WO2009099553A2 - Use of kinase inhibitor in treatment of atherosclerosis - Google Patents

Abstract

Methods and compositions are provided for the treatment of cardiovascular disease. In the methods of the invention, a small molecule inhibitor of cFMS is administered to a patient in a dose effective in reducing the clinical indicia of atherosclerosis.

Description

USE OF KINASE INHIBITOR IN TREATMENT OF ATHEROSCLEROSIS

GOVERNMENT RIGHTS

[01] This invention was developed in part under NIH Award No. HL030568. The government has certain rights in this invention.

FIELD OF THE INVENTION

[02] Embodiments of the present invention are directed to inhibiting macrophage-colony stimulating factor (M-CSF) cell signaling and, in particular, the diseases caused by such signaling, by inhibiting or otherwise interfering with the biological activity of the M-CSF receptor, the product of the proto-oncogene c-fms.

INTRODUCTION

[03] Cardiovascular disease is a major health risk throughout the industrialized world. Atherosclerosis, the most prevalent of cardiovascular diseases, is the principal cause of heart attack, stroke, and gangrene of the extremities, and thereby the principle cause of death in the United States. Atherosclerosis is a complex disease involving many cell types and molecular factors. The process, in normal circumstances, is a protective response to insults to the endothelium and smooth muscle cells (SMCs) of the wall of the artery, consists of the formation of fibrofatty and fibrous lesions or plaques, preceded and accompanied by inflammation. The advanced lesions of atherosclerosis may occlude the artery concerned, and result from an excessive inflammatory-fibroproliferative response to numerous different forms of insult. For example, shear stresses are thought to be responsible for the frequent occurrence of atherosclerotic plaques in regions of the circulatory system where turbulent blood flow occurs, such as branch points and irregular structures.

[04] The first observable event in the formation of an atherosclerotic plaque occurs when blood-borne monocytes adhere to the vascular endothelial layer and transmigrate through to the sub-endothelial space. Adjacent endothelial cells at the same time produce oxidized low density lipoproteins (LDL). These oxidized LDL's are then taken up in large amounts by the monocytes through scavenger receptors expressed on their surfaces. In contrast to the regulated pathway by which native LDL (nLDL) is taken up by nLDL specific receptors, the scavenger pathway of uptake is not regulated by the monocytes.

[05] These lipid-filled monocytes are called foam cells, and are the major constituent of the fatty streak. Interactions between foam cells and the endothelial and SMCs which surround them lead to a state of chronic local inflammation which can eventually lead to smooth muscle cell proliferation and migration, and the formation of a fibrous plaque. [06] Atherosclerotic plaques can occlude arteries gradually, leading to ischemia. Often, this type of plaque growth occurs gradually over time with a compensatory remodeling of the vessel lumen to maintain adequate perfusion. A large percentage of plaques do not result in what would be considered a significant angiographically measureable occlusion. If such plaques contain a thin fibrous cap and prominent macrophage foam cell core, they are characterized as unstable and prone to rupture. Plaque rupture (via the activity of proteases produced by infiltrating inflammatory macrophages, endothelial/SMC cell apoptosis or hemodynamic events) will trigger a rapid thrombosis as the tissue factor and other pro- coagulation factors present in plaques contact the blood stream resulting in a heart attack (if occurring within the coronary arteries) or stroke. The rupture of unstable plaques can occur with no prior warning or symptoms or can be preceded by unstable angina. Overall, it is important to understand that plaques can lead to vessel occlusion and ischemia (when the vessel can no longer compensate by expanding lumen diameter) or a seemingly modest plaque (in terms of the amount protruding into the lumen) can rupture and trigger a very rapid thrombotic event.

[07] Ischemia is a condition characterized by a lack of oxygen supply in tissues of organs due to inadequate perfusion. Such inadequate perfusion can have number of natural causes, including atherosclerotic or restenotic lesions, anemia, or stroke, to name a few. Many medical interventions, such as the interruption of the flow of blood during bypass surgery, for example, also lead to ischemia. In addition to sometimes being caused by diseased cardiovascular tissue, ischemia may sometimes affect cardiovascular tissue, such as in ischemic heart disease. Ischemia may occur in any organ, however, that is suffering a lack of oxygen supply.

