MXPA00001275A - METHOD TO PREVENT ACCELERATED ATHEROSCLEROSIS USING (sRAGE) SOLUBLE RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS - Google Patents

Abstract

The present invention provides for a method to prevent accelerated atherosclerosis in a subject predisposed thereto which comprises administering to the subject a polypeptide derived from soluble receptor for advanced glycation endproduct in an amount effective to prevent accelerated atherosclerosis in the subject. The present invention also provides for a method to prevent a macrovessel disease in a subject predisposed thereto which comprises administering to the subject a polypeptide derived from soluble receptor for advanced glycation endproduct in an amount effective to prevent macrovessel disease in the subject.

Description

METHOD TO PREVENT ACCELERATED ATEROSCLEROSIS USING SOLUBLE RECEIVER (sRAGE) FOR FINAL PRODUCTS OF ADVANCED GLICATION Technical Field The invention described here was done with the support of the Government under NIH Concession No. HL56881 and AG00602 of the Department of Human Services and Health. Accordingly, the Government of the United States has certain rights over this invention.

Background of the Invention Through this application, reference is made to several publications by author and date within the text. The full citations for these publications can be found listed alphabetically at the end of the specification immediately after the Sequence Listing and the claims. The descriptions of those publications in their entirety were therefore incorporated, as reference in this application, to more fully describe the state of the art as known to those skilled in the art to the date of the invention described and claimed herein. Ischemic heart disease is a leading cause of morbidity and mortality in the general population, but especially in patients with diabetes. The prevalence of coronary artery disease is as high as 55% in adult patients with diabetes (Robertson and Strong, 1968). In fact, the data from the Framingham Cardiac Study show that mortality from cardiovascular disease in non-insulin-dependent diabetes (NIDDM) is more than double in diabetic men and more than quadruple in diabetic women, when compared with control subjects. diabetics (Kannel and McGee, 1979). In addition to the increased prevalence, studies have shown that atherosclerosis in diabetic patients is clearly more accelerated and extensive. In a series of autopsies, for example, it was found that patients with diabetes have a more severe disease of the left anterior descending coronary artery (aller et al., 1980), and a higher incidence of two- and three-vessel disease.

(Crall and Roberts, 1978), and a greater diffusion of the distribution of atherosclerotic lesions (Hamby et al., 1976). These findings were confirmed by coronary angiography in symptomatic patients (Pyorala et al., 1978). The reasons for accelerated atherosclerosis in the diabetes scenario are numerous. However, even after the correction of dyslipidemia, hypertension and obesity, multivariate analysis studies have indicated that diabetic patients have an excessive risk of cardiovascular disease in relation to non-diabetic subjects (Kannel and McGee, 1979). For example, in the Nurses' Health Study of 1,500 diabetic subjects among a total of 115,000 women, the incidence of cardiovascular disease was 5 times higher in diabetic subjects, regardless of their cholesterol levels (Anson et al., 1991). These data suggest that factors unique to the diabetic population play an important role.

Brief Description of the Invention The present invention provides a method for preventing accelerated atherosclerosis in a subject predisposed thereto, which comprises administering to the subject a polypeptide derived from soluble receptor for the final product of advanced glycation in an amount effective to prevent Accelerated atherosclerosis in the subject. The present invention also provides a method for preventing a macrovascular disease in a subject predisposed thereto, which comprises administering to the subject a polypeptide derived from the soluble receptor for the final product of glycation, in an amount effective to prevent macrovascular disease in the subject.

Brief Description of the Figures Figures IA, IB. Approximate appearance of the proximal aorta of mice with apolipoprotein E (0) under dissection microscopy. Aortic specimens were subjected to retrograde injection of methylene blue in mice with apolipoprotein E (0) with diabetes (16-week-old mice; 10-week diabetes, Figure IA) or non-diabetic controls compared by age (16 weeks of age, Figure IB). Figure 2. Treatment of mice with apolipoprotein E (0) diabetics with sRAGE suppresses accelerated atherosclerosis. Mice with apolipoprotein E (0) became diabetic with stz. 2 weeks after diabetes, mice were treated with sRAGE (20 μg / day, intraperitoneally) or equimolar amounts of mouse serum albumin (40 μg / day, intraperitoneally) for 6 more weeks. The area of the average lesion in the diabetic mice treated with sRAGE, 150,046 ± 18,549 μm2 was significantly lower than that observed in mice treated with mouse serum albumin, 271,008 ± 16,721 um2, p, 0.02. Figures 3A, 3B. Approximate appearance of the proximal aorta with mice with apolipoprotein E (0) diabetics treated with mouse serum albumin (left panel) or soluble mouse RAGE (right panel) under dissection microscopy. Mice with apolipoprotein E (0) they became diabetic with stz. After 2 weeks of diabetes, the mice were treated with sRAGE (20 μg / day, intraperitoneally) or equimolar amounts of mouse serum albumin (40 μg / day, intraperitoneally) for 6 more weeks. Close inspection of the proximal aorta revealed an almost complete absence of lesions in the second and third branches of the proximal aorta in mice treated with sRAGE compared with those treated with mouse serum albumin. There was also a marked decrease in lesions at the first branch point and in the aortic arch in mice treated with sRAGE.

