almost as effective as the reconstituted particles containing apo-HDL. and "Example 2 Factors that affect the stimulation mediated by LPS of TNF-a, while preserving the integrity of the interaction between plasma proteins, and the cellular elements of the blood, can be appropriately studied in a complete human blood system, in vi tro. Such a system was used to determine which of the lipoprotein components is important in the neutralization of LPS. The tested materials were reconstituted high-density lipoprotein (R-HDL), natural plasma lipoproteins (VLDL, LDS, HDL), lipoprotein deficient serum (LPDS), and triglyceride-rich 20% INTRALIPID® emulsion (a mixture of triglycerides and phospholipids). The blood was collected in a heparinized tube, diluted with Hank's Balanced Salt Solution (hereinafter "HBSS"), or the material to be tested, dissolved in HBSS. The resulting material was transferred to Starstedt tubes (250 μl / tube). LPS was dissolved in pyrogen-free saline containing 10 mM HEPES, and added (2.5 μl) to a final concentration of 10 ng / ml. After the , incubation for four hours at 37 ° C, the tubes were cooled to 4 ° C, followed by centrifugation at 10,000 x g for 5 minutes. The supernatant was collected, and tested for the determination of TNF-a using commercially available ELISA. Table 1, below, compares the compositions of the tested materials. Figures 5A and 5B present the results. The data are shown graphically as the amount of TNF-α produced, shown graphically against the concentration of the aggregated protein (Figure 5A), and the phospholipid (Figure 5B). Logarithmic scales were used, in order to show the wide range of concentrations used, with 10 ° equal to 1 mg / ml. All incubations of whole blood contained 10 ng / ml of LPS of E. coli 0111: B4, supplemented with one of the compositions, as shown by the keys for figures 5A and 5B. The fact that the materials differ in effectiveness when the protein content is shown graphically (Figure 5A), while being very similar when graphically showing the phospholipid content (Figure 5B) suggests that the phospholipid is the important component. This is confirmed by the finding that a protein-free lipid emulsion is more effective than natural HDL, but less effective than HDL-R. The protein does not seem important for neutralization.

Composition of natural lipoproteins and reconstituted HDL Example 3 As the next step, protein-free lipid emulsions containing different amounts of neutral lipid were tested in human whole blood. The same test was used in vi tro of human whole blood, that the one that is described in the example 2. All the particles described in the present were done by means of the same protocol, which involved the mixing of a phospholipid, sphingomyelin or phosphatidylcholine, triolein, and / or ester ester of unesterified cholesterol, dissolved in chloroform, and weighing it into a flask. Vitamin E (0.02% w / v) was added as an antioxidant. An anhydrous lipid film was then prepared by blowing nitrogen gas or argon onto the sample. A volume of non-pyrogenic salt solution was then added to the flask, followed by mixing on a vortex mixer until all the lipid was suspended. The solution was then homogenized in a high pressure homogenizer. Samples containing phosphatidylcholine (PC), with or without triolein, were cyclized through the homogenizer 10 times at 1406.13 kg / cm2 (20,000 psi). Samples containing cholesterol ester with one or more other lipids were cycled through 15-20 times at 2109.20 kg / cm2 (30,000 psi). The homogenized solutions were filtered through 0.45 μm syringe filters, and the filtrate was stored at room temperature until it was used (within three days). Figures 6A and 6B present these results. In these studies, the production of LPS-dependent TNF-α is shown graphically against aggregated triglyceride concentration (Figure 6A) or aggregated phospholipid (Figure 6B). The compositions, as indicated by the code, contained (by weight) 7% triglyceride ("TG"), 45% TG, 89% TG, 94% TG, HDL-R, or phospholipid without TG, ( shown in Figure 6B only). An 89% composition of TG is a formulation of 10% INTRALIPID®, while 94% of TG refers to 20% INTRALIPID®. In all other tests, egg phosphatidylcholine (PC), and triolein were used. These results show that the protein-free compositions, when compared via the triglyceride content, are very different. These are very similar when tested via the phospholipid (PC) content. This confirms the role of the phospholipid, especially since the phospholipid is only effective, but less so than emulsions containing up to 45% TG.

Example 4 The work then proceeded to the experiments in vi in a mouse model, which is accepted as a reliable system to predict efficiency in humans. In these experiments, mice were injected, in bolus form, with sufficient quantities of the formulations described in Example 3, as well as others (pure phosphatidylcholine, 7% TG, 25% TG, 45% TG, 71% TG, 81% TG, 89% TG, 94% TG), or a saline control, to provide phospholipid doses (either 200 mg / kg or 400 mg / kg), together with 25 mg / kg of LPS of E. coli 0111: B4. The control group received intravenous physiological saline in a volume sufficient to equalize the emulsion volume. Survival after 72 hours is presented in figure 7. Of the 344 animals in the control groups, 155 survived. PC only had a modest protective effect, statistically not significant at a confidence level of 95%, while compositions of 7%, 45% and 71% of TG significantly improved survival. Compositions of 80% and 89% of TG were marginally effective, while TG 94% decreased survival. When the dose was increased to provide 400 mg / kg of CP, 89% and 94% TG emulsions significantly decreased the survival time, probably due to TG poisoning, as explained below.

Example 5 The work described in examples 2-4? - established that phospholipids are an active agent useful in the inhibition of endotoxemia. The fact that non-polar lipids other than triglycerides can form emulsions with phospholipids other than PC, suggested that others can be tested. Examples are espingomyelin (another phospholipid), and unesterified cholesterol (a neutral polar lipid), and mixtures of these. Thus, also, esterified cholesterol (a non-polar ester), squalene (a hydrocarbon), and vitamin E (a non-polar antioxidant) can be used. A series of experiments were designed to test these, using the whole human blood test of example 2, above, and the mouse survival test of example 4. Emulsions were prepared, in the manner described above, using pure phosphatidylcholine, phosphatidylcholine with 10% (weight / weight) of unesterified cholesterol, 10% (weight / weight) of sphingomyelin, or 10% total of a mixture of both. The emulsions were added to the whole blood, at a concentration of 100 mg / dl, with reference to PC, and 10 ng / ml of LPS. The mixture was incubated, and measured in TNF-α released. The results are shown in figure 8.

The production of TNF-a was substantially reduced with PC alone. Emulsions containing unesterified cholesterol, sphingomyelin, or the mixture of both, were also suppressors of TNF-a release.

Example 6 The whole blood assay was also used to determine the effect of unesterified cholesterol and / or sphingomyelin for solutions containing neutral lipids. Again, the emulsions were added to 100 mg / dl of PC. The various compositions (weight / weight) are described in the following table.

Figures 9A and 9B show the results.

PC emulsions made with either neutral lipid, with or without additional polar lipids, demonstrated inhibition. Again, the The concentration of LPS used was 10 ng / ml, which is a clinically relevant concentration of endotoxin. Emulsions containing cholesterol ester are less effective than emulsions that '' contain TG, while those emulsions containing 0 not esterified cholesterol do not suppress the TNF-a, as well as those emulsions which do not contain it. The addition of sphingomyelin to the emulsions appeared to improve the suppression of TNF-α production. 15 Example 7 and Emulsions containing cholesterol ester were tested in an in vi ve model (e.g., That used in example 4), with a lethal dose of endotoxin. The emulsions were prepared with PC and TG, or with PC and cholesterol ester (CE), and were administered to provide a single bolus dose of 200 mg / kg PC, together with 25 xiig / kg of 5 E LPS. coli 0111: B4 (a lethal dose), through the vein of the tail. The control groups received physiological saline, intravenously, in one volume to equalize the volume of the emulsion. In Figure 10, the data compare the results of emulsions containing EC and TG.

Each emulsion was tested in a minimum of two experiments, using a total of 16 or more animals. ** 'As shown, emulsions containing 0% or 45% EC (% by weight) significantly improved survival. These results, taken with those of Example 6, show that CE can be replaced by TG to create emulsions that neutralize endotoxin. 5 Example 8 Free phospholipid protein emulsions with triglyceride effectively block the production of TNF-a in whole blood stimulated with LPS. In theory, these emulsions can also be effective in vi, if they can be administered safely in doses that provide protective concentrations of phospholipid in plasma. Previous experiments with HDL-R suggest that the minimum dose of phospholipid is approximately 200 mg / kg. Using this dose and a plasma volume of 4.5% of body weight, the expected triglyceride concentration in the plasma can be calculated after the administration of a series of emulsions with increasing triglyceride content. The result is shown in Figure 11 as a smooth line that curves in an upward direction with the increasing weight percent of TG. Plasma TG concentrations rarely rise above 1000 mg / dL in healthy adults, even after a fatty diet. Pancreatitis is reported in patients with plasma TG above 2000 mg / dl (Far er et al., Amer. J. Med. 54: 161-164 (1973); Krauss et al., Amer. J. Med. 62: 144-149 (1977); Glueck et al., J. Lab. Clin. Med. 123: 59-61). Plasma TG above 4000 mg / dl is extre rare and causes serious disorders. The last two thresholds are shown by horizontal lines in the previous figure. The administration of either 10% or 20% INTRALIPID® in a dose to provide 200 mg / kg of phospholipid, is expected to raise TG plasma concentrations (see the two open circles) well above the safe limits. In contrast, the administration of emulsions containing 7%, 45%, 71% or 78% (solid tables from left to right) elevates the plasma TG to 136, 477, 1300 or 2000 mi / di respectively. Emulsions with TG content of up to about 50% are expected to be free of TG toxicity.

Example 9 The effectiveness of combinations of phospholipid and bile acid, for example, sodium cholate, was tested in the same type of experiment described in the previous examples. The procedure by which the formulations administered to the test animals was prepared, however, differed In this example, and in the following examples, the formulations were prepared using a high pressure homogenizer, Microfluidizer This apparatus facilitates the scaling up of either liquid triolein or liquid soy triglyceride in an appropriate amount of water, or water plus 9 mM, 18 mM, or 36 mM sodium cholate, weighing granular phosphatidylcholine It was then added, slowly, to the solution, while stirring.This requires always 3 to 5 minutes to disperse the liquid.After dispersion, the materials were emptied into the microfluidizer. The device uses hydraulic pressure to drive a pump which, in turn, directs two opposing jets of sample towards each other, the pressure can be as high as 25,000 pounds per inch (1757.67 kg / cm2). square) After the collision, the jets are forced through a hole in the shape of a plus sign, whereby the sample is homogenized. The sample was recirculated through the microfluidizer, with "one step" which is defined as the amount of time it takes to pump the entire sample through the machine. The sample was circulated through 20 steps to produce a homogenized sample. Dextrose was added to a final concentration of 5%. The purified endotoxin of E. coli 0111: B4 (40 mg / kg), and the emulsion, as discussed below (200 mg of phosphatidylcholine / kg), was mixed at room temperature, and immediately administered to C57BL6 / J mice (weight between 19 and 30 g), via intravenous injection through the vein of the tail. Those mice that received cholate alone, were administered a volume of sodium cholate equal to the cholate / eml preparation (emulsion) at the same cholate concentration. The control mice received the same volume of 5% dextrose, to equalize the osmolality of the plasma. The results are described in the following Table, immediately. The emulsion is the phosphatidylcholine / 7% triglyceride emulsion, described in the preceding examples. When sodium cholate was used, it was added to the indicated concentration to the raw materials, before the emulsification of the materials.

For convenience, the weight percentage of the emulsions is as follows. When 9 mM cholate was used, the percentages by weight relative to the emulsion are 7% cholate, 6.1% triglyceride, and 86.9% phosphatidylcholine. At 18 mM cholate, the percentages by weight are 13.1% cholate, 5.7% triglyceride, and 81.2% phosphatidylcholine. At 36 mM cholate, the relative values are 23.2% cholate, 5% triglyceride and 71.8% phosphatidylcholine. It should be noted that the amount of LPS administered in these experiments (40 mg / kg) is much higher than the amount used for the lethality studies in the previous experiments. The attempt of these higher doses is to reduce any protective effect attributable to phosphatidylcholine and / or triglyceride. Thus, the conclusion to be reached after these experiments is that there is a protective effect attributable to the salt of bile acid, sodium cholate. Not presented here, are the studies carried out using other salts of bile acid and bile salts containing taurine. Additional examples of bile acids include allodesoxycholic acid, lithocholic acid, hiodeoxycholic acid, hiocholic acid, α, β and β -uricolic acids, walldeoxycholic acid, ursodeoxycholic acid, ursocholic acid, and all salts thereof, such as their sodium salts or conjugates of taurine or glycine. See Hoffmann, supra.