[08] The most common cause of ischemia in the heart is atherosclerotic disease of epicardial coronary arteries. By reducing the lumen of these vessels, atherosclerosis causes an absolute decrease in myocardial perfusion in the basal state or limits appropriate increases in perfusion when the demand for flow is augmented. Coronary blood flow can also be limited by arterial thrombi, spasm, and, rarely, coronary emboli, as well as by ostial narrowing due to luetic aortitis. Congenital abnormalities, such as anomalous origin of the left anterior descending coronary artery from the pulmonary artery, may cause myocardial ischemia and infarction in infancy, but this cause is very rare in adults. Myocardial ischemia can also occur if myocardial oxygen demands are abnormally increased, as in severe ventricular hypertrophy due to hypertension or aortic stenosis. The latter can be present with angina that is indistinguishable from that caused by coronary atherosclerosis. A reduction in the oxygen- carrying capacity of the blood, as in extremely severe anemia or in the presence of carboxy- hemoglobin, is a rare cause of myocardial ischemia. Not infrequently, two or more causes of ischemia will coexist, such as an increase in oxygen demand due to left ventricular hypertrophy and a reduction in oxygen supply secondary to coronary atherosclerosis. [09] There are very limited options on the market today for patients with heart disease. They may be prescribed drugs to lower blood pressure or prescribed statins to improve their cholesterol profile. Very frequently these patients will undergo a coronary bypass or balloon angioplasty to open up a vessel. However, in no way are any of these drugs (including statins) or treatments an actual cure for the disease. Inevitably the bypass will become occluded after a period of years and require a second bypass. The process of inserting a ballon/stent in an angioplasty procedure damages the artery wall and frequently triggers a rapid smooth muscle cell mediated restenosis. New types of drug-coated stents have come on the market to deal with the problem of restenosis. These drug-coated stents are inserted in a very invasive procedure and cannot cure heart disease.

[10] Currently, only a few new drugs are on the horizon for atherosclerosis. One drug works to block cholesterol absorption (Zetia) but was shown to have significant risks in some studies. The others fall into a class of drugs that attempt either to elevate HDL levels ("good cholesterol" that can remove cholesterol from cells and has anti-inflammatory properties) or are peptides components of ApoAI (the primary component of HDL).

[11] Unstable angina is a particular manifestation of atherosclerosis where the methods of the invention would be particularly effective. A patient with chest pain at rest is described as having unstable angina. This condition is very serious, and indicates that the patient has an unstable cornary plaque on the verge of rupture. Such a plaque is highly inflamed and can rupture due to the activity of macrophages within the lesion. Such a patient will be monitored closely, undergo angiography, very likely resulting in balloon angioplasty or even bypass surgery. Short of these highly invasive procedures, few therapeutic options exist for this disease.

[12] M-CSF was first identified as a factor that stimulates monoctye/macrophage colonies in semi-solid bone marrow cultures. It regulates monocyte/macrophage survival, differentiation, proliferation, migration and anti-infective potential by activating a signaling cascade mediated by its receptor, the tyrosine kinase proto-oncogene c-fms. Inflammatory disorders which display elevated levels of M-CSF include arthritis, obesity, and atherosclerosis. The potential involvement of M-CSF in atherosclerosis was suggested by the observation that M-CSF is induced in endothelial cells by treatment with oxidized lipids and is expressed at high levels in atherosclerotic lesions. However, these data do not address whether the molecule has a causative effect in these diseases, and several significant issues remain unresolved as to the role of M-CSF in these diseases. Publications.

[13] Rajavashisth et al. J Clin Invest. 1998; 101 :2702-2710. Murayama et al. Circulation. 1999; 99:1740-1746. Rosenfeld et al. Am J Pathol. 1992; 140:291 -300. Shi et al. Circ Res. 2000;86:1078-1084. Levine et al. J Clin Invest. 1998; 101 : 1557-1564.

[14] US patent 20020176847; US patent application 20060258666; US patent application 20040002145.

SUMMARY OF THE INVENTION

[15] The present invention provides methods and compositions for the treatment of cardiovascular disease, including but not limited to, atherosclerosis, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation, particularly atherosclerosis. In the methods of the invention, a small molecule inhibitor of cFMS is administered to a patient in a dose effective in reducing the clinical indicia of atherosclerosis. In some embodiments the administration is oral. The inhibitor inhibits the ligand mediated signal transduction pathway that results in macrophage migration, survival, differentiation and proliferation, and thus decreases macrophage-mediated inflammation within the artery wall, resulting in decreased atherosclerosis, a more stabile atherosclerotic plaque less prone to rupture, and decreased restenosis after coronary angioplasty procedures.