Detailed Description of the Invention The present invention provides a method for preventing accelerated atherosclerosis in a subject predisposed thereto, which comprises administering to the subject a polypeptide derived from a soluble receptor for the advanced glycation end product in an effective amount to prevent Accelerated atherosclerosis in the subject. The subject can be a mammal. The mammal can be a human. The subject can be a diabetic subject. The subject may suffer from a deficiency of apolipoprotein, or hyperlipidemia. Hyperlipidemia can be hypercholesterolemia or hypertriglyceridemia. The subject may have a glucose metabolism disorder. The subject can be an obese subject. In one embodiment of the invention, the polypeptide may comprise at least a portion of the natural soluble receptor for the final product of advanced glycation. The polypeptide may comprise a "V" domain of natural soluble receptor for the final product of advanced glycation. The polypeptide may comprise a 10 kilodalton domain of natural soluble receptor for the final product of advanced glycation. The polypeptide may comprise a sequence less than or equal to 20 amino acids in length, which sequence is within the natural soluble receptor sequence for the final product of advanced glycation. For example, the sequence may be 5 amino acids long, 3 amino acids long, 8 amino acids long or 11 amino acids long. The length can also be any other length between 2 and 20 amino acids. In one embodiment of the invention, the length can be of an amino acid. The polypeptide can be a mimetic peptide, a synthetic polypeptide or a polypeptide analogue. The n The polypeptide may be an unnatural polypeptide having a chirality not found in nature, ie D-amino acids or L-amino acids. In another embodiment of the present invention, the method may further comprise administering to the subject a pharmaceutically acceptable carrier during administration of the polypeptide. The administration may comprise intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; liposome-mediated release; or topical, nasal, oral, ocular or otic release. The polypeptide can be released every hour, daily, weekly, monthly, annually (for example in a temporary release form) or as a temporary release. The release may be a continuous release over a period of time, for example, intravenous release. The effective amount of the polypeptide may comprise from about 0.000001 mg / kg of body weight to about 100 mg / kg of body weight. In one embodiment, the effective amount may comprise from about 0.001 mg / kg of body weight to about 50 mg / kg of body weight. In another embodiment / the effective amount may range from about 0.01 mg / kg of body weight to about 10 mg / kg of body weight. The actual effective amount used will be based on the size of the polypeptide, the biodegradability of the polypeptide, the bioactivity of the polypeptide and the bioavailability of the polypeptide. If the polypeptide does not degrade rapidly, is bioavailable and highly active, a smaller amount will be required to be effective. The effective amount will be known to one skilled in the art; and it will also depend on the form of the polypeptide, the size of the polypeptide and the bioactivity of the polypeptide. One skilled in the art could routinely perform tests of empirical activity for a polypeptide to determine the bioactivity in bioassays and thereby determine the effective amount. The present invention also provides a method for preventing a macrovascular disease in a subject predisposed thereto, which comprises administering to the subject a polypeptide derived from a soluble receptor for the final product of advanced glycation in an amount effective to prevent macrovascular disease in the subject. The subject can be a human or an animal. The subject can be a diabetic subject. The subject may suffer from an apolipoprotein deficiency. The subject may suffer from hyperlipidemia. Hyperlipidemia can be hypercholesterolemia or hypertriglyceridemia. The subject may have a glucose metabolism disorder. The subject can be an obese subject. In one embodiment of the invention, the polypeptide comprises at least a portion of the natural soluble receptor for the end product of advanced glycation (RAGE). The polypeptide may comprise a "V" domain of natural soluble receptor for the final product of advanced glycation. The polypeptide may comprise a 10 kilodalton domain of natural soluble receptor for the final product of advanced glycation. The polypeptide may comprise less than or equal to 20 amino acids in length, which sequence is within the natural soluble receptor sequence for the final product of advanced glycation. The polypeptide can be a mimetic peptide, a synthetic polypeptide or a polypeptide analogue. In another embodiment of the present invention, the method may further comprise administering a pharmaceutically acceptable carrier to the subject during administration of the polypeptide. The administration may comprise intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; liposome-mediated release; or topical, nasal, oral, ocular or otic release. The sRAGE polypeptide can be administered hourly, daily, weekly, monthly, annually (for example in a temporary release form) or as a temporary release. The release or administration may be a continuous release over a period of time, for example intravenous release. The following abbreviations were used here: AGE - end products of advanced glycation; RAGE -receptor for the end products of advanced glycation; sRAGE - soluble receptor for the final products of advanced glycation. The polypeptide can be a peptide, a mimetic peptide, a synthetic polypeptide, a derivative of a natural polypeptide, a modified polypeptide, a labeled polypeptide, or a polypeptide which includes non-natural peptides. The mimetic peptide can be identified by selecting large libraries of different compounds that are mimetic peptides to determine a compound, which is capable of preventing accelerated atherosclerosis in a subject predisposed thereto. The polypeptide can be a soluble receptor derivative for the final product of advanced glycation (sRAGE). The polypeptide can be a soluble extracellular portion of a receptor for the final product of advanced glycation, an antibody or a portion thereof, where the antibody is capable of specifically binding to the receptor for the final product of advanced glycation. The antibody can be a monoclonal antibody or a polyclonal antibody. A portion of the antibody can be a Fab or a region that determines complementarity or a variable region. The polypeptide may be capable of specifically binding to β-amyloid peptide. The polypeptide can bind to β-amyloid peptide at the site where the receptor interacts for the final product of advanced glycation. In addition to the natural forms of the polypeptides derived from sRAGE, the present invention also encompasses other polypeptides such as sRAGE polypeptide analogues. Such analogs include fragments of sRAGE. Following the procedures of the application published by Alton et al. (WO 83/04053), genes encoding the microbial expression of polypeptides having primary conformations differing from those specified herein can be designed and manufactured easily in terms of the identity of the location of one or more residues (e.g. , additions and terminal and intermediate deletions). Alternatively, modifications of cDNA and genomic genes can be easily effected by well-known site-directed mutagenesis techniques and employed to generate analogues and derivatives of the sRAGE polypeptide. Such products share at least one of the biological properties of the sRAGE but may differ in others. As examples, the products of the invention include those which are shortened in size for example, by deletions; or those which are more stable to hydrolysis (and, therefore, may have more pronounced or longer lasting effects than natural ones); or that have been altered to suppress or add one or more potential sites for O-glycosylation and / or N-glycosylation or that have one or more cysteine residues deleted or replaced by eg alanine or serine residues and are potential way, more easily isolated in active form of microbial systems; or that they have one or more tyrosine residues replaced by phenylalanine and bind more or less rapidly to target proteins or to target cell receptors. Also included are polypeptide fragments that reproduce only a part of the continuous amino acid sequence or secondary conformations within sRAGE, fragments which possess a property of sRAGE and not others. It is surprising that the activity is not necessary for one or more of the polypeptides of the invention to have utility. * 13 z. therapeutic or utility in other contexts, such as in sRAGE antagonism assays. Competitive antagonists can be very useful in, for example, cases of sRAGE overproduction. Applicable to the polypeptide analogs of the invention are the reports of the immunological property of synthetic peptides that substantially reproduce the amino acid sequence existing in natural proteins, glycoproteins and nucleoproteins. More specifically, relatively low molecular weight polypeptides have been shown to participate in immune reactions, which are similar in duration and degree to the immune reactions of physiologically significant proteins, such as viral antigens, polypeptide hormones and the like. Included among the immune reactions of such polypeptides are the provocation of the formation of specific antibodies in immunologically active animals [Lerner et al., Cell, 23, 309-310 (1981); Ross et al., Nature, 294, 654-658 (1981); Walter et al., Proc. Nati Acad. Sci. USA, 78, 4882-4886 (1981); Wong et al., Proc. Nati Sci. USA, 79, 5322-5326 (1982); Baron et al., Cell, 28, 395-404 (1982); Dressman et al., Nature, 295, 185-160 (1982); and Lerner, Scientific American, 248, 66-74 (1983). See also, Kaiser et al. [Science, 223, 249-255 (1984)] related to the biological and inmonological properties of synthetic peptides, which share approximately secondary structures of peptide hormones but do not share their primary structure conformation. The polypeptide of the present invention may be a peptidomimetic compound, which may be at least partially natural. The peptidomimetic compound may be a small molecule that indicates a portion of the amino acid sequence of sRAGE. The compound may have greater stability, efficacy, potency and bioavailability by virtue of imitation. In addition, the compound may have lower toxicity. The peptidomimetic compound may have higher mucosal intestinal permeability. The compound can be prepared synthetically. The compound of the present invention may include L, D or non-natural amino acids, alpha amino acids, disubstituted at the alpha position, N-alkyl amino acids, lactic acid (an isoelectronic analogue of alanine). The peptide backbone of the compound can have at least one linkage replaced with PSI- [CH = CH] (Kempf et al., 1991). The compound may further include trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, poly-L-propargylglycine, poly-D, L-allylglycine, poly-L-allylglycine. One embodiment of the present invention is a peptidomimetic compound having the biological activity to prevent accelerated atherosclerosis in a subject where the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic. Examples of non-natural amino acids which may be suitable 5-amino acid mimics include β-alanine, La-amino butyric acid, L-α-amino butyric acid, La-amino isobutyric acid, Le-amino caproic acid, 7-amino heptanoic acid , L-aspartic acid, L-glutamic acid, cysteine (acetaminomethyl), N-e-Boc-N-a-CBZ-L-lysine, N-e-Boc-N-a-10 Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N- -Boc-N-dCBZ-L-ornithine, N-d-Boc-N-a-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, Boc-L-thioproline. (Blondelle, et al., 1994; Pinilla, et al., 1995). The subject can be a mammal or a non-mammal. 15 The subject can be a human. The subject can be a mouse, a cow, a monkey, a horse, a pig or a dog. The subject can be a diabetic subject. The subject may suffer from an apolipoprotein deficiency. The subject may have a glucose metabolism disorder. The subject can be an obese subject. The subject may have genetically mediated or diet-induced hyperlipidemia. Form AGEs in environments enriched with lipids still in euglycemia. Administration in this modality may be intralesional, intraperitoneal, intramuscular or intramuscular injection. intravenously; infusion; release mediated by liposomes; topical, nasal, oral, anal, ocular or otic release. The administration may be constant for a certain period of time or periodically and at specific intervals. The carrier can be a diluent, an aerosol, a topical carrier, an aqueous solution, a non-aqueous solution or a solid carrier. In the practice of any of the methods of the invention or in the preparation of any of the pharmaceutical compositions, a "therapeutically effective amount" is an amount which is capable of preventing accelerated atherosclerosis in a subject predisposed thereto. Consequently, the effective amount will vary with the subject being treated, as well as the condition to be treated. For the purposes of this invention, methods of administration include, but are not limited to, cutaneous, subcutaneous, intravenous, parenteral, oral or topical administration, or aerosol administration. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutically acceptable carriers, such as phosphate-buffered saline, water, emulsions such as an oil / water emulsion or a triglyceride emulsion, various types of agents moisturizers, tablets, coated tablets and capsules. An example of an acceptable triglyceride emulsion useful in the intravenous and intraperitoneal administration of the compounds is the triglyceride emulsion commercially known as Intralipid®. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, fats or vegetable oils, gums, glycols, or other known excipients. Such carriers may also include flavoring additives and colorants or other ingredients. This invention also provides pharmaceutical compositions that include therapeutically effective amounts of compositions and polypeptide compounds, capable of preventing accelerated atherosclerosis in the subject, by inhibiting the binding of a β-amyloid peptide with a receptor for the final product of advanced glycation, together with diluents , preservatives, solubilizers, emulsifiers, adjuvants and / or suitable carriers. Such compositions may be liquid or lyophilized or otherwise dry formulations and include diluents of various buffer contents (eg, Tris-HCl., Acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent surface absorption, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (eg, Thimerosal, benzyl alcohol, parabens), volume-enhancing substances or tonicity modifiers (eg, lactose, mannitol), the covalent attachment of polymers such as polyethylene glycol to the compound, complexation with metal ions, or the incorporation of the compound in or on particulate preparations of polymeric compounds, such as polylactic acid, polyglycolic acid, hydrogels, etc., or on the iposomas, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocytic spectra or spheroplasts. Such compositions will influence the physical state, solubility, stability, release rate in vivo, and rate of in vivo elimination of the compound or composition. The choice of compositions will depend on the physical and chemical properties of the compound capable of preventing accelerated atherosclerosis in a subject predisposed to it. Controlled or sustained release compositions include formulations in lipophilic deposits (for example, fatty acids, waxes, oils). Also comprised by the invention are the particulate compositions coated with polymers (e.g., poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors. Other embodiments of the compositions of the invention incorporate particulate forms of protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. Portions of the polypeptide or composition of the invention may be "labeled" by association with a detectable labeling substance (eg, radiolabeled with 125 I or biotinylated) to provide reagents useful in the detection and quantification of the compound or its cells that contain the receptor or its derivatives in solid and fluid tissue samples such as blood, cerebrospinal fluid or urine. When administered, the compounds are often rapidly eliminated from the circulation and therefore can produce a relatively short-lived pharmacological activity. Consequently, frequent injections of relatively large doses of bioactive compounds may be required to sustain therapeutic efficacy. It is known that compounds modified by the covalent attachment of water-soluble polymers, such as polyethylene glycol, polyethylene glycol and propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone or polyproline exhibit substantially larger half-lives in the blood after intravenous injection than the corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications can also increase the solubility of the compound in aqueous solution, eliminate aggregation, increase the physical and chemical stability of the compound and greatly reduce the immunogenicity and reactivity of the compound. As a result, in vivo biological activity can be achieved by administering such polymer compound adducts less frequently or at lower doses than with the unmodified compound. The binding of polyethylene glycol (PEG) to compounds is particularly useful because PEG has very low toxicity in mammals (Carpenter et al., 1971). For example, a PEG adduct of adenosine deaminase was approved in the United States for use in humans for the treatment of Severe Combined Immunodeficiency Syndrome. A second advantage offered by the PEG conjugation is that it effectively reduces the immunogenicity and antigenicity of the heterologous compounds. For example, a PEG adduct of a human protein may be useful for the treatment of diseases * "in other mammalian species without the risk of activating a severe immune response The polypeptide or composition of the present invention can be released in a microencapsulation device to reduce or prevent a host immune response against the polypeptide or against cells that can The polypeptide or composition of the present invention can also be released microencapsulated in a membrane, such as a liposome Polymers such as PEG can conveniently be attached to one or more residual reactive amino acids in a protein such as the alpha group -amino of the amino-terminal amino acid, the amino-epsilon groups of the side chains of lysine, the sulfhydryl groups of the side chains of cysteine, the carboxyl groups of the side chains of aspartyl and glutamyl, the carboxyl-alpha group of the carboxy-terminal amino acid, the tyrosine side chains or activated chain derivatives of glycosyl linked to certain asparagine, serine or threonine residues. Numerous activated forms of PEGs have been described to direct the reaction with proteins. PEG reagents useful for the reaction with protein amino groups include active esters of carboxylic acid or carbonate derivatives, particularly those in which the leaving groups are N-hydroxysuccinamide, p-nitrophenol, imidazole or l-hydroxy-2-nitrobenzene -4-sulfanate. PEG derivatives containing maleimido and haloacetyl groups are useful reagents for the modification of free proteins of sulfhydryl groups. Likewise, PEG reagents containing hydrazine or amino hydrazide groups are useful for the reaction with aldehydes generated by periodate oxidation of carbohydrate groups in the proteins.

