METHOD FOR TREATING AND PREVENTING ATHEROSCLEROSIS

Abstract: A method for treating or preventing atherosclerosis in a mammal is described. An agent for inhibiting interaction between P-selectin and a ligand of P-selectin is provided. The agent is administered to a mammal in need of such treatment to cause this inhibition to occur.

Description: METHOD FOR TREATING AND PREVENTING ATHEROSCLEROSIS This application is a continuation-in-part application of pending application Serial No. 08/253,663, filed on June 3, 1994, and entitled METHOD FOR TREATING AND PREVENTING ATHEROSCLEROSIS. The entire contents of the parent application are hereby expressly incorporated by reference. The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant Nos. 7F32 HL08908 and POl HL42443 awarded by the National Institutes of Health, and ROl HL53756 awarded by the National Institutes of Health, National Heart, Lung and Blood Institute. Field of the Invention This invention relates to treatment and prevention of atherosclerosis. Background of the Invention Atheroεcleroεis is a principal cause of heart attacks, strokes and gangrene of the extremities. It has been reported that approximately 50% of all deaths in the United States, Europe and Japan are due to atherosclerosis. Atherosclerotic lesions can result from an excessive inflammatory-fibroproliferative response to various forms of insult to the endothelium and smooth muscle cells of the artery wall. It is believed that the earliest type of atherosclerotic lesion is formed by binding of monocytes and T lymphocytes (CD4 + and CD8 + ) to the surfaces of endothelial cells in the lumen of the artery wall. These migrating cells proceed to penetrate beneath the arterial surface. The monocytes become macrophages, accumulate lipid, and become foam cells. These cells, together with the T lymphocytes, form a lesion called the fatty streak. The fatty streak subsequently develops into a fibrofatty intermediate lesion which is composed predominantly of layers of smooth muscle cells together with lipid-filled macrophages and T cells. These lesions in turn develop into complex occlusive lesions called fibrous plaques. The fibrous plaques can increase in size by projecting into the arterial lumen, and may thereby impede the flow of blood. Sudden death from myocardial infarctions can result from ruptures in the fibrous cap of the plaque, causing hemorrhage into the plaque, thrombosis and occlusion of the artery. Current treatments for atherosclerosis include bypass grafting, endarterectomy, and angioplasty. These methods are high-risk invasive surgical procedures. Moreover, the failure rate of such treatments can often be high due to restenosis, which is thought to result from further inflammation, smooth muscle accumulation and thrombosis. Summary of the Invention It is an object of the invention to provide a safe, effective, easy and inexpensive method for treating or preventing atherosclerosis. It is yet another object of the invention to provide a method for treating or preventing atherosclerosis which does not involve an invasive procedure. It is yet another object of the invention to provide a simple method for treating cr preventing atherosclerosis such as administering a pill, administering an injection or inserting an implant. It is yet another object of the invention to treat or prevent atherosclerosis by administering to a mammal an agent which inhibits interaction between P-selectin and a ligand of P-selectin, so as to reduce formation of atherosclerotic lesions in the arteries. Still another object of the invention is to provide a method fo treating or preventing atherosclerosis by administering an agent which inhibits P-selectin function, and in which P-selectin function is restored upon depletion of the agent . According to the invention, a method for treating or preventing atherosclerosis in a mammal is provided. An agent is provided for inhibiting interaction between P-selectin and a ligand of P-selectin. The agent is administered to a mammal in need of such treatment to cause this inhibition to occur. In certain embodiments, the P-selectin is on a cell, preferably an endothelial cell or a platelet. The ligand preferably is a carbohydrate, e.g., sialyl-Lewis x, sialyl-Lewis a, sialyl-Lewis x-pentasaccharide, polylactosaminoglycan, a carbohydrate containing 2,6 sialic acid, Lewis x 3 '-0-sulfate, or heparin oligosaccharides, or a glycoprotein, e.g., PSGL-1, 160 kD monospecific P-selectin ligand, or lyεosomal membrane glycoproteins. The ligand can be on, e.g., monocytes, neutrophils, eosinophils, CD4 T cells, CD8 + T cells or natural killer cells. The agent can be, e.g., a soluble form of at least a portion of P-selectin or the ligand or mixtures thereof; an inhibitory protein, e.g., an antibody, e.g., a polyclonal or a monoclonal antibody, against at least a portion of P-selectin or the ligand or mixtures thereof; an inhibitory peptide, e.g. , consisting of at least a portion of one of the binding sites on P-selectin or the ligand or mixtureε thereof; an inhibitory carbohydrate, e.g., sialyl-Lewis x or its analogs, sialyl-Lewis a or its analogs, heparin oligosacchardies or carbohydrates containing 2,6 sialic acids; an inhibitory glycoprotein, e.g., PSGL-1, 160 kD monospecific P-εelectin ligand, lysosomal membrane glycoprotein or glycoprotein containing sialyl Lewis X; an inhibitory sulfatide; analogε of P-εelectin or the ligand or mixtures thereof; substances derived from natural products, e.g., snake venoms or plant extracts; inhibitors of granular release; or inhibitors of a molecule required for the synthesiε, poεt-translational modification, or functioning of P-selectin or the ligand. In certain embodiments, the agent inhibits interaction between P-selectin and the ligand so as to at least partially prevent formation of, or to at least partially reverse a