[16] In some embodiments of the invention the inhibitor has the structure (GW2580, SPG- 1 ):

[17] where R₁ and R₂ are independently selected from H, from a protecting group, e.g. Boc; a cleavable moiety useful in prodrug formulation, including a cleavable oligopeptide, an ester linkage, a cleavable carbohydrate, and the like; and a polymeric carrier, e.g. PEG, PLGA, and the like. R₃ is selected from CH₃, and a polymeric carrier, e.g. PEG, PLGA, and the like. In some embodiments, the inhibitor is GW2580 or a prodrug derivative therefore. Such derivatives may be ester derivatives, where the attached ester substituents include one or more amino or carboxylate groups.

BRIEF DESCRIPTION OF THE DRAWINGS

[18] Figure 1. Histochemical analysis of macrophage content in M-CSF deficient mice. Secondary antibody alone (negative control) (A). Lesional macrophages in female M-CSF^+/+ LDLR^"'^" animals (B) and M-CSF⁺'^" LDLR^"'^" animals (C) were detected with a MOMA-2 antibody (red). (D) Aortic root atherosclerotic lesion area positive for macrophages was measured and expressed as a percentage of intimal thickening area in each respective section.). Lintima, M: media, (magnification 20Ox)

[19] Figure 2. Effect of an M-CSF deficiency on atherosclerotic lesions in the proximal aorta. Lesions in aortic root sections of M-CSF^+/+ and ^+/" mice were quantitated by lipid staining (A) and after bone marrow transplantations (C). (B) Representative lesion sections stained with Movat Pentachrome show collagen in yellow, SMCs in red and proteoglycans in blue. The lesion data are presented as box plots. See Methods for a description of box plots. [20] Figure 3. Inflammatory gene expression is suppressed in atherosclerotic lesions of M- CSF deficient mice. The expression levels of M-CSF, SRA, CSF-1 R and F4/80 were examined by quantitative PCR of aortic RNA from mice after a Western diet. Data are presented as means +/-SEM and 5 to 8 mice of each genotype were tested. [21] Figure 4. Quantitation of apoptosis in the aortic root of M-CSF deficient mice. Apoptotic nuclei (green, indicated with arrows) were detected with a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay in M-CSF +/+ LDLR-/- lesions (A), M-CSF +/- LDLR-/- lesions (B). Apoptotic cells were within areas of macrophage infiltration (see black circles), hintima, M: media, (magnification 400x).

[22] Figure 5. Effect of the M-CSF receptor kinase inhibitor on atherosclerotic lesions in the proximal aorta and on plasma PON activity. Lesions in aortic root sections of LDLR -/- mice on a Western diet treated twice a day with 80mg/kg of the M-CSF receptor kinase inhibitor for 8 weeks were quantitated by lipid staining (A). The lesion data are presented as box plots. See Methods for a description of box plots. Relative plasma PON activity after the 8-week treatment was determined in (B).

[23] Figure 6. Aortic and hepatic inflammatory gene expression is suppressed in LDLR-/- mice treated with the M-CSF receptor kinase inhibitor GW2580. The relative expression level of MMP-9, ICAM-1 , E-Selectin, CSF-1 R, SRA, and F4/80 was examined by quantitative PCR of aortic RNA from LDLR-/- mice on a Western diet treated with GW2580 (A). Aortic uPAR expression was examined in (B) while hepatic F4/80, CSF-1 R, CSF-1 , VCAM-1 , IL-6, and IL-1 beta expression is shown in (C). Data are presented as means +/-SEM and 6 mice from each category were tested.

[24] Figure 7. Effect of an M-CSF deficiency on atherosclerotic lesions in the ascending and thoracic and abdominal aorta. The ascending and thoracic aorta was removed and cleared of all connective tissue before being fixed and pinned out. Lipids were stained for with Oil red O (red). En face staining of the thoracic and abdominal aorta from such animals is shown in (A). The lesion data are presented as box plots (B). Lesions in aortic root sections of M-CSF +/+ and +/- mice were quantitated by lipid staining (C). Lesional macrophages were detected with a MOMA-2 antibody and aortic root atherosclerotic lesion area positive for macrophages was measured and expressed as a percentage of intimal thickening area in each respective section (D). Apoptotic nuclei within areas of macrophage infiltration were detected by a TUNEL assay and quantitated (E-F).