CLINICAL ASPECTS In one embodiment of the present invention, the object may suffer from clinical aspects such as those described below and as described, moreover, in the Biochemistry of Harper, R.K. Murray, et al. (Editors) 21st edition, (1988) Appelton & Lange, East Norwalk, CT. Such clinical aspects may predispose the subject to atherosclerosis or accelerated atherosclerosis. Thus, such subjects would benefit from the administration of a polypeptide derived from sRAGE in an effective amount for an effective time. The subject of the present invention can demonstrate clinical signs of atherosclerosis, hypercholesterolemia or other disorders as discussed below.

Clinically, hypercholesterolemia can be treated by interrupting the enterohepatic circulation of bile acids. It is reported that significant reductions in plasma cholesterol can be effected by this procedure, which can be achieved by the use of cholestyramine resin or surgically by operations to exclude the ileum. Both procedures produce a blockage in the reabsorption of bile acids. Then, due to the release of feedback regulation normally exerted by bile acids, the conversion of cholesterol to bile acids increases greatly in an effort to maintain the reserve of bile acids. LDL (low density lipoprotein) receptors in the liver are upregulated, causing increased LDL consumption with the consequent decrease in plasma cholesterol.

Cholesterol, Atherosclerosis, and Coronary Heart Disease. Many researchers have shown a correlation between increased serum lipid levels and the incidence of coronary heart disease and atherosclerosis in humans. Of serum lipids, cholesterol has been one of the most frequently isolated because it is mainly related in the relationship. However, other parameters - such as triacylglycerol concentration - show similar correlations. Patients with arterial disease may have any of the following abnormalities: (1) high concentrations of VLDL (very low density lipoproteins) with normal LDL concentrations; (2) LDL elevated with normal VLDL; (3) elevation of both lipoprotein fractions. There is also an inverse relationship between the concentrations of HDL (high density lipoproteins) (HDL2) and coronary heart disease, and some consider that the most predictive relationship is the ratio of LDL cholesterol: HDL. This relationship is "explainable in terms of the proposed roles of LDL in the transport of cholesterol to tissues and HDL that act as the cholesterol scavenger." Atherosclerosis is characterized by the deposit of cholesterol and cholesteryl ester of lipoproteins that contain apo-B-100 in the connective tissue of the arterial walls, diseases in which prolonged elevated levels of VLDL, IDL or LDL occur in the blood (for example, diabetes mellitus, lipid nephrosis, hypothyroidism, and other conditions of the hyperlipidemia) are often accompanied by premature or more severe atherosclerosis.