formed, atherosclerotic fatty streak, and/or an intermediate lesion, and/or a fibrous plaque, or so as to at least partially prevent growth of an atherosclerotic lesion after a surgical procedure for preventing reεtenosis. Variations of this method of this invention include administering the agent prior to formation of an atherosclerotic lesion, administering the agent subsequent to formation of an atheroεclerotic lesion, and administering the agent to a human. Another aspect of the invention is a therapeutic agent in a dosage form and concentration εuitable for treating or preventing atherosclerosis in a mammal in need of such treatment, the agent being effective to inhibit interaction between P-selectin and a ligand of P-selectin. The above and other objects, features and advantages of the present invention will be better understood from the following specification. Detailed Description This invention provides a method for treating or preventing atherosclerosis in a mammal. An agent is provided which inhibits interaction between P-selectin and a ligand of P-selectin. The agent is administered to a mammal in need of such treatment to cause this inhibition to occur. Atheroscleroεiε iε a condition which iε meant to include the preεence of any one or more types of atherosclerotic lesions on the surface of an arterial wall. Such lesions include fatty streakε, fibrofatty intermediate lesions and fibrous plaqueε. Atherosclerosis develops in many mammals. By mammals is meant human as well as non-human mammals. Treating atheroεclerosiε iε meant to include preventing, arresting, altering, and reversing formation of atheroεclerotic leεionε. P-εelectin iε a cell surface adhesion receptor. A receptor is a transmembrane protein with three major domainε. The extracellular domain has an active site on the exterior side of the membrane which recognizes and binds to a ligand. A short hydrophobic domain makes up the tranε embrane portion, and an intracellular cytoplasmic domain transmitε a signal to the cell that the ligand has bound to the receptor. The extracellular domain of P-selectin includes a Ca '-dependent C-type lectin domain, an epidermal growth factor-like domain, and a serieε of consensus repeats related to those of complement-binding proteins . P-selectin is expresεed in variouε cellε, including endothelial cells and platelets. P-selectin mediates adhesion of different types of cellε to each other. For example, P-εelectin typically mediates heterotypic interactions of plateletε or endothelial cellε with blood cellε. Cellε which bind to P-εelectin include monocyteε, neutrophilε, eoεinophilε, CD4 T cellε, CD8 T cellε and natural killer cells. The binding of P-selectin to another cell can result from recognition of a ligand for P-selectin on that cell. By ligand is meant a moiety which binds to P-selectin, the moiety being either alone or attached to another molecule. P-selectin ligands include carbohydrate groupε, e.g., sialyl-Lewiε X (Foxall et al . , J. Cell Biol., 117(4): 895-902, 1992; Polley et al . , Proc. Nat' 1 Acad. Sci., USA, 88:6224-6228, 1991) sialyl-Lewis a (Berg et al. , J. Biol. Chem. , 266: 14869-14875, 1991), sialyl-Lewiε x pentasaccharide (Mulligan et al., Nature 364: 149-151, 1993), polylactoεaminoglycan, carbohydrate containing 2,6 εialic acid (Larεen et al . , J. Biol. Chem. 267: 11104-11110, 1992), Lewiε x 3 '-0-sulf te (Yuen et al., Biochemistry 31: 9126-9133, 1992) and heparin oligosaccharides (Nelson et al . , Blood 82: 3253-3258, 1993). P-selectin ligands are also meant to include glycoproteins which contain a carbohydrate structure. For example, a P-εelectin carbohydrate ligand can be linked to a mucin-like molecule. (Sako et al . , Cell 75(6): 1179-1186, 1993; Linter et al . , J. Biol. Chem. 125: 471-481, 1994). By mucin is meant serine- and threonine-rich proteinε that are heavily O-glycoεylated and have an extended structure. Other glycoprotein ligands include PSGL-1, 160 kD monoεpecific P-selectin ligand (Linter et al . , J. Biol. Chem. 125: 471-481, 1994) and lyεosomal membrane glycoproteinε (Fukuda, J. Biol. Chem. 266: 21327-21332, 1991). Analogε of the above ligandε which can bind to P-selectin, e.g., where fucose iε replaced, e.g., by a diol group, or derivatives of the sialyl-Lewis x compounds which carry a SO_ group instead of sialic acid, or contain a εialic acid in a 2,6 linkage, are alεo meant to be included aε P-εelectin ligandε. It is known that P-selectin is involved in cellular reεponεes to inflammation resulting from injury or infection. This invention demonstrates that P-selectin can also be involved in the formation of atherosclerotic lesions. Example 1 shows that the presence in mice of a homozygous null mutation in P-εeiectin significantly decreases the size of the atherosclerotic lesionε that are formed when τ:he mice are fed a high fat diet, as compared to wild-type mice fed a high fat diet. The general health of these homozygous P-selectin deficient mice appear normal up to at leaεt two yearε of age. The fact that theεe P-seiectin deficient mice are viable, fertile, of normal size and vigor, and free of obvious signε of infection or diεeaεe, demonεtrateε that P-εelectin iε not required for normal development. The mouεe fed a high fat diet, or variouε genetically engineered mice, are generally accepted aε a good model for atheroεcleroεiε in humanε . (Luεiε, Trends in Cardiovascular Medicine, 3: 135-143, 1993; Stoltzfus and Rubin, Trends in Cardiovascular Medicine, 3: 130-134, 1993; Ishida and Paigen, In Genetic Factorε in Atherosclerosiε; Monogr. Hum. Gen., Vol. 12: 189-222, 1989). Significantly, Example 4 εhowε that the preεence in mice of a homozygous null P-selectin mutation also causeε a significant reduction in the size of atherosclerotic lesionε that are formed when the mice have in addition an LDL receptor-deficient mutation. Mice which lack LDL are a model εystem for the human disease called homozygous familial hypercholesterolemia (see Ishibaεhi et al . , J. Clin. Invest. , 93:1885-1893 (1994)), in which functional LDL receptor is absent, and as a consequence cholesterol-rich lipoproteinε accumulate in the plasma, resulting in atherosclerotic lesions in childhood. The agent of this invention can inhibit interaction between P-selectin and a ligand of P-selectin. By inhibiting interaction is meant, e.g., that P-εelectin and itε ligand are unable to properly bind to each other to effect proper formation of atheroεclerotic leεionε. Such inhibition can be the result of any one of a variety of events, including, e.g. , preventing or reducing interaction between P-selectin and the ligand, inactivating P-selectin and/or the ligand, e.g. , by cleavage or other modification, altering the affinity of P-εelectin and the ligand for each other, diluting out P-εelectin and/or the ligand, preventing surface, plasma membrane, expression of P-selectin or reducing syntheεiε of P-εelectin and/or the ligand, synthesizing an abnormal P-εelectin and/or ligand, synthesizing an alternatively spliced P-selectin and/or ligand, preventing or reducing proper conformational folding of P-selectin and/or the ligand, modulating the binding propertieε of P-εelectin and/or the ligand, interfering with εignalε that are required to activate or deactivate P-selectin and/or the ligand, activating or deactivating P-selectin and/or the ligand at the wrong time, or interfering with other receptorε, ligandε or other molecules which are required for the normal syntheεis or functioning of P-selectin and/or its ligand. Examples of agents include soluble formε of P-εelectin or the ligand, inhibitory proteins, inhibitory peptideε, inhibitory carbohydrates, inhibitory glycoproteins, inhibitory glycopeptides, inhibitory sulfatides, synthetic analogs of P-selectin or the ligand, certain subεtanceε derived from natural productε, inhibitorε of granular releaεe, and inhibitorε of a molecule required for the synthesiε or functioning of P-selectin or the ligand. The soluble form of either P-selectin or the ligand, or a portion thereof, can compete with its cognate molecule for the binding site on the complementary molecule, and thereby reduce or eliminate binding between the membrane-bound P-selectin and the cellular ligand. The εoluble form can be obtained, e.g. , from purification or εecretion of naturally occurring P-εelectin or ligand, from recombinant P-εelectin or ligand, or from εyntheεized P-εelectin or ligand. Soluble formε of P-selectin or ligand are also meant to include, e.g., truncated soluble secreted forms, proteolytic fragments, other fragments, and chimeric conεtructε between at leaεt a portion of P-εelectin or ligand and other moleculeε. Soluble forms of P-selectin are described in Mulligan et al . , J. Immunol., 151: 6410-6417, 1993, and soluble formε of P-εelectin ligand are deεcribed in Sako et al., Cell 75(6): 1179-1186, 1993. Inhibitory proteinε include, e.g., anti-P-εelectin antibodieε (Palabrica et al . , Nature 359: 848-851, 1992; Mulligan et al . , J. Clin. Invest. 90: 1600-1607, 1992; Weyrich et al . , J. Clin. Invest. 91: 2620-2629, 1993; Winn et al., J. Clin. Invest. 92: 2042-2047, 1993); anti-P-selectin ligand antibodies (Sako et al . , Cell 75(6): 1179-1186, 1993); Fab_ fragments of the inhibitory antibody generated through enzymatic cleavage (Palabrica et al . , Nature 359: 848-851, 1992); P-selectin-IgG chimeras (Mulligan et al . , Immunol. 151: 6410-6417, 1993); and carrier proteins expresεing a carbohydrate moiety recognized by P-εelectin. The antibodieε can be directed againεt P-εelectin or the ligand, or a εubunit or fragment thereof. Both polyclonal and monoclonal antibodieε can be used in this invention. Preferably, monoclonal antibodies are used. Most preferably, the antibodies have a constant region derived from a human antibody and a variable region derived from an inhibitory mouse monoclonal antibody. Antibodies to human P-selectin are described in Palabrica et al . , Nature 359: 848-851, 1992; Stone and Wagner, J.C.I. , 92: 804-813, 1993; and to mouse P-selectin are described in Mayadas et al . , Cell, 74: 541-554, 1993. Antibodieε to human ligand are deεcribed in Sako et al., Cell 75(6): 1179-1186, 1993. Antibodieε that are commercially available againεt human P-selectin include clone AC1.2 monoclonal from Becton Dickinson, San Jose, CA. An inhibitory peptide can, e.g., bind to a binding site on the P-selectin ligand εo that interaction aε by binding of P-εelectin to the ligand iε reduced or eliminated. The inhibitory peptide can be, e.g., the εame, or a portion of, the primary binding site of P-selectin, (Geng et al . , J. Biol. Chem., 266: 22313-22318, 1991, or it can be from a different binding site. Inhibitory peptideε include, e.g., peptides or fragments thereof which normally bind to P-selectin ligand, synthetic peptides and recombinant peptides. In another embodiment, an inhibitory peptide can bind to a molecule other than P-εelectin or its ligand, and thereby interfere with the binding of P-selectin to its ligand because the molecule iε either directly or indirectly involved in effecting the synthesis and/or functioning of P-selectin and/or its ligand. Inhibitory carbohydrates include oligosaccharides containing sialyl-Lewis a or sialyl-Lewis x or related structures or analogs, carbohydrates containing 2,6 sialic acid, heparin fractions depleted of anti-coagulant activity, heparin oligoεaccharideε, e.g., heparin retraεaccharides or low weight heparin, and other sulfated polysaccharides . Inhibitory carbohydrateε are deεcribed in Nelεon et al . , Blood 82: 3253-3258, 1993; Mulligan et al . , Nature 364: 149-151, 1993; Ball et al., J. Am. Chem. Soc. 114: 5449-5451, 1992; De Freeε et al. , J. Am. Chem. Soc. 115: 7549-7550, 1993. Inhibitory carbohydrates -hat are commercially available include, e.g., 3 '-sialyl-Lewiε x, 3 '-sialyl-Lewis a, lacto-N-fucopentose III and 3 '-sialyl-3-fucosyllactoεe, from Oxford GlyeoSys erns, Rosedale, NY. Inhibitory glycoproteinε, e.g., PSGL-l , 160 kD monoεpecific P-εelectin ligand, lysosomal membrane glycoproteinε, glycoprotein containing εialyl-Lewis x, and inhibitory εulfatides (Suzuki et al., Biochem. Biophys . Res. Commun. 