Example 10 Further studies were subsequently carried out, the first of which was a survival study, using mice as the subject animals. In the survival study, the subject animals were divided into four groups. The first group received a 5% dextrose solution, and acted as a control. The second group received an emulsion of 93% (by weight) of phosphatidylcholine and 7% (by weight) of triglyceride, prepared as described above. The emulsion contained 5% dextrose, and soy phospholipids at approximately 50 mg / ml lipid. In the third and fourth groups, the animals received an emulsion similar to that given to the second group, supplemented either with 18 mM sodium cholate, or 18 mM sodium deoxycholate. In this experiment, the protocol used was identical to that described in example 9. Survival was measured 72 hours after the challenge, and is summarized in the following table: Table 1. Effect of the Addition of Bile Acid to 7% Triglyceride Emulsion in the Survival at 72 hours in Mice Note that the statistical significance between the group survival comparisons was tested using the generalized Wilcox method, using a computer program. Comparisons against controls in group I are listed under "1", comparisons against animals in group 2, treated with 7% emulsion, listed under "2", and comparisons against animals in group 3, treated with emulsion plus sodium cholate, are listed under "3". The percentage of survival, and the statistical analysis show the clear and unexpected superiority of the formulations containing salts of bile acids.

Example 11 A second group of experiments used a rabbit model. In this model, the release of TNF (tumor necrosis factor) -a was measured. The rabbits were divided into three groups, and received 5% dextrose solution, the phospholipid and triglyceride emulsion (93% / 7%), discussed above, or a 93% / 7% emulsion, which also contained acid. 18 mM colic. All emulsions were adjusted to 5% dextrose, as in Example 10. The rabbits received a bolus of emulsion priming and, two hours later, were challenged with 100 μg of LPS of E. coli 0111: B4. After the priming bolus, the formulations were administered to provide a continuous maintenance infusion, via intravenous administration, of 50 mg of lipid per kilogram of body weight per hour. Intravenous administration was continued for three hours after the challenge. Blood was taken from the rabbits at the baseline, 30 minutes after the administration of the primary bolus, and every hour for five hours of administration. In the following table, the peak values of TNF-a are presented. These occurred two hours after the administration of endotoxin. Statistical significance was determined, using the well-known Student's test. As shown in the table, the TNF-α values were significantly reduced after administration of 18 mM cholic acid.

Table 2. Effect of Emulsions on the Production of TNF-a in Rabbits The above examples detail the invention which involves, in one aspect, the alleviation or prevention of endotoxemia in a subject, by means of the administration of an effective amount of a phospholipid with which an endotoxin is associated. The association of the phospholipid and the endotoxin is then eliminated from the subject by means of standard biological processes well known to someone familiar with the processes, by means of which the lipoprotein particles are eliminated. The association of endotoxin with phospholipid, inactivates it. The examples also show that the administration of a member of the family of the colanoic acids or salts of colanoic acid, such as a bile acid or a salt of bile acid, can also be used to achieve the same end as the phospholipids, for example , the relief or prevention of endotoxemia. Thus, peptide and protein free compositions containing one, or both, of a bile acid / bile acid salt and a phospholipid, can be used to treat endotoxemia. The colanoic acids are described by, for example, Hoffmann, Hepatology 4 (5): 4S-14S (1984), incorporated by reference. Particular attention is drawn to page 5S, figures 1 and 2, incorporated by reference, which shows the characteristic structures of the colanoic acids. The subject to be treated is preferably a human, but the practice of the invention is equally applicable also in a veterinary context. "Relief" as used herein, refers to treatment to facilitate the burden of endotoxemia caused by any of the various endotoxins produced by, for example, gram negative bacteria (S. tiphimuri um, E. coli, etc.). Prophylaxis can be achieved by administering the agent to a point where the subject is in, or close to being in, a situation where exposure to endotoxin can result. Classically, this happens during surgery. Thus, a subject who is close to undergoing a surgical procedure may have the active ingredient administered preparatory to the procedure. The effective amount of the combination of phospholipid and bile acid necessary for the treatment of the subject may vary. In general, a dose of up to about 200 mg total to about 800 mg of phospholipid per kilogram of subject body weight is preferred, although the amount may decrease, or increase, depending on the severity of endotoxemia or the degree of risk in the context of prophylaxis. For the colanoic acids and salts, such as bile acids and their salts, a dose of from about 10 mg to about 300 mg / kg of body weight, more preferably 15 mg to about 275 mg per kilogram of body weight is used. It is desirable to administer the bile acid / bile acid salt and the phospholipids in compositions that also contain neutral lipids, but this is not necessary, that neutral lipid free emulsions of the phospholipids are also considered. The desire for the combined administration of phospholipids results from the fact that neutral lipids and phospholipids are associated in particles which resemble lipoproteins, but differ from these in that they do not contain protein of peptide components, which of course , are always present in lipoproteins. Especially desirable forms of treatment are those where the phospholipid is a phosphatidylcholine, such as egg yolk phosphatidylcholine, soy-based phosphatidylcholine or a sphingolipid. For bile acid / bile acid salt, cholic acid and / or its salts are preferred, such as sodium cholate, sodium deoxycholate, and sodium chenodeoxycholate. With respect to neutral lipids, it is preferred to use cholesterol ester or triglyceride, but other neutral lipids, such as squalene or other hydrocarbon oils, di- and mono-glycerides and antioxidants such as vitamin E may also be used. which compositions can be administered, can vary, with a bolus or other intravenous forms that are especially preferred. When a bolus form is used, and the composition contains triglyceride, for example, some care must be taken in the dosage. It is clearly well known that triglycerides are toxic if they are administered in too large a quantity. The person skilled in the art, however, can easily formulate the compositions, so that the risk of triglyceride poisoning is reduced, or eliminated. In general, when a bolus form is used, the compositions should contain no more than about 80 weight percent triglyceride or other neutral lipid, preferably not more than 70 weight percent. More preferably, the compositions should contain no more than about 50 weight percent neutral lipid when a bolus is administered. When non-bolus forms are used, however, such as other intravenous forms, the risk of poisoning is decreased. In spite of everything, the intervals outlined above are Preferred for intravenous administrations, or other forms of administration, although it should be understood that these are not required. For bile acids and bile acid salts, the doses are preferably from about 25 mg / kg of weight Body weight up to about 500 mg / kg of body weight with especially preferred doses ranging from about 50 mg / kg of body weight to about 100 mg / kg of body weight.

For phospholipids, a dose of from about 100 mg / kg of body weight to about 1000 mg / kg of body weight is preferred. The doses are general, however, and will vary depending on the subject and the mode of administration. As indicated above, the free protein and peptide formulations require that at least one phospholipid or bile acid / bile acid salt be present. For phospholipids, it is preferred that at least one neutral lipid, such as a triglyce, diglyce or monoglyce be present. The compositions may include additional matls such as sterols (e.g., cholesterol, β-sitosterol), estied or unestied lipids (e.g., cholesterol ester or unestied cholesterol), hydrocarbon oils such as squalene, antioxidants such as vitamin E, but these are not required. Of course, more than one phospholipid, and / or more than one neutral lipid, can be used in any such formulations. When the combinations of neutral lipid and phospholipid are used, the neutral lipid should be present from about 3% to about 50% by weight relative to the total amount of the lipid in the composition. In the case of bile acid / bile acid salts, these can be used separately, or in combination a phospholipid, a neutral lipid, or both. With respect to these additional matls (for example phospholipids and neutral lipids), the preferred species are those described and mentioned above. Additional optional ingredients include those listed above. Also a part of the invention are the compositions useful in the treatment of endotoxemia. One embodiment of this feature of the invention is a composition containing at least one of each a bile acid / bile acid salt, a phospholipid and a neutral lipid, wherein the composition as a whole contains an amount that alleviates endotoxemia, active ingredient. This composition preferably contains, in percent by weight, from about 5% to about 30% by weight of bile acid / bile acid salt, from about 3% to about 50% by weight of neutral lipid, and from about 10% to about approximately 95% phospholipid. Especially preferred are compositions containing from about 10 to 15% by weight of bile acid / bile acid salt, from about 5% to about 10% by weight of neutral lipid, and the remainder of the composition which is phospholipid. It should be noted that these percentages by weight are relative to compositions consisting of three components. When the three-component system is combined with, for example, a carrier, - "adjuvant, optional ingredients, such as those discussed above, the percentage by weight with respect to the complete composition will decrease. Therapeutic compositions are always protein-free and peptide-free In the case of compositions that do not contain a bile acid or a bile acid salt, such protein-free and peptide-free compositions preferably contain at least about 3% in weight of a neutral lipid, up to about 50% by weight of neutral lipid, the remainder being at least one phospholipid.Preferably, the neutral lipid is a triglyce, but can be any of the additional neutral lipids described above. preferably a phosphatidylcholine Other aspects of the invention will be clear to the person skilled in the art and not necessarily They will be reiterated here. It will be understood that the specification and the examples are illustrative but not limiting of the present invention, and that other embodiments within the spirit and scope of the invention will be suggested to those skilled in the art.

LIST OF SEQUENCES GENERAL INFORMATION: (i) APPLICANT: Levine, Daniel M. Parker, Thomas S. Rubin, Albert L. Gordon, Bruce R. Saal, Stuart D. (ii) TITLE OF THE INVENTION: Useful Methods in Prophylaxis and Endotoxin-Based Therapy (iii) NUMBER OF SEQUENCES: 9 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Felfe & Lynch (B) STREET: 805 Third Avenue (C) CITY: New York (D) STATE: New York (E) COUNTRY: E. U. A. (F) ZIP CODE: 10022 (v) COMPUTER LEGIBLE FORM: (A) MEDIA TYPE: 5.25 inch floppy disk, 360 kb storage r- (B) COMPUTER: IBM PS / 2 (C) OPERATING SYSTEM: PC-DOS (D) SOFTWARE: Wordperfect (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: '(C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: PCT / US93 / 07453 (B) DATE OF SUBMISSION: AUGUST 9, 1993 (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: 07 / 928,930 (B) DATE OF SUBMISSION: 12-AUGUST-1992 (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Hanson, Norman D. (B) REGISTRATION NUMBER: 30,946 (C) REFERENCE NUMBER / CASE: ROGO 211.4- PCT (ix) INFORMATION FOR TELECOMMUNICATIONS: (A) TELEPHONE: (212) 688-9200 (B) TELEFAX: (212) 838-3884 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: sp Trp Leu Lys Wing Phe Tyr Asp Lys Val Wing Glu Lys 5 10 Leu Lys Glu Wing Phe 15 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Lys Trp Leu Asp Wing Phe Tyr Lys Asp Val Wing Lys Glu 5 10 Leu Glu Lys Wing Phe 15 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (B) TYPE: amino acid • y (D) TOPOLOGY: linear 0 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Ala Glu Lys Leu 5 10 Lys Glu Ala Phe 5 15 r (2) INFORMATION FOR SEQ ID NO: 4 (i) CHARACTERISTICS OF THE SEQUENCE: 0 (A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Pro Lys Leu Glu Glu Leu Lys Glu Lys Leu Lys Glu Leu 5 10 Leu Glu Lys Leu Lys Glu Lys Leu Wing 15 20 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Val Ser Ser Leu Lys Glu Tyr Trp Ser Ser Leu Lys Glu 5 10 Ser Phe Ser 15 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Val Ser Ser Leu Leu Ser Ser Leu Lys Glu Tyr Trp Ser 5 10 Ser Leu Lys Glu Ser Leu Ser 5 15 20 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: 0 (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: 5 Val Ser Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu Lys 5 10 r? Glu Tyr Trp Ser Ser Leu Lys Glu Ser Glu Ser 15 20 0 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 amino acids (B) TYPE: amino acid 5 (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Glu 5 10 Leu Glu Ala Leu Lys Gln Lys Met Lys 15 20 2) INFORMATION FOR SEQ ID NO: 9 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: Pro Leu Ala Glu Asp Leu Gln Thr Lys Leu Asn Glu Asn 5 10 Val Glu Asp Leu Arg Lys Gln Leu Val 15 20 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (67)

1. A method for treating a subject for endotoxemia, characterized in that the method comprises administering to said subject an effective amount of a peptide-free and protein-free composition containing a sufficient amount of a colanoic acid or an acid salt colanoic, and a phospholipid, to alleviate endotoxemia in said subject.

2. The method according to claim 1, characterized in that the colanoic acid or the salt of colanoic acid is a bile acid or a salt of bile acid.

3. The method according to claim 2, characterized in that the phospholipid is a phosphatidylcholine.

4. The method according to claim 1, characterized in that the composition further comprises a neutral lipid.

5. The method according to claim 4, characterized in that the neutral lipid is a triglyceride.

6. The method according to claim 1, characterized in that the bile acid is cholic acid.

7. The method of compliance with / - claim 1, characterized in that the acid salt 10 bile is a cholate salt.

8. The method according to claim 7, characterized in that the cholate salt is a sodium cholate.

9. The method according to claim 2, characterized in that the composition further comprises a neutral lipid.

10. The method according to claim 9, characterized in that the phospholipid is a phosphatidylcholine and the neutral lipid is a triglyceride.

11. The method according to claim 1, characterized in that it comprises the intravenous administration of said composition.

12. The method according to claim 1, characterized in that it comprises the oral administration of said composition.

13. The composition of interest, useful in the treatment of endotoxemia, characterized in that it comprises a therapeutically effective amount of (i) a colanoic acid or salt of colanoic acid, (ii) a neutral lipid and (iii) a phospholipid, wherein the composition it is free of protein and free of peptide.

14. The composition according to claim 13, characterized in that the colanoic acid or the salt of the colanoic acid is a bile acid or a salt of bile acid.

15. The composition according to claim 14, characterized in that the bile acid salt is sodium cholate.

16. The composition according to claim 13, characterized in that the neutral lipid is a triglyceride.

17. The composition according to claim 13, characterized in that the phospholipid is a phosphatidylcholine.

18. The composition according to claim 13, characterized in that the bile acid salt is a cholate, the neutral lipid is a triglyceride and the phospholipid is a phosphatidylcholine.

19. The protein-free and peptide-free composition useful in the treatment of endotoxemia, characterized in that it comprises: a) at least one neutral lipid in an amount equal to about 3% to about 50% by weight of total lipid in said composition, and b) at least one phospholipid.

20. The protein-free and peptide-free composition according to claim 19, characterized in that the neutral lipid is a triglyceride.

21. The protein-free and peptide-free composition according to claim 19, characterized in that the phospholipid is a phosphatidylcholine.

22. The protein-free and peptide-free composition according to claim 19, characterized in that at least one neutral lipid comprises cholesteryl ester.

23. The protein-free and peptide-free composition according to claim 19, characterized in that it also comprises sphingosine.

24. A method for alleviating endotoxemia in a subject in need thereof, characterized in that the method comprises administering to said subject an effective amount of a peptide-free and protein-free composition, which comprises at least one phospholipid with which they associate endotoxins associated with endotoxemia.

25. The method according to claim 24, characterized in that the composition further comprises a neutral lipid.

26. The method according to claim 24, characterized in that it comprises administering said composition in the form of a bolus.

27. The method according to claim 24, characterized in that it comprises administering said composition intravenously.

28. The method according to claim 24, characterized in that it comprises administering said composition in an amount sufficient to provide up to about 800 mg of phospholipid per kilogram of body weight of the subject.

29. The method according to claim 28, characterized in that it comprises administering said composition in an amount sufficient to provide up to about 400 mg of phospholipid per kilogram of body weight of the subject.

30. The method according to claim 28, characterized in that it comprises administering said composition in an amount sufficient to provide up to about 200 mg of phospholipid per kilogram of body weight of the subject.

31. The method according to claim 28, characterized in that it comprises the administration of said composition in an amount sufficient to provide up to about 100 mg of phospholipid per kilogram of body weight of the subject.

32. The method according to claim 24, characterized in that the phospholipid is a phosphatidylcholine.

33. The method according to claim 24, characterized in that the phospholipid is a sphingolipid.

34. The method according to claim 25, characterized in that the neutral lipid is a triglyceride.

35. The method according to claim 25, characterized in that the neutral lipid is a cholesterol ester.

36. The method according to claim 25, characterized in that the composition comprises a neutral lipid in an amount up to about 80% by weight of the composition.

37. The method according to claim 36, characterized in that the neutral lipid is present in an amount up to about 70% by weight of the composition.

38. The method according to claim 37, characterized in that the neutral lipid is present in an amount of up to about 50% by weight of the composition.

39. The method according to claim 38, characterized in that the neutral lipid is present in an amount of up to about 10% by weight of the composition.

40. The method according to claim 24, characterized in that the composition further comprises a sterol.

41. The method according to claim 40, characterized in that the sterol is β-sitosterol or a plant sterol.

42. The method according to claim 25, characterized in that the neutral lipid is an esterified lipid.

43. The method according to claim 25, characterized in that the neutral lipid is a non-esterified lipid.

44. The method according to claim 42, characterized in that the esterified lipid is esterified cholesterol.

45. The method according to claim 43, characterized in that the non-esterified lipid is unesterified cholesterol.

46. The method for alleviating endotoxemia in a subject in need thereof, characterized in that it comprises administering to said subject an effective amount of a peptide-free and protein-free composition, which comprises at least one phospholipid with which they associate endotoxins associated with endotoxemia.

47. The method according to claim 46, characterized in that the composition further comprises at least one neutral lipid.

48. The method according to claim 46, characterized in that it comprises the administration of said composition in the form of a bolus.

49. The method according to claim 46, characterized in that it comprises administering said composition intravenously.

50. The method according to claim 46, characterized in that it comprises administering the composition in an amount sufficient to provide up to about 800 mg of phospholipid per kilogram of body weight of said subject.

51. The method according to claim 50, characterized in that it comprises the administration of the composition in an amount sufficient to provide up to about 400 mg of phospholipid per kilogram of body weight of the substrate.

52. The method according to claim 50, characterized in that it comprises administering the composition in an amount sufficient to provide up to about 200 mg of phospholipid per kilogram of body weight of the subject.

53. The method according to claim 50, characterized in that it comprises the administration of the composition in an amount sufficient to provide up to about 100 mg of phospholipid per kilogram of body weight of the subject.

54. The method according to claim 46, characterized in that the phospholipid is a phosphatidylcholine.

55. The method according to claim 46, characterized in that the phospholipid is a sphingolipid.

56. The method according to claim 47, characterized in that the neutral lipid is a triglyceride.

57. The method according to claim 47, characterized in that the neutral lipid is a cholesterol ester.

58. The method according to claim 47, characterized in that the composition comprises a neutral lipid in an amount up to about 80% by weight of the composition.

59. The method according to claim 58, characterized in that the neutral lipid is present in an amount up to about 70% by weight of the composition.

60. The method according to claim 59, characterized in that the neutral lipid is present in an amount up to about 50% by weight of the composition.

61. The method according to claim 60, characterized in that the neutral lipid is present in an amount of up to about 10% by weight of the composition.

62. The method according to claim 46, characterized in that the composition also comprises a sterol.

63. The method according to claim 62, characterized in that the sterol is β-sitosterol or a plant sterol.

64. The method according to claim 47, characterized in that the neutral lipid is an esterified lipid.

65. The method according to claim 47, characterized in that the neutral lipid is a non-esterified lipid.

66. The method according to claim 65, characterized in that the esterified lipid is esterified cholesterol.

67. The method according to claim 66, characterized in that the non-esterified lipid is unesterified cholesterol.