Experiments on the induction of atherosclerosis in animals indicate a wide variation in susceptibility per species. Rabbits, pigs, monkeys, and humans are species in which atherosclerosis can be induced by feeding cholesterol. Rats, dogs, mice and cats are resistant. Thyroidectomy or treatment with thiouracil drugs will allow the induction of atherosclerosis in dogs and rats. Low blood cholesterol is a characteristic of hyperthyroidism. Hereditary factors play the major role in determining individual blood cholesterol concentrations, but of the dietary and environmental factors that lower blood cholesterol, the replacement in the diet of polyunsaturated fatty acids by some of the saturated fatty acids It has been the most intensely studied. Natural oils that contain a high proportion of linoleic acid are beneficial in lowering plasma cholesterol and include peanut oil, cottonseed oil, corn and soybean oil, while milk fat, beef fat and coconut oil, which contain a high proportion of saturated fatty acids, raise the level. Sucrose and fructose have a greater effect on the elevation of blood lipids, particularly triacylglycerols, than other carbohydrates. The reason for the cholesterol lowering effect of polyunsaturated fatty acids is not yet clear. However, several hypotheses have advanced towards the explanation of the effect, including the stimulation of the excretion of cholesterol in the intestine and the stimulation of cholesterol oxidation of bile acids. It is possible that the cholesteryl esters of polyunsaturated fatty acids are metabolized more rapidly by the liver and other tissues, which can increase their rate of turnover and excretion. There is other evidence that the effect is largely due to a deviation of the distribution of plasma cholesterol to tissues, due to a higher catabolic rate of LDL. Saturated fatty acids cause the formation of smaller VLDL particles that contain relatively more cholesterol, and can be used by extrahepatic tissues at a lower rate than larger particles. All these tendencies can be considered atherogenic. Additional factors that are considered to play a part in coronary heart disease include high blood pressure, smoking, obesity, lack of exercise, and drinking carbonated water instead of hard water. The elevation of free fatty acids in plasma will also lead to x "l increase the secretion of VLDL by the liver, involving the delivery of triacylglycerol and extra cholesterol to the circulation. Factors that lead to higher or fluctuating levels of free fatty acids include emotional tension, nicotine from smoking cigarettes, drinking coffee, and taking large meals instead of more continuous feeding. Premenopausal women appear to be protected against many of these harmful effects, possibly because they have higher HDL concentrations than men and postmenopausal women.

Hypolipidemic Drugs When dietary measures fail to achieve reduced serum lipid levels, it may refer to the use of hypolipidemic drugs. Several drugs are known that block the formation of cholesterol in several stages in the biosynthetic pathway. Many of these drugs have dangerous effects, but the fungal inhibitors of HMG-CoA reductase, compactin and mevinolma, reduce LDL cholesterol levels with few adverse effects. Sitosterol is a hypocholesterolemic agent that works by blocking the absorption of cholesterol in the gastrointestinal tract. Resins such as colestipol and cholestyramine (Questran) prevent the reabsorption of bile salts by combining them, thereby increasing their fecal loss.

Neomycin also inhibits the reabsorption of bile salts. Clofibrate and gembivrozil exert at least part of their hypolipidemic effect by diverting the hepatic flow of free fatty acids from the esterification routes to those of oxidation, to decrease in this way the secretion of triacylglycerol and VLDL containing cholesterol by the liver. In addition, it facilitates the hydrolysis of triacylglycerols of VLDL by lipoprotein lipase. Probucol appears to increase the catabolism of LDL via pathways independent of the receptor. Nicotinic acid reduces the flow of FFA by inhibiting the lipolysis of adipose tissue, thereby inhibiting the production of VLDL by the liver.

Disorders of Plasma Lipoproteins (Dislipoproteinemias) A few individuals in the population exhibit elevated defects in their lipoproteins, leading to the primary condition of hypo or hyperlipoproteinemia. Many others that have defects such as diabetes mellitus, hypothyroidism, and atherosclerosis show abnormal lipoprotein patterns that are very similar to one or the other of the primary inherited conditions. Virtually all of these primary conditions are due to a defect at one stage or another in the course of the formation, transport or destruction of lipoprotein. Not all abnormalities are dangerous.

Hypoliproteinemia: 1. Abetalipoproteinemia - This is a rare hereditary disease, characterized by the absence of β-lipoprotein (LDL) in the plasma. Blood lipids are present in low concentrations - especially acylglycerols, which are virtually absent, because chylomicrons or VLDL do not form. Both the intestine and the liver accumulate acylglycerols. Abetalipoproteinemia is due to a defect in the synthesis of apoprotein B. 2. Hypobetalipoproteinemia Familiar - In hypobetalipoproteinemia, the LDL concentration is between 10 and 50% of normal, but the formation of chylomicrons occurs. It should be concluded that apo-B is essential for the transport of triacylglycerol. Most individuals are healthy and live a long time. 3. Familial alpha-lipoprotein deficiency (Tangier's disease) - In homozygous individuals, there is almost no absence of HDL in plasma and accumulation of cholesteryl esters in the tissues. There is no damage in the formation of chylomicrons or VLDL secretion by the liver. However, in electrophoresis, there is no pre-β- 3 $ lipoprotein, but a broad ß band containing the endogenous triacylglycerol is found. This is because the pre-β normal band contains other apoproteins normally provided by HDL. Patients tend to develop hypertriacilglicerolemia as a result of the absence of apo-C-II, which normally activates lipoprotein lipase.

Hyperlipoproteinemia: 1. Family lipoprotein lipase deficiency (type I) - This condition is characterized by the very slow elimination of chylomicrons from the circulation, which leads to abnormally high levels of chylomicrons.

VLDL can be increased, but there is a decrease in LDL and HDL. In this way, the condition is induced by fat. It must be corrected by reducing the amount of fat and increasing the proportion of complex carbohydrates in the diet. A variation of this disease is caused by a deficiency in apo-C-II, required as a cofactor for lipoprotein lipase. 2. Familial hypercholesterolemia (type II) - Patients are characterized by hyperbetalipoproteinemia (LDL), which is associated with an increase in total cholesterol in plasma. There is also a tendency for VLDL of type Ilb to rise. Therefore, the patient may have somewhat elevated triacylglycerol levels, but plasma - as is not true in other types of hyperlipoproteinemia - remains clean. The deposition of lipids in tissue (for example, xanthomas, atheromas) is common. A type II pattern can also arise as a secondary result of hypothyroidism. The disease appears to be associated with reduced rates of LDL clearance from the circulation due to defective LDL receptors and is associated with an increased incidence of atherosclerosis. The reduction of cholesterol and saturated fats in the diet can be useful in the treatment. A disease that produces hypercholesterolemia, but due to a different cause, is Wolman's disease (storage disease of cholesteryl ester). This is due to a deficiency of cholesteryl hydrolase ester in the lysosomes of cells such as fibroblasts that normally metabolize LDL. 3. Familial type III hyperlipoproteinemia (broad beta disease, remnant removal disease, familial dysbetalipoprotememia) - This condition is characterized by an increase in both the chylomicrons and the remnants of VLDL; these are lipoproteins with a density of less than 1019 but appear as a broad band on electrophoresis (ß-VLDL). The causes hypercholesterolemia and hypertriacilglicerolemia. Xanthomas and atherosclerosis are present in peripheral and coronary arteries. Weight loss treatment and diets containing complex carbohydrates, unsaturated acids and low cholesterol are recommended. The disease is due to a deficiency in the metabolism remaining in the liver caused by an abnormality in apo-E, which is normally present in 3 isoforms, E2, E3 and E4. Patients with type III hyperlipoproteinemia have only Ea, which does not react with the E receptor. 4. Familial hyperthercylglycelaemia (type IV) - This condition is characterized by high levels of triacylglycerol (VLDL) produced endogenously. Cholesterol levels rise in proportion to hypertriacilglicerolemia, and glucose intolerance is frequently present. Both LDL and HDLn are in lower than normal amounts. This lipoprotein pattern is also commonly associated with coronary heart disease, non-insulin-dependent diabetes mellitus type II, obesity, and many other conditions, including alcoholism and consumption of progestational hormones. The treatment of primary hyperlipoproteinemia of type IV is by? S * 3 weight reduction; the replacement of soluble carbohydrates in the diet with complex carbohydrates, unsaturated fat, diets low in cholesterol; and also hypolipidemic agents. 5. Familial Hyperlipoproteinemia type V - The lipoprotein pattern is complex, since chylomicrons and VLDL are elevated, causing both triacylglycerol and cholesterolemia. The concentrations of LDL and HDL are low. Often xanthomas are present, but the incidence of atherosclerosis is apparently not strange. Glucose tolerance is abnormal and is often associated with obesity and diabetes. The reason for the condition, which is familiar, is unclear. The treatment consisted of weight reduction followed by a diet not very high in carbohydrates or fats. It has been suggested that one more cause of hypolipoproteinemia is the overproduction of apo-E, which may have an influence on the plasma concentration of VLDL and LDL. 6. Familial hyperalphalipoproteinemia - This is a rare condition associated with an increase in HLL concentrations apparently beneficial to health.

Lecithin Efficiency: Cholesterol Acyltransferase (LCAT) Familiar: In affected subjects, the plasma concentration of cholesteryl esters and lysolecithin is low, while the concentration of cholesteryl and lecithin is increased. Plasma tends to be cloudy. There are also abnormalities in lipoproteins. A fraction of HDL contains structures in the form of discs in stacks or cartridges that are clearly nascent HDL unable to absorb cholesterol due to the absence of LCAT. Also present as an abnormal LDL subfraction is lipoprotein-X, in other circumstances found only in patients with cholestasis. VLDL are also abnormal, migrating as ß-lipoproteins after electrophoresis (ß-VLDL). Patients with parenchymal liver disease also show a decrease in LCAT activity and abnormalities in serum lipids and lipoproteins.

Drugs with Carriers In a preferred embodiment the pharmaceutical carrier can be a liquid and the pharmaceutical composition will be in the form of a solution. In another equally preferred embodiment, the pharmaceutically acceptable carrier is a solid and the composition is in the form of a powder or tablet. In one more modality, the pharmaceutical carrier is a gel and the composition is in the form of a suppository or cream. In a further embodiment the active ingredient can be formulated as part of a transdermal pharmaceutically acceptable patch. A solid carrier may include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers or fillers, glidants, compression aids, binders or tablet disintegrating agents; It can also be an encapsulating material. In the powders, the carrier is a finely divided solid which is mixed with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression properties in the proper proportions and compacted in the desired shape and size. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low melting point waxes, and ion exchange resins. Liquid carriers are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, or a mixture of both or pharmaceutically acceptable oils or fats. The liquid carriers may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral or parenteral administration include water (which partially contains additives as above, for example, cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, for example glycols) and their derivatives, and oils (for example, coconut oil and fractionated arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in the form of sterile liquid compositions for parenteral administration. The liquid carrier for the pressurized compositions may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. xx%? Liquid pharmaceutical compositions which are sterile solutions or suspensions may be used for example, for intramuscular, intrathecal, epidural, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The active ingredient can be prepared with a sterile solid composition, which can be dissolved or suspended at the time of administration using sterile water, saline, or other sterile, appropriate injectable medium. The carriers are intended to necessarily include inert binders, suspending agents, lubricants, flavors, sweeteners, preservatives, dyes and coatings. The active ingredient of the present invention (ie, the polypeptide derived from sRAGE, or composition) can be administered orally in the form of a sterile solution or suspensions containing other solutes or suspending agents, for example, enough saline or glucose to make the isotonic solution, bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (sorbitol oleate esters and their anhydrides copolymerized with ethylene oxide) and the like. The active ingredient can also be administered orally either in the form of liquid or solid composition. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets and powders, and liquid forms, such as solutions, syrups, elixirs and suspensions. Useful forms for parenteral administration include sterile solutions, emulsions and suspensions.

Atherosclerosis In one embodiment of the present invention, the subject may be predisposed to atherosclerosis. This predisposition may include genetic predisposition, environmental predisposition, metabolic predisposition, or physical predisposition. There have been recent reviews of atherosclerosis and cardiovascular disease. For example: Keating and Sanguinetti, (May 1996) Molecular Genetic Insights into Cardiovascular Disease, Science 272: 681-685 is incorporated by reference in its entirety in the present application. The authors reviewed the application of molecular tools to inherited forms of cardiovascular diseases such as arrhythmias, cardiomyopathies and vascular diseases. Table 1 * of this reference includes heart disease and the aberrant protein associated with each disease. The listed diseases are LQT disease, familial hypertrophic cardiomyopathy, and§ & muscular dystrophy of Duchenne and Becker; deficiencies of Acyl-CoA dehydrogenase from Barth syndrome; mitochondrial disorders; familial hypercholesterolemia; hyperbetalipoproteinemia; homocystinuria; Type III hyperlipoproteinemia; supravalvular aortic stenosis; Ehler-Danlos IV syndrome; Marfa syndrome; hereditary hemorrhagic telangiectasia. These conditions are included as possible predispositions of a subject to atherosclerosis. In addition, after reviewing a model of atherosclerosis in Breslow mice (1996) Mouse Models of Atherosclerosis, Science 272: 685. Reference is also incorporated by reference in its entirety in the present application. Breslow also includes a table (Table 1), which exposes several mouse models and the atherogenic stimulus. For example, mouse models include C57BL / 6; Apo E deficiency; ApoE injury; ApoE R142C; LDL receptor deficiency; and HuBTg. One embodiment of the present invention is where a subject has a predisposition to atherosclerosis according to what is shown by the mouse models presented in the Breslow publication. Gibbons and Dzau reviewed vascular diseases in Molecular Therapies for Vascular Disease, Science Vol. 272, pages 689-693. In one embodiment of the present invention, the subject may manifest the pathological events described in Table 1 of the Gibbons and Dzau publication. For example, the subject may have endothelial dysfunction, endothelial damage, cellular activation and phenotypic modulation, deregulated cell growth, deregulated apoptosis, thrombosis, plaque rupture, abnormal cell migration or modification of the extracellular or intracellular matrix. In another embodiment of the present invention, the subject may have diabetes. The subject may demonstrate complications associated with diabetes. Some examples of such complications include activation of endothelial and macrophage AGE receptors, altered lipoproteins, matrix, and altered basal membrane proteins; altered contractility and hormonal response of the vascular smooth muscle; altered endothelial cell permeability, sorbitol accumulation; decrease in neural myoinositol or altered Na-K ATPase activity. Such complications were discussed in a recent publication by Porte and Schwartz, Diabetes Complications: Why is Glucose Potential Toxic ?, Science, Vol. 272, pages 699-700. This invention is illustrated in the following Experimental Details section. These sections were established to help understand the invention but are not intended to be limiting to any • ir ~ - i r 4% of the invention, as set forth in the subsequent claims.

EXPERIMENTAL DETAILS Example 1: Suppression of Accelerated Diabetic Atherosclerosis by the Soluble Receptor for the Advanced Glycation End Products (sRAGE) The central aspect of diabetes is the presence of hyperglycemia. An important complication of the interaction of glucose with proteins / lipids is the irreversible formation of Advanced Glycation End Products, or AGEs (Brownlee, 1992). AGEs accumulate in plasma and tissue during normal aging, and to an accelerated degree in patients with diabetes. AGEs have been linked to the pathogenesis of diabetic complications. It has been shown that the interaction of AGEs with their cellular receptor RAGE (Receptor for AGE) on monocytes and endothelial cells results in the development of a proinflammatory environment in which increased migration / activation of monocytes, endothelial hyperpermeability and expression Increased adhesion molecules and tissue factor on endothelial cells result in the generation of a conducive environment for the development of vascular lesions (Schmidt et al, 992; Neeper et al, 1992; Schmidt et al, 1994; Schmidt et al, 1995). It has also been shown that the extracellular portion of RAGE (called soluble or sRAGE), composed of an immunoglobulin domain of type "V" followed by two domains of type "C" interferes with the ability of AGEs to bind to and activate cellular RAGE (Schmidt et al., 1994). In vivo, administration of sRAGE blocks hyperpermeability in diabetic rats (Wautier et al., 1996). As discussed below, a model of accelerated atherosclerosis in diabetes was developed and used to test the hypothesis that chronic administration of sRAGE (elimination t0 / 2 in diabetic rodents for 22 hours) prevents the development of atherosclerosis. accelerated These studies demonstrated that the daily intraperitoneal injection of sRAGE prevents the accelerated development of atheroclerosis in mice deficient (or destitute) of apolipoprotein E diabetic patients with streptozoticin.

Materials and Methods: Animals and induction of diabetes. Mice with apolipoprotein E (0) on the background C57B1 / 6J (NIO, 10 backcrossed generations with a> 99% homology) were obtained from Jackson Laboratories. At the age of 7 weeks, diabetes was induced in certain male mice with multiple intraperitoneal injections of streptozotocin (55 mg / kg) in 4 daily injections in sterile citrate buffer (0.05 M, pH 4.5). The control mice were treated with vehicle (buffer only). The plasma glucose concentration was then determined by colorimetric assay (Sigma ") using the blood obtained from the tail vein.The mice were considered diabetic if the plasma glucose levels exceeded 300 mg / dl on two separate occasions. All mice were maintained on a normal food diet.

Quantification of atherosclerotic lesions. Mice were sacrificed at the time points indicated later after the induction of diabetes or control treatment. The quantitative analysis of atherosclerotic lesions was performed on sections of the aortic sinus. After a humanitarian sacrifice, the hearts were fixed in formalin (10%), embedded in gelatin (25%) and frozen. Cryostat sections 10 microns thick were cut, stained with red oil or counterstained with hematoxylin and light green. The area of the fatty lesion was /? 4 He then determined by computer-aided image analysis (Zeiss Image, Media Cybernetics) in five consecutive sections or cuts, each separated by 80 microns. The area of the average lesion was quantified for each group.

Preparation of soluble mouse RAGE. A construct containing soluble mouse RAGE cDNA was prepared and contrasted with baculovirus DNA according to the manufacturer's instructions (PharMingen). Sf9 cells were then infected with the construct for three days in Grace's insect medium containing fetal bovine serum (10%), followed by three days in Grace's insect medium without serum (Gibco). At the end of the second three days, the cells were separated from the supernatant using centrifugation (1200 rpm x 20 mins.) And the supernatant was dialyzed against a buffer containing sodium phosphate (0.02M, pH 7.4) and NaCl (0.05M). . After dialysis, the supernatant was applied to a SP sepharose resin (5 ml) using the FPLC system (Pharmacia®). The soluble RAGE of mouse was eluted using a linear gradient of sodium chloride (0.05 M to 0.6 M). The SDS-PAGE revealed that the material is single-band. Before introduction into mice, soluble mouse RAGE - £. purified was passed through an endotoxin removal column (Detoxigel, Pierce). The final product was devoid of endotoxin as determined by the test in the amoebocyte assay (Sigma®), dialyzed against phosphate-buffered saline and stored in aliquots at 80 ° C.

Treatment of diabetic mice with soluble RAGE. After the induction of diabetes, certain diabetic mice were treated once a day with soluble mouse RAGE (20 μg / day, intraperitoneally) or with equimolar concentrations of mouse serum albumin (40 μg / day, intraperitoneally) starting from two weeks after the induction of diabetes and continuing for six weeks. At the end of that time, the mice were sacrificed and the aorta was subjected to quantitative morphometric analysis of atherosclerotic lesions.

Analysis of lipoproteins. The mice fasted four hours before obtaining the plasma for lipoprotein analysis. Plasma cholesterol and triglyceride concentrations were measured using commercial equipment (Boehringer Mannheim®).

VLDL (very low density lipoproteins), IDL (intermediate density lipoproteins) / LDL (low density lipoproteins), and HDL (high density lipoproteins) were separated by ultra density centrifugation as well as by FPLC.

RESULTS After treatment with streptozotocin (stz), the average plasma glucose concentration was approximately 350-500 mg / dl, compared to 130-160 mg / dl in the controls. Consistent with previous studies in apo E (0) mice (Plump et al., 1962), fatty lesions were observed in both diabetic mice and control at the first time points (4 weeks). Lesions appeared for the first time in the aortic root and in the lesser curvature of the aortic arch, progressing to each of the main branches of the thoracic aorta, starting soon. At each time point, the lesions were consistently larger in size and more extensive in the diabetic mice compared to the controls. For example, after ten weeks of diabetes, the mice showed discrete lesions at each of the thoracic branching sites, such as near-complete occlusion of the vessels (Figure IB). This contrasted markedly with control mice treated with citrate, compared by age, in which only moderate fatty streaks were visualized, mainly in the aortic root (Figure IA). The quantitative analysis of the lesions revealed that after eight weeks of diabetes, the average lesion area in the diabetic mice was approximately 3.7 times higher than that observed in the non-diabetic controls. Visualization with red oil O / light green hematoxylin demonstrated advanced atherosclerotic lesions with evidence of fibrous cap formation after eight weeks of diabetes. A similar experiment revealed an increase of approximately 3 times in the area of average lesion after 6 weeks of diabetes. The analysis of the lipid profile indicated that the The induction of diabetes resulted in an approximately 2-fold increase in VLDL levels, an increase of approximately 1.4-fold in LDL levels and no changes in HDL levels, compared to control mice treated with citrate. There was no differences in plasma triglyceride levels in diabetic and non-diabetic mice. Consistent with the hypothesis presented here that the AGE-RAGE interaction was increased was important in the pathogenesis of accelerated atherosclerosis in mice diabetics, the treatment of diabetic mice with sRAGE (20 μg / day, intraperitoneally) resulted in an increase of approximately 1.8 times in the area of average lesion compared to diabetic mice treated with mouse serum albumin, 150,046 ± 18,549 vs 271,008 ± 16,721 μm2, respectively, p < 0.02 (Figure 2). Visual inspection of the aortic tree of a typical diabetic mouse after 8 weeks of diabetes treated with mouse serum albumin revealed evidence of extensive atherosclerotic plaques at the major branch points and in the aortic arch (Figure 3A), which decreased markedly in diabetic mice treated with sRAGE (Figure 3B). Importantly, mice treated with sRAGE showed no alterations and plasma glucose levels. In addition, mice treated with sRAGE did not show differences in the lipid profile (total cholesterol, total triglyceride, as well as lipoprotein fractionation by FPLC analysis), compared to diabetic mice treated with mouse serum albumin. These data suggest that treatment with sRAGE decreased accelerated diabetic atherosclerosis in an independent manner from glucose and lipids.

DISCUSSION As discussed in detail here, the development of one of the first models of accelerated atherosclerosis in a diabetic mouse after treatment with streptozotocin has been demonstrated. The initial and more advanced atherosclerotic lesions were demonstrated in diabetic mice compared to controls compared by age. An important role for increased AGE-RAGE interaction in the development of accelerated diabetic atherosclerosis, the treatment of diabetic mice with sRAGE, a competitive inhibitor of AGE interaction with cellular RAGE, resulted in a statistically significant decrease in the area of the average atherosclerotic lesion after 8 weeks of diabetes. Taken together, these data indicate that the administration of soluble RAGE may be a novel and important means by which the chronic complications of diabetes, such as accelerated atherosclerosis, are prevented.

Example 2: An Accelerated Atherosclerosis Model in Diabetic Mice Overexpressing Human Apo B: Suppression by the Soluble Receptor of the End Products of Advanced Glycation. The lipid independent mechanism contributes to accelerated cardiovascular disease in diabetes (D). In persistent hyperglycemia, non-enzymatic glycation / protein / lipid oxidation forms irreversible Advanced Glycation End Products (AGE), which accumulate in the plasma / tissue D e interact with different cellular receptors, such as RAGE, activating the vascular cellular disturbance. In vitro, soluble RAGE (sRAGE), the two extracellular thirds of RAGE, binds to AGE and blocks its ability to interact with, and activate cellular RAGE. Female mice overexpressing human apoB became diabetics with streptozotocin; D and control mice (C) were fed normal food for 6 weeks. Certain D mice were treated once daily with sRAGE (20 μg / day, intraperitoneally) or mouse serum albumin (MSA, 40 μg / d). At 6 weeks, morphometric analysis revealed a 26-fold increase in the area of average lesion (MLA) in D vs. D mice. C (919 ± 38 v. 35 ± 15 μm2, p <0.0005). In D vs. V mice C, cholesterol levels (TC) (113 ± 25 v 73 ± 19 mg / dl, p = 0.08), tr glyceride (TG) (102 ± 21 v. 79 ± 7 mg (dl; p = 0.33), and apoB (62 ± 7 v. 70 ± 5 mg / dl; p = 0.39) did not differ. Analysis by FPLC revealed identical lipid profiles. MLA decreased 8-fold in mice treated with sRAGE vs. MSA (124 ± 18 v. 993 ± 48 μm2, p <0.0005) without significant differences in TC levels (102 ± 5 v. 106 ± 7 mg / dl; p = 0.65), TG (122 Sl) ± 8 v. 112 ± 9 mg / dl; p = 0.46) or apoB (61 ± 2 V. 63 ± 3 mg / dl; P = 0.66) in mice treated with sRAGE vs. treated with MSA; the profiles of FPL were identical. Hyperglycemia (hbAlc) persisted in both groups. Thus, diabetic mice that overexpress human apoB may be an ideal model with which the contribution of lipid-independent mechanisms underlying accelerated diabetic atherosclerosis can be dissected. These data identify the AGE-RAGE interaction as a novel objective to design therapeutic agents to prevent diabetic vasculopathy. Thus, sRAGE suppresses the development of accelerated atherosclerotic injury in diabetic mice overexpressing the human polyprotein B gene. This mouse model system is useful for testing possible compounds for the treatment of diabetic microvascular disease. One embodiment of this invention is a method for identifying a compound, which improves diabetic microvascular disease in a subject, which comprises administering the compound to the subject (e.g., this mouse model) and comparing the amount of development of atherosclerotic lesion. accelerated in that mouse with the amount of development of the lesion in a mouse to which the compound was not administered, a decrease in the development of the lesion in the first mouse indicate that the compound is useful for improving * - diabetic microvascular disease .

Example 3 - Maintenance of Vascular Structure Integrity in mice without Diabetic LDL Receptor Treated with soluble Receptor for AGE (sRAGE). Vascular structure integrity (VSI) decreased in diabetes; probably as a result of receptor-independent mechanisms (such as increased formation and cross-linking of the Advanced Glycation End Products, products of non-enzymatic protein glycoxidation) and mechanisms dependent on interactions with specific receptors. The best characterized receptor for AGE (RAGE) interacts with AGE structures that lead to vascular hyperpermeability, increased endothelial expression (EC) of adhesion molecules, and macrophage attraction (MP) and activation with production of cytokines such as TNF-a and IL-lß. Mice without LDLR (LDLR 0) were diabetic with streptozotocin. To study the role of this interaction in diabetic vascular disease, LDLR 0 diabetic mice were treated with soluble RAGE (sRAGE; the extracellular two-thirds of RAGE to bind to AGEs and interferes with their ability to interact with and activate cellular RAGE) or equimolar concentrations of vehicle, mouse serum albumin (MSA). The tensile strength (TS) of the aorta was measured as an indicator of the VSI. After six weeks administered daily sRAGE (20 ug / day), TS of aorta was 101 ± 6.9 Newtons (N) (mean ± SEM), compared with 40 ± 7.3 N in LDLR mice 0 diabetics treated with MSA (p = 0.004) (2.53 times). The TS of the control LDLR 0 mice was 91 + 13 N; there was no significant difference between the sRAGE and control mice (p = 0.57). There was a significant difference between TS of the control aorta and diabetic LDLR (p = 0.02). Zymography of aortic extracts revealed increased gelatinase activity in diabetic mice treated with MSA vs. SRAGE (1.6 times). Taken together, these data suggest that multiple mechanisms, such as the formation of AGEs and destructive inflammatory processes mediated by the receptor, act by decreasing vascular structure and integrity in diabetes. The beneficial effects of sRAGE indicate a role for the AGE-RAGE interaction in this process, and identify RAGE as an important therapeutic target for diabetic vascular disease.

Example 4- Suppression of Accelerated Atherosclerosis in Diabetic LDL-Receptor-Free Mice by the Soluble Receptor for AGE (sRAGE) Previously it was demonstrated that mice without diabetic LDL-receptor (LDLR 0) with streptozotocin (stz) showed an increase in the development of lesions vascular after six weeks of normal food compared to non-diabetic LDLR or control. The analysis of the lipid content did not reveal significant differences in the level or profile of cholesterol or triglyceride in plasma, suggesting the contribution of lipid-independent mechanisms. To delineate a possible role for the interaction of the Advanced Application Final Products (AGEs) with its cellular receptor RAGE, we treated LDLR 0 diabetic mice with soluble RAGE (sRAGE) or equimolar concentrations of mouse serum albumin (MSA), both by administration intraperitoneal once a day. After six weeks of daily treatment with dietary food, the area of the atherosclerotic lesion (MALA) was 8803 ± 287 μm2 in mice treated with MSA. In contrast, MALA in mice treated with 20 μg of sRAGE was 2412 ± 184 μm2 (p <0.0001 compared to MSA), 1312 ± 73 μm2 in mice treated with sRAGE (40 μg) (p < 0.0001) compared with MSA). No significant differences were observed in the lipids between the groups sRAGE (20 μg) and MSA (cholesterol: RAGE - 216 + 19 mg / dl and MSA - 220 ± 20 mg / dl (p = 0.53)), (triglycerides: sRAGE - 81 ± 7 mg / dl and MSA - 77 ± 8 mg / dl (p = 0.3)). For FPLC, there were no differences in the lipid profile. To the sacrifice, the average levels of glycosylated hemoglobin f- were 9.69 + 9 and 9.23 +7 in the mice treated with Msa and sRAGE (20 μg), respectively; p = 0.3. Taken together these data suggest that the interaction of AGEs with cellular RAGE may be an important component in the development of accelerated vascular injury in diabetic LDLR mice and identifies this interaction as a feasible target for the prevention of diabetic vascular disease.

REFERENCES Brownlee M. Glycation products and the pathogenesis of diabetic disorders. Diab. Care 15 (12): 1835-1842, 1992. Carpenter, et al. (1971) Toxicol. Appl. Pharmacol., 18: 35-40. Crall FVJ and WC Roberts. The extramural and mtramural coronary artepas m juvenile diabetes mellitus: analysis of nine necropsy patients aged 19 to 38 years with onset of diabetes before age 15 years. Am. J. Med. 64: 221-230, 1978. Hamby Rl et al. Reappraisal of the role of the diabetic state in coronary artery disease. Chest 2: 251-257, 1976.

Kannel WB and DL MaGee. Diabetes and cardiovascular disease: the Framingharm study. J. Am. Med. Assoc. 241: 2035-2038, 1979. Kannel WB and DL McGee. Diabetes and glucose tolerance as risk factors for cardiovascular disease: the Framingham study. Diab. Care 2: 120-126, 1979. Manson JE et al. A prospective study of maturity-onset diabetes mellitus and risk ot coronary heart disease and stroke in women. Arch. Of. Int. Med. 151: 1141-1137, 1991. Neeper M, AM Schmidt, J Brett, SD Yan, F Wang, YC Pan, K Elliston, D Stern and A Shaw. Cloning and expression of RAGE: a cell surface receptor for advanced glycocylation end products of proteins. J. Biol. Chem. 267: 14998-15004, 1992. Plump, AS et al. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell 71: 343-353, 1992. Pyorala KM, M Laasko and M Uusitupa. Diabetes and atherosclerosis: an epidemiologic view. Diab. Metab. Rev. 3: 463-524, 1987. Robertson WB and JB Strong. Atherosclerosis in persons with hypertension and diabetes mellitus. Lab. Invest. 18: 538-551, 1968.

Schmidt AM, Vianna M, Gerlach M, Brett J, Ryan Ryan, J Kao, Esposito C, Hegarty H, Hurley W, Olauss M, Wang E, Pan YC, Tsang TC, and D Stern. Isolation and characterization of binding proteins for advanced glycosylation end products from lung tissue whi ch are present on the endothelial cell surface. J. Biol. Chem. 267: 14987-14997, 1992. Schmidt AM, 0 Hori, J Brett, SD Yan, JL Wautier, and D Stern. Cellular receptors for advanced glycation endproducts: implications for oxidative stress and cellular dysfunction in the pathogenesis of vascular lesions. Arterioscl. And Thromb. 14: 1521-1528, 1994. Schmidt AM, SD Yan, and D Stern. The dark side of glucose (News and Views). Nat. Med. 1: 1002-1004, 1995. Schmidt AM, M Hasu, D Popov, JH Zhang, SD Yan, J Brett, R Cao, K Kuwabara, G Costache, N Simionescu, and D Stern. The receptor for Advanced Glycation Endproducts (RAGE) has a central role in vessel wall interactions and gene activation in response to AGEs in the intravascular space. PNAS (USA) 91: 8807-8811, 1994. Waller BE et al. Status of the coronary artery at necropsy in diabetes mellitus with onset after age 30 yrs: analysis of 229 diabetic patients with and without clinical evidence of coronary heart disease and comparison to 183 control subjects. Am. J. Med. 69: 498-506, 1980.

Wautier JL, O ZouJ§ourian, O Chappey, MP Wautier, PJ Guillausseau, R Cao, O Hon, D Stern, and AM Schmidt. Recepter-mediated endothelial cell dysfunction in diabetic vasculopathy: soluble receptor for advanced glycation endproducts blocks hyperpermeability. J. Clin. Invest. 97: 238-243, 1996. 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.

Claims (35)

$ 3 CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for preventing accelerated atherosclerosis in a subject predisposed to this, characterized in that it comprises administering to the subject a polypeptide derivative of the soluble receptor for the final product of the advanced glycation in an amount effective to prevent accelerated atherosclerosis in the subject.

2. The method of compliance with the claim 1, characterized in that the subject is a mammal.

3. The method of compliance with the claim 2, characterized in that the mammal is a human.

4. The method according to claim 1, characterized in that the subject is a diabetic subject.

5. The method according to claim 1, characterized in that the subject suffers from an apolipoprotein deficiency.

6. The method according to claim 1, characterized in that the subject suffers from hyperlipidemia.

7. The method according to claim 6, characterized in that the hyperlipidemia is hypercholesterolemia or hypertriglyceridemia.

8. The method according to claim 1, characterized in that the subject has a disorder in the metabolism of glucose.

9. The method according to claim 1, characterized in that the subject is an obese subject.

The method according to claim 1, characterized in that the polypeptide comprises at least a portion of the natural soluble receptor for the final product of the advanced glycation.

The method according to claim 1, characterized in that the polypeptide comprises a domain V of natural soluble receptor for the final product of the advanced glycation.

The method according to claim 1, characterized in that the polypeptide comprises a 10 kilodalton domain of natural soluble receptor for the final product of advanced glycation.

13. The method according to claim 1, characterized in that the polypeptide comprises a sequence less than or equal to 20 amino acids in length, which sequence is within the natural soluble receptor sequence for the final product of advanced glycation.

The method according to claim 1, characterized in that the polypeptide is a mimetic peptide, a synthetic polypeptide or a polypeptide analogue.

15. The method according to claim 1, characterized in that it comprises administering to the subject a pharmaceutically acceptable carrier during administration of the polypeptide.

16. The method according to claim 1, characterized in that the administration comprises intralesional, intraparitoneal, intramuscular or intravenous injection; infusion; liposome-mediated release; or topical, nasal, oral, ocular or otic release.

17. The method according to claim 1, characterized in that the polypeptide is administered daily.

18. The method according to claim 1, characterized in that the effective amount of polypeptide comprises from about 0.000001 mg / kg of body weight to about 100 mg / kg of body weight.

19. A method for preventing macrovascular disease in a subject predisposed thereto, characterized in that it comprises administering to the subject a polypeptide derived from the soluble receptor of the final product of advanced glycation in an amount effective to prevent macrovascular disease in the subject.

20. The method according to claim 19, characterized in that the subject is a human.

21. The method according to claim 19, characterized in that the subject is a diabetic subject.

22. The method according to claim 19, characterized in that the subject suffers from an apolipoprotein deficiency.

23. The method according to claim 19, characterized in that the subject suffers from hyperlipidemia.

24. The method according to claim 23, characterized in that the hyperlipidemia is hypercholesterolemia or hypertriglyceridemia.

25. The method according to claim 19, characterized in that the subject has a disorder of glucose metabolism.

26. The method according to claim 19, characterized in that the subject is an obese subject. ""3

27. The method according to claim 19, characterized in that the polypeptide comprises at least a portion of the natural soluble receptor for the final product of the advanced glycation.

28. The method according to claim 19, characterized in that the polypeptide comprises a domain V of natural soluble receptor for the final product of the advanced glycation.

29. The method according to claim 19, characterized in that the polypeptide comprises a 10 kilodalton domain of natural soluble receptor for the final product of advanced glycation.

30. The method according to claim 19, characterized in that the polypeptide comprises less than or equal to 20 amino acids in length, which sequence is within the natural soluble receptor sequence for the final product of the advanced glycation.

31. The method according to claim 19, characterized in that the polypeptide is a mimetic peptide, a synthetic polypeptide or a polypeptide analogue.

32. The method according to claim 19, characterized in that it further comprises administering a pharmaceutically acceptable carrier to the subject during administration of the polypeptide.

33. The method according to claim 19, characterized in that the administration comprises intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; liposome-mediated release; or topical, nasal, oral, ocular or otic release.

34. The method according to claim 19, characterized in that the sRAGE polypeptide is administered daily.

35. The method according to claim 19, characterized in that the effective amount of polypeptide comprises from about 0.000001 mg / kg of body weight to about 100 mg / kg of body weight.