190: 426-434, 1993; Todderud et al . , J. Leuk. Biol. 52: 85-88, 1992) that inhibit P-selectin interaction with itε ligand can alεo be uεed in this invention. Synthetic analogs or mimetics of P-selectin or the ligand also can serve as agents. P-selectin analogs or mimetics are substanceε which reεemble in εhape and/or charge diεtribution P-selectin. An analog of at least a portion of P-selectin can compete with its cognate membrane-bound P-selectin for the binding site on the ligand, and thereby reduce or eliminate binding between the membrane-bound P-selectin and the ligand. Ligand analogs or mimetics include substances which resemble in shape and/or charge distribution the carbohydrate ligand for P-selectin. An analog of at least a portion of the ligand can compete with its cognate cellular ligand for the binding εite on the P-εelectin, and thereby reduce or eliminate binding between P-εelectin and the cellular ligand. In certain embodiments which use a ligand analog, the εialic acid of a carbohydrate ligand iε replaced with a group that increases the stability of the compound yet still retainε or increaεes its affinity for P-selectin, e.g. a carboxyl group with an appropriate spacer . An advantage of increaεing the εtability iε that it allowε the agent to be adminiεtered orally. Sialyl-Lewiε x analog with glucal in the reducing end and a bivalent sialyl-Lewiε x anchored on a galactoεe reεidue via β-l,3- and β-1,6- linkageε also inhibit P-selectin binding (DeFrees et al . , J. Am. Chem. Soc, 115: 7549-7550, 1993). Agents are also meant to include substances derived from natural products, such as snake venoms and plant extracts, that inhibit P-selectin interaction with itε ligand. Such εubεtanceε can inhibit this interaction directly or indirectly, e.g. , through specific proteolytic cleavage or other modification of P-selectin or its ligand. An inhibitor of granular release also interferes with P-selectin expresεion on the cell εurface, and therefore interferes with P-selectin function. By granular releaεe iε meant the εecretion by exocytosis of storage granules containing P-selectin: Weibel-Palade bodies of endothelial cells or α-granules of plateletε. The fuεion of the granular membrane with the plaεma membrane results in expresεion of P-εelectin on the cell εurface. Exampleε of εuch agents include colchicine. (Sinha and Wagner, Europ. J. Cell. Biol. 43: 377-383, 1987). Agents also include inhibitors of a molecule that is required for syntheεiε, post-translational modification, or functioning of P-selectin and/or the ligand, or activators of a molecule that inhibits the synthesiε or functioning of P-εelectin and/or the ligand. Agentε include cytokineε, growth factorε, hormoneε, signaling components, kinaεes, phosphataεeε, homeobox proteins, transcription factors, translation factors and post-translation factors or enzymes. Agents are also meant to include ionizing radiation, non-ionizing radiation, ultrasound and toxic agentε which can, e.g., at least partially inactivate or deεtroy P-selectin and/or the ligand. An agent is alεo meant to include inhibitors which are not entirely P-selectin specific. For example, an agent may inhibit other selectin interactionε in addition to P-εelectin interactionε, e.g. , L and/or E εelectin interactions. Such overlapping specificity may provide additional therapeutic advantage. Administration of the agent can be accomplished by any method which allowε the agent to reach the target cells. These methods include, e.g., injection, deposition, implantation, suppositorieε, oral ingestion, inhalation, topical administration, or any other method of administration where access to the target cells by the agent is obtained. Injections can be, e.g., intravenous, intradermal, subcutaneouε, intramuscular or intraperitoneal . Implantation includes inserting implantable drug delivery systems, e.g., microεphereε, hydrogelε, polymeric reεervoirε, choleεterol matriceε, polymeric syεterns, e.g., matrix erosion and/or diffusion syεtemε and non-polymeric εyεtemε, e.g., compreεεed, fuεed or partially fused pellets. Suppositories include glycerin suppoεitories . Oral ingestion doseε can be enterically coated. Inhalation includeε adminiεtering the agent with an aeroεol in an inhalator, either alone or attached to a carrier that can be absorbed. Administration of the agent can be alone or in combination with other therapeutic agents. In certain embodiments, the agent can be combined with a suitable carrier, incorporated into a liposome, or incorporated into a polymer releaεe εyεtem. Preferably, protein agentε are administered by intravenouε or intramuεcular injection; peptide agents by intravenous or intramuscular injection or by glycerin suppository; carbohydrate or εulfatide agents by intravenous or intramuscular injection, or with an aeroεol in an inhalator; and synthetic analog agents by intravenouε or intramuscular injection, or with an aerosol in an inhalator, or orally. In certain embodiments of the invention, the administration can be designed so as to result in sequential exposures to the agent over εome time period, e.g., hours, days, weeks, months or years. Thiε can be accompliεhed by repeated administrations of the agent by one of the methods described above, or alternatively, by a controlled release delivery system in which the agent is delivered to the mammal over a prolonged period without repeated administrations. By a controlled release delivery system is meant that total release of the agent does not occur immediately upon administration, but rather is delayed for some time period. Release can occur in bursts or it can occur gradually and continuously. Administration of such a system can be, e.g., by long acting oral dosage forms, bolus injections, transdermal patcheε and sub-cutaneous implants. Examples of systems in which release occurs in burstε include, e.g., systems in which the agent is entrapped in liposomeε which are encapsulated in a polymer matrix, the liposomes being senεitive to a specific stimuli, e.g., temperature, pH, light or a degrading enzyme, and syεtemε in which the agent iε encapεulated by an ionically-coated microcapεule with a microcapsule core-degrading enzyme. Exampleε of systemε in which releaεe of the agent is gradual and continuous include, e.g. , eroεional systems in which the agent is contained in a form within a matrix, and diffusional systemε in which the agent permeateε at a controlled rate, e.g., through a polymer. Such εustained release syεtemε can be, e.g., in the form of pellets or capsuleε. The agent can be suspended in a liquid, e.g. , in disεolved form or colloidal form. The liquid can be a εolvent, partial εolvent or non-εolvent. In many caεeε water or an organic liquid can be used. The agent can be administered prior to or subsequent to fibrous plaque formation. In certain embodiments, the agent iε adminiεtered to patients, e.g., after angioplasty, stenting procedure, atherectomy, or bypaεs surgery or other vesεel-corrective techniqueε, to aid in preventing reεtenoεis. The agent alεo can be adminiεtered, preferably on a daily basis, to patients with familial hypercholesteremia, an early debilitating diseaεe, who develop atherosclerotic lesionε at a young age, often reεulting in arterial narrowing and death. The agent is administered to the mammal in a therapeutically effective amount. By therapeutically effective amount is meant that amount which iε capable of at leaεt partially preventing or reversing plaque formation. A therapeutically effective amour.- can be determined on an individual basis and will be based, at least in part, on consideration of the specieε of mammal, the mammal 'ε εize, the agent uεed, the type of delivery εyεtem uεed, the time of administration relative to plaque formation, and whether a single, multiple, or controlled release doεe regimen iε employed. A therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation. Preferably, the concentration of an inhibitory protein, peptide, glycoprotein or glycopeptide if applied syεtemically, is at a doεe of about 0.1 to about 500 mg/kg body weight. Moεt preferably the dose is about 0.1 to about 5 mg/kg. The specific concentration partially depends upon the particular inhibitory protein, glycoprotein, peptide or glycopeptide used, as some are more effective than others, Preferably, the concentration of a carbohydrate or a synthetic analog, if applied syεtemically is at a dose of about 0.01 to about 200 mg/kg body weight. Most preferably, the dose is about 0.1 to about 5 mg/kg. Preferably, the concentration of a sulfatide, if applied syste mically iε at a dose of about 1 to about 100 mg/kg body weight. Preferably, the concentration of a soluble form of P-selectin or ligand, if applied syεtemically iε at a doεe of about 1 to about 100 mg/kg body weight. Most preferably, the dose is about 1 to about 5 mg/kg. The dosage concentration of the agent that iε actually administered is dependent at least in part upon the final concentration that is desired at the site of action, the method of administration, the efficacy of the particular agent, the longevity of the particular agent, and the timing of adminiεtration relative to the formation of the atherosclerotic lesion. Preferably, the dosage form is εuch that it does not subεtantially deleteriously affect the mammal . The dosage can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation. The agents of the invention are meant to include reverεible and non-reverεible agentε. If an agent is reversible, the inhibition of the interaction between P-selectin and its ligand will be reversed at some point after administration of the agent ceaseε. A reverεible agent is preferable in that it permits diεcontinuation of adminiεtration of the agent during periodε of infection or woundε . P-εelectin function iε thereby reεtored and able to act in itε inflammation-reεponεe capacity to aid in fighting infectionε or in wound repair. The invention alεo includeε a therapeutic agent in a dosage form and concentration suitable for treating or preventing atherosclerosis in a mammal in need of such treatment, the agent being effective to inhibit interaction between P-selectin and itε liσand. EXAMPLES Example 1: P-Selectin-Deficient Mice Fed a High Fat Diet Have Significantly Smaller Atheroεclerotic Leεionε Than Wild-Type Mice This example illuεtrateε that P-εelectin plays an important role in the formation of atherosclerotic lesionε in blood veεselε. Compariεons were made of atherosclerotic lesionε in wild-type and P-εelectin-deficient mice fed a high fat diet. The P-εelectin deficient mice contain a homozygouε null mutation in P-εelectin and were generated by homologous recombination in embryonic stem cellε aε described in Mayadas et al., Cell 74: 541-554, 1993. Age-matched female wild-type and P-selectin deficient mice were used. (C57BL and 129 mixed background; both of these strainε are εusceptible to aortic lesion formation upon 14 week exposure to a high fat diet.). The mice were anesthetized and bled from the retroorbital venous plexus at the initiation of the prescribed diets. They were divided into two groupε, each conεiεting of wild-type and P-εelectin deficient mice. The control low fat group waε fed Purina mouεe chow containing 4.5% (w/w) animal fat, 0.03% (w/w) choleεterol, no εodium cholate and no caεein. The other group waε fed a high fat diet containing 15% (w/w) fat (from butter), 1.15-1.25% (w/w) choleεterol, 0.5% (w/w) sodium cholate and 20% caεein (Rubin et al., Nature 353: 265-267 (1991)). The mice were εtarted on the dietε at 12-16 weekε of age and maintained on the dietε for 19-21 weekε, at which time blood was drawn and the mice were sacrificed. The total cholesterol levelε in the blood plasma increased by comparable amountε in both P-εelectin-deficient and wild-type mice. The p value iε 0.46, indicating that there waε no εtatiεtical difference in cholesterol levels in response to the high fat diet in the two setε of mice. The measured cholesterol value increases were similar to those reported by Paigen et al . , Atherosclerosiε, 57: 65-75, 1985. The heartε were processed according to Paigen et al . , Atherosclerosiε, 68: 231-240 (1987). The heart and attached aorta were placed in 0.9% εaline for 1 hour to remove erythrocyteε and allow muεcle relaxation. The hearts were then fixed in 10% buffered formalin and embedded in gelatin. For quantitative evaluation, the hearts were embedded in O.C.T. (optimal cooling temperature) compound, frozen and εectioned on a cryoεtat. Sectionε were diεcarded until reaching the junction of the heart muscle and aorta where the valve cusps become visible and the aorta is rounded. Unstained sectionε were regularly examined to locate the area of interest. This area of the aorta waε shown previously to consistently result in lesionε in C57BL/6 mice following 14 weekε expoεure to the high fat diet. (Paigen et al. , Atheroεclerosis, 68: 231-240, 1987). Once the area waε localized, four conεecutive 10 μm sections were collected for each slide. Sectioning continued for approximately 350 μm (9-10 εlideε/heart) towardε the aortic arch and exiting the valve region. Sections were collected onto gelatin coated glasε εlideε and odd numbered εlideε were εtained with oil red-0 and hematoxylin. Tissues were then counterstained with light green. One εection on each of the odd numbered εlideε waε aεεeεsed. Where posεible, the same section on each of the five εlideε was used for quantitation. Thus, five εectionε, each 80 μm apart, were examined. If a εection on a εlide waε folded or damaged, then the εection immediately following or preceding replaced the flawed εection. The slides were coded and the examiner was unaware of the genotype of the animal from which the sectionε originated. The size of the lesion was quantified using an ocular micrometer (net grid with 100 squareε; each εquare 25 x 25 μm using 40x objective). Lesions less than 0.1 square using the 40x objective (400x magnification) were not counted. Lesions for each section were totaled. As shown in Table 1, the average size of the lesionε in the P-εelectin deficient mice fed a high fat diet waε 3.6 times smaller than for the wild-type mice fed a high fat diet. No aortic lesionε were presenr. in wild-type or P-εelectin deficient mice (one each) fed the low fat control diet. TABLE 1 SIZE (μm 2 ) OF ATHEROSCLEROTIC LESIONS IN WILD TYPE AND P-SELECTIN-DEFICIENT MICE (five values per mouse, each 80 microns apart) Wild Type P-Selectin-Deficient 562.50 406.25 1375.00 1000.00 2000.00 562.50 562.50 687.50 1062.50 937.50 4750.00 0.00 2937.50 312.50 13000.00 1250.00 1375.00 187.50 0.00 750.00 0.00 62.50 2250.00 125.00 2250.00 437.50 2937.50 1125.00 0.00 750.00 1250.00 218.75 375.00 0.00 437.50 187.50 625.00 0.00 250.00 0.00 437.50 0.00 250.00 187.50 187.50 93.75 125.00 187.50 125.00 218.75 0.00 500.00 0.00 625.00 0.00 9625.00 0.00 2812.50 0.00 437.50 TABLE 1 continued: Wild Type P-Selectin-Deficient 812.50 0.00 875.00 187.50 4875.00 125.00 3812.50 187.50 4437.50 562.50 0.00 0.00 0.00 0.00 62.50 0.00 187.50 0.00 1312.50 0.00 5875.00 2625.00 6000.00 6812.50 7875.00 4750.00 5937.50 5687.50 9125.00 437.50 Statistical comparison of the lesion formation in the wild-type and P-εelectin-deficient mice waε done uεing the εtudent t-teεt. Each mouεe provided five individual values for statiεtical evaluation. Other inveεtigatorε have previously determined that leεionε 80 μm equidistant apart are likely to represent separate events and can therefore be computed separately. (Paigen et al, Atherosclerosiε, 68: 231-240, 1987). Aε Table 2 demonεtrateε, analyεiε of the atheroεclerotic leεion data shows that the obtained t-statistic could have occurred by chance two timeε out of a thouεand, and therefore the difference in the εize of the leεions in the wild-type and P-selectin-deficient mice are highly statistically different. TABLE 2 t-TEST: TWO-SAMPLE ASSUMING EQUAL VARIANCES Wild Type P-Selectin-Deficient Mean 2,212.50 618.75 Variance 8,306,760.20 2,405,408.65 Observationε 50.00 40.00 Pooled Variance 5,691,388.49 Hypotheεized Mean Difference 0 df 88 t Stat 3.149 P(T<=t) two-tail 0.002 Example 2: Treating Atheroεcleroεiε in a Human with Sialyl Lewis x This example illustrates a method for treating atheroscleroεis in a human with an agent which inhibits interaction between P-selectin and its ligand. The patient is given an intramuscular injection of sialyl-Lewiε x, once a day for a period of six months. (Mulligan et al . , Nature 364: 149-151, 1993). The dose concentration per day iε 1 mg/kg body weight. Thiε treatment interfereε with further development of atheroεclerotic leεions. Example 3 Treating Atheroscleroεis in a Human With an Analog of Sialyl-Lewis x This example illustrateε a method for treating atheroεcleroεiε in a human with an agent which inhibits interaction between P-selectin and its ligand. The patient is given a synthetic analog of a carbohydrate ligand orally, in the form of a pill, once a day. The compound is a mimetic of a carbohydrate ligand for P-εelectin εimilar in εize and charge distribution to sialyl-Lewis x. The analog is εyntheεized uεing both enzymatic (uεe of highly purified glycosyltranεferases and glycosidaεes) and conventional chemical methods. The compound has a rigid structure to fix its conformation to that of the highest affinity for P-selectin. The original position of the sialic acid is occupied by a carboxyl group, and that of the fucose by a hydrogen donor, a triol. The dose concentration per day is 1 mg/kg body weight. Administration iε carried out for a period of three monthε. Thiε treatment interferes with further development of atherosclerotic leεionε. Example 4 : Mice Lacking LDL Receptor, a Mouεe Model for Human Homozygous Familial Hypercholesterolemia, Develop Significantly Smaller Atherosclerotic Lesionε If They Are Alεo Deficient in P-Selectin Thiε example illuεtrateε that the abεence of P-selectin can significantly attenuate the severe phenotype of heart disease in mice lacking LDL receptor — a situation genetically identical to a human diseaεe called homozygous familial hypercholesterolemia (FH) . In humans with FH, the absence of functional LDL receptor leads to the accumulation of cholesterol-rich lipoproteins in plaεma. Aε a conεequence, macrophages loaded with cholesteryl esters are deposited throughout the body and atherosclerotic lesions of the aortic root and coronary arteries develop in childhood. (See Goldstein and Brown, Familial Hypercholesterolemia. In The Metabolic Basis of Inherited Disease, eds . Scriver et al., McGraw Hill Inc., N.Y. 1215-1250 (1989)). To examine whether the absence of P-selectin can influence the development of the extensive atherosclerotic lesions in FH, the P-selectin-deficient mice described in Example 1 (Mayadas et al. , Cell 74:541-554 (1993)) were bred with LDL receptor-deficient mice developed through gene targeting technology described in Ishibaεhi et al., J. Clin. Inveεt., 92:883-893 (1993). The phenotype _f the LDL receptor-deficient mice iε remarkably εimilar to the phenotype of human homozygouε FH when the animalε are fed an atherogenic diet: rich in cholesterol, saturated fat, and cholic acid (Ishibaεhi et al . , J. Clin. Inveεt . , 93:1885-1893 (1994)). Through the above-described breeding, a colony of mice deficient for LDL receptor and either wild-type for P-selectin or homozygous-deficient for P-εelectin have been obtained. Twelve mice which are LDL receptor-deficient and wild-type for P-selectin (P-selectin-poεitive) , and 11 mice deficient for both LDL receptor and P-εelectin (P-εelectin-negative) , were put on an atherogenic diet for 8 weekε. Their hearts were then procesεed aε described in Example 1. Within two weeks of the onset of the diet, their plasma cholesterol reached levels above 1,000 mg/dl , as compared to 200 mg/dl prior to the diet administration. At the time of sacrifice, a large εample of blood was collected for individual cholesterol, triglyceride and lipoprotein profile analysis. No differences were detected between the P-εelectin-negative and P-εelectin-poεitive mice. After 8 weekε on the high choleεterol diet, the mice had practically no HDL, and most of the cholesterol waε in the LDL-VLDL region, in agreement with results reported by others (Isibaεhi et al . , J. Clin. Inveεt., 93:1885-1893 (1994)). Importantly, there was no difference in the total plasma choleεterol levelε between the P-εelectin-positive and negative mice — both groups gave approximately 1000 mg/dl (levelε comparable to those seen in human FH) (Table 3). TABLE 3 CHOLESTEROL LEVELS IN LDLR-DEFICIENT MICE AFTER 8 WEEKS ON HIGH-FAT DIET (mg/dl) P-Selectin Wild Type P-Selectin-Deficient 842 1068 1066 1053 1088 1228 1021 1076 940 1176 1241 1025 1135 795 1046 1114 926 1036 1842 1024 1438 1283 1280 Statistics: P-Selectin P-Selectin Wild Type Deficient mean 1155.42 1079.82 standard deviation 272.09 127.94 n 12 11 P value 0.411 The results shown in Table 3 confirmed that the diet had the desired effect on plasma cholesterol level and also that the mice were correctly genotyped as LDL receptor-deficient. As deεcribed in Example 1, five sections of the aorta in the cuspε regionε were aεεesse . The mean area of the lesion was determined and this εingle value for each animal (Table 4) waε uεed for statistical analysiε (Tableε 5 and 6) . TABLE 4 MEAN ATHEROSCLEROTIC LESION SIZE (mm 2 ) IN LDL RECEPTOR-DEFICIENT MICE (one value per mouse which is the mean from 5 sectionε, 80 microns apart) P-Selectin P-Selectin Positive Negative (total) (total) 0 .267 0 .082 0 .149 0 .189 0, .283 0, .207 *0, .256 0, .241 0, ,276 0, ,154 0 .182 *0, ,240 0, .253 *0 , ,079 *0, .436 *0 , ,157 *0, ,279 *0 , ,209 *0, ,177 0, ,100 0 ,097 *0, ,044 *0, ,336 * indiates males TABLE 5 t-TEST FOR P-SELECTIN-POSITIVE MICE AND P-SELECTIN-NEGATIVE MICE: TWO-SAMPLE ASSUMING EQUAL VARIANCES P-Selectin P-Selectin Positive Negative (total) (total) Mean 0.249 0.155 Variance 0.008 0.005 Observationε 12.000 11.000 Pooled Variance 0.006 Hypothesized Mean Difference 0.000 df 21.000 t Stat 2.810 P(T<=t) two-tail 0.010 TABLE 6 t-TEST FOR P-SELECTIN-POSITIVE MALE MICE AND P-SELECTIN-NEGATIVE MALE MICE: TWO-SAMPLE ASSUMING EQUAL VARIANCES P-Selectin P-Seiectin Positive Negative (males) (males) Mean 0.296 0.146 Variance 0.009 0.007 Observations 5.000 5.000 Pooled Variance 0.008 Hypothesized Mean Difference 0.000 df 8.000 t Stat 2.632 P(T<=t) two- J tail 0.030 As shown in Table 4, the mean εize of the atheroεclerotic leεionε in the P-εelectin-poεitive mice waε very large. Deεpite the overwhelming εize of the atheroεclerotic lesions in this FH model, the absence of P-selectin caused a εignificant reduction in leεion εize (Table 5) . Thiε reεult waε eεpecially notable in maleε, where the leεionε in the P-selectin-positive mice were twice the size of those found in P-selectin-negative animals (Table 6). Those εkilled in the art will be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Theεe and all other equivalen ε are intended to be encompaεεed by the following claimε. What iε claimed iε:

Claims: CLAIMS 1. A method for treating or preventing atherosclerosiε in a mammal, comprising: providing an agent for inhibiting interaction between P-selectin and a ligand of P-selectin, and administering said agent to a mammal in need of such treatment to cause such inhibition to occur. 2. The method of claim 1 wherein said P-εelectin iε on a cell. 3. The method of claim 2 wherein said cell is an endothelial cell. 4. The method of claim 2 wherein said cell is a platelet. 5. The method of claim 1 wherein said ligand comprises a carbohydrate. 6. The method of claim 1 wherein said ligand compriseε a glycoprotein. 7. The method of claim 1 wherein said ligand is εelected from the group conεiεting of εialyl-Lewis x, sialyl-Lewis a, sialyl-Lewis x-pentasaccharide, polylactoεa inoglycan, carbohydrate containing 2,6 sialic acid, Lewis x 3 '-0-sulfate, heparin oligosaccharideε, PSGL-1, 160 kD monospecific P-selectin ligand and iysoεomal membrane glycoproteinε . 8. The method of claim l wherein εaid ligand iε on a cell selected from the group consiεting of monocyteε, neutrophilε, eoεinophiis, CD4 + T cellε, CD8 T cellε, and natural killer cellε. 9. The method of claim l wherein said ligand is on a leukocyte. 10. The method of claim 9 wherein said leukocyte is a neutrophil. 11. The method of claim 9 wherein εaid leukocyte iε a monocyte. 12. The method of claim 1 wherein εaid P-εelectin can bind to εaid ligand in the abεence of εaid agent. 13. The method of claim 1 wherein εaid agent iε εelected from the group conεiεting of a soluble form of at least a portion of said P-selectin and a soluble form of at least a portion of said ligand and mixtures thereof. 14. The method of claim 1 wherein said agent iε an inhibitory protein. 15. The method of claim 14 wherein said inhibitory protein iε εelected from the group conεisting of an antibody against at least a portion of said P-selectin and an antibody againεt at least a portion of said ligand and mixtures thereof. 16. The method of claim 15 wherein said antibody iε a monoclonal antibody. 17. The method of claim 1 wherein εaid agent iε an inhibitory peptide. 18. The method of claim 17 wherein εaid P-εelectin haε a firεt binding εite for said ligand and εaid ligand has a second binding site for said P-selectin, and wherein said inhibitory peptide iε a peptide εelected from the group conεiεting of at leaεt a portion of εaid firεt binding εite and at leaεt a portion of εaid εecond binding εite and mixtureε thereof. 19. The method of claim 1 wherein εaid agent iε an inhibitory carbohydrate. 20. The method of claim 19 wherein said inhibitory carbohydrate is selected from the group consisting of sialyl-Lewis x and its analogε, εialyl Lewiε a and its analogs, heparin oligosaccharides and carbohydrates containing 2,6 εialic acid. 21. The method of claim 1 wherein εaid agent iε an inhibitory glycoprotein. 22. The method of claim 21 wherein εaid inhibitory glycoprotein iε selected from the group consiεting of PSGL-1, 160 kD monoεpecific P-selectin ligand, lysoεomal membrane glycoprotein and glycoprotein containing sialyl-Lewis x. 23. The method of claim 1 wherein said agent is an inhibitory sulfatide. 24. The method of claim 1 wherein said agent is selected from the group consiεting of an analog of said P-selectin and an analog of. said ligand and mixtures thereof. 25. The method of claim 1 wherein said agent is a εubstance derived from snake venom or a plant extract. 26. The method of claim 1 wherein said agent is an inhibitor of granular release. 27. The method of claim 1 wherein said agent iε an inhibitor of a molecule required for the synthesiε, poεt-tranεlational modification or functioning of εaid P-selectin or said ligand. 28. The method of claim 1 wherein said agent inhibitε interaction between said P-selectin and said ligand so as to at least partially prevent formation of an atherosclerotic fatty streak. 29. The method of claim 1 wherein said agent inhibits interaction between said P-selectin and said ligand so as to at least partially prevent formation of an atherosclerotic intermediate lesion. 30. The method of claim 1 wherein said agent inhibits interaction between said P-selectin and said ligand so as to at least partially prevent formation of an atheroεclerotic fibrouε plaque. 31. The method of claim 1 wherein εaid agent inhibitε interaction between εaid P-εelectin and εaid ligand εo aε to at leaεt partially prevent growth of an atheroεclerotic leεion after a εurgical procedure for at leaεt partially preventing reεtenoεiε. 32. The method of claim 1 wherein said agent inhibits interaction between said P-selectin and said ligand so as to at least partially reverse a formed atherosclerotic fatty streak. 33. The method of claim 1 wherein said agent inhibits interaction between said P-selectin and said ligand so aε to at least partially reverse a formed atheroεclerotic intermediate leεion. 34. The method of claim 1 wherein εaid agent inhibitε interaction between εaid P-εelectin and εaid ligand so as to at least partially reverse a formed atheroεclerotic fibrouε plaque. 35. The method of claim 1 wherein εaid adminiεtering occurε prior to formation of an atheroεclerotic leεion. 36. The method of claim 1 wherein said administering occurs subεequent to formation of an atherosclerotic lesion. 37. The method of claim 1 wherein said mammal iε a human, 38. A therapeutic agent in a doεage form and concentration εuitable for treating or preventing atheroεcleroεiε in a mammal in need of εuch treatment, εaid agent being effective to inhibit interaction between P-εelectin and a ligand of P-εelectin.

Inventors: JOHNSON ROBERT C, WAGNER DENISA D, JOHNSON, ROBERT, C., WAGNER, DENISA, D.
Applicants: BLOOD RES CENTER, CENTER FOR BLOOD RESEARCH, INC.
Application year: 1995
Main classification: A61K39
IPC codes: A61K31, A61K33, A61K35, A61K36, A61K38, A61K39, A61K45, A61P9, A61P43, C07K14, C07K16