[25] Figure 8. Histochemical analysis of cell proliferation within aortic root lesions of M- CSF deficient mice. Proliferating cells within lesions of female M-CSF +/+ LDLR-/- animals (B) and M-CSF +/- LDLR-/- animals (C) were detected with a Ki67 antibody (red). Nuclei are stained by DAPI (blue). Secondary antibody alone (negative control) in (A). Proliferating cell nuclei in each lesion were counted and normalized by the total number of nuclei in the lesion (D). l:intima, M: media, (magnification 20Ox)

[26] Figure 9. Determination of monocyte/macrophage migration in response to M-CSF. Neutralizing antibodies to M-CSF were added to an artery wall coculture in which oxidized- LDL mediated monocyte migration was measured (A). See Methods for details. Thioglycolate elicited peritoneal cells, highly enriched in macrophages, were quantitated in M-CSF +/+ LDLR -/- and M-CSF +/- LDLR -/- mice (B). A representative example of a leukocyte flow cytometric scattergram for the resident (CD115+/Ly6C-low) and inflammatory (CD115+/Ly6C- high) monocyte subset is shown in (C). Three to five animals per group were examined. [27] Figure 10. Effect of the M-CSF receptor kinase inhibitor GW2580 on monocyte migration. The M-CSF kinase inhibitor was added to an artery wall coculture in which oxidized-LDL mediated monocyte migration was measured in (A) or MCP-1 mediated monocyte migration was measured in (B). The ability of HDL (HDL Exp.) from inhibitor treated mice or control mice (HDL Cont.) to block monocyte migration or LDL to promote migration were measured in (C). See Methods for details. ^*P<0.05

[28] Figure 11. Plasma cholesterol levels in LDLR-/- mice treated with the M-CSF receptor kinase inhibitor GW2580. Total cholesterol levels after 8 weeks of a Western diet were determined in LDLR -/- treated with the cFMS kinase inhibitor.

[29] Figure 12. Quantitation of apoptosis in the aortic root of M-CSF deficient mice. This is an enlargement of Figure 4. Apoptotic nuclei (green, indicated with arrows) were detected with a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay in M-CSF +/+ LDLR-/- lesions (A), M-CSF +/- LDLR-/- lesions (B). Apoptotic cells were within areas of macrophage infiltration (see black circles), hintima, M: media, (magnification 40Ox).

DEFINITIONS

[30] Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein. [31] As used herein, "atherosclerosis" refers to a disease of the arterial blood vessels that results in hardening or furring of the arteries caused by the formation of multiple atheromatous plaques within the arteries. Pathologically, the atheromatous plaque includes nodular accumulation of a soft, flaky, yellowish material at the center of large plaques, composed of macrophages nearest the lumen of the artery, sometimes with underlying areas of cholesterol crystals, and possibly also calcification at the outer base of older/more advanced lesions. The atheromatous plaques, though compensated for by artery enlargement, eventually lead to plaque ruptures and stenosis (i.e., narrowing) of the artery and, therefore, an insufficient blood supply to the organ it feeds. Alternatively, if the compensating artery enlargement process is excessive, then a net aneurysm results. The complications associated with atherosclerosis are chronic, slowly progressing and cumulative. Most commonly, the rupture of a soft plaque causes the formation of a blood clot (e.g., thrombus) that will rapidly slow or stop blood flow, e.g. 5 minutes, leading to death of the tissues fed by the artery. A common recognized scenario is coronary thrombosis of a coronary artery causing myocardial infarction (i.e., a heart attack). Another common scenario in advanced disease is claudication from insufficient blood supply to the legs, typically due to a combination of both stenosis and aneurysmal segments narrowed with clots. Kidney, intestinal and other arteries are also typically involved. [32] As used herein, "subject," "individual," or "patient" refers to any mammal, including humans, bovines, ovines, porcines, canines and felines, in need of treatment. In certain embodiments, the patient is a human. In general, the methods of the invention are applicable to pediatric and adult patients.

[33] The term "concentration in blood," refers to a concentration of an agent obtained in whole blood or in a fluid obtained from blood, such as plasma or serum. [34] As used herein, reference to "variants" or "derivatives" of GW2580 embraces compounds which differ from the structure of GW2580 but which have substantially the same activity as native GW2580.

[35] As used herein, "treatment" or "treating" refers to inhibiting the progression of a disease or disorder, e.g., atherosclerosis, or delaying the onset of a disease or disorder, whether physically, e.g., stabilization of a discernible symptom, physiologically, e.g., stabilization of a physical parameter, or both. As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or condition, or a symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease or disorder and/or adverse affect attributable to the disease or disorder. "Treatment," as used herein, covers any treatment of a disease or disorder in a mammal, such as a human, and includes: decreasing the risk of death due to the disease; preventing the disease of disorder from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; inhibiting the disease or disorder, i.e., arresting its development (e.g., reducing the rate of disease progression); and relieving the disease, i.e., causing regression of the disease. Therapeutic benefits of the present invention include, but are not necessarily limited to, reduction of risk of onset or severity of disease or conditions associated with atherosclerosis.

[36] As used herein, a "therapeutically effective amount" refers to that amount of the compound sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of a compound that provides a therapeutic benefit in the treatment or management of a disease or disorder. Further, a therapeutically effective amount with respect to a compound of the invention means that amount of compound alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease or disorder. The term can encompass an amount that improves overall therapy, reduces or avoids unwanted effects, or enhances the therapeutic efficacy of or synergies with another therapeutic agent.

[37] As used herein, a "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a "pharmaceutical composition" is sterile, and free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, subcutaneous, intradermal, intratracheal and the like. In some embodiments the composition is suitable for administration by an oral route.

[38] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient. Said carrier medium is essentially chemically inert and nontoxic. [39] As used herein, the phrase "pharmaceutically acceptable" means approved by a regulatory agency of the Federal government or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly for use in humans.

[40] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such carriers can be sterile liquids, such as saline solutions in water, or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is an example of a suitable carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The carrier, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin. Examples of suitable pharmaceutical carriers are a variety of cationic polyamines and lipids, including, but not limited to N-(1(2,3-dioleyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTMA) and diolesylphosphotidylethanolamine (DOPE). Liposomes are suitable carriers for gene therapy uses of the invention. Such pharmaceutical compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration.

[41] As used herein, "pharmaceutically acceptable derivatives" of a compound of the invention include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization. The compounds produced may be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs. [42] As used herein, the phrase "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable, essentially nontoxic, acids and bases, including inorganic and organic acids and bases. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[43] As used herein, an "immediate release" formulation of GW2580 refers to a drug composition or mixture of drug compositions in which there is no carrier that regulates the bioavailability of the drug's active ingredient(s) to tissues at the site of drug administration in the patient's body. It will be understood that any component of the formulation that limits or impairs access of the drug's active ingredient(s) to tissues at the site of drug administration in the patient's body is a carrier that regulates the bioavailability of the active ingredient(s) so affected for purposes of the foregoing definition.

[44] "In combination with" as used herein refers to uses where, for example, the first compound is administered during the entire course of administration of the second compound; where the first compound is administered for a period of time that is overlapping with the administration of the second compound, e.g. where administration of the first compound begins before the administration of the second compound and the administration of the first compound ends before the administration of the second compound ends; where the administration of the second compound begins before the administration of the first compound and the administration of the second compound ends before the administration of the first compound ends; where the administration of the first compound begins before administration of the second compound begins and the administration of the second compound ends before the administration of the first compound ends; where the administration of the second compound begins before administration of the first compound begins and the administration of the first compound ends before the administration of the second compound ends. As such, "in combination" can also refer to regimen involving administration of two or more compounds. "In combination with" as used herein also refers to administration of two or more compounds which may be administered in the same or different formulations, by the same of different routes, and in the same or different dosage form type.

[45] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[46] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[47] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[48] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[49] It must be noted that as used herein and in the appended claims, the singular forms "a", "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an individual" includes one or more individuals, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[50] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention. The invention will now be described in more detail.

DETAILED DESCRIPTION OF THE INVENTION

[51] The present invention provides methods and compositions for treating a subject having atherosclerosis. The invention provides a method comprising administering to a subject having atherosclerosis an effective amount of an inhibitor of CSF-1 , including the inhibitor GW2580 or a derivative thereof, wherein the administering is effective to treat atherosclerosis in the subject.

[52] Subjects suitable for treatment with the methods disclosed herein include subjects that suffer from atherosclerosis, particularly those having or at risk of an atherosclerotic disease event, such as stroke, acute coronary syndrome including heart attack, heart disease including congestive heart failure, peripheral artery occlusive disease, and the like. [53] Atherosclerosis is characterized by the deposition of atheromatous plaques containing cholesterol and lipids on the innermost layer of the walls of large and medium-sized arteries. Atherosclerosis encompasses vascular diseases and conditions that are recognized and understood by physicians practicing in the relevant fields of medicine. Atherosclerotic cardiovascular disease including restenosis following revascularization procedures, coronary heart disease (also known as coronary artery disease or ischemic heart disease), cerebrovascular disease including multi-infarct dementia, and peripheral vessel disease including erectile dysfunction are all clinical manifestations of atherosclerosis and are therefore encompassed by the terms "atherosclerosis" and "atherosclerotic disease." [54] The term "atherosclerotic disease event" as used herein is intended to encompass disease events arising from complications associated with atherosclerosis, including but not limited to, coronary heart disease events, cerebrovascular events, an acute coronray syndrome, and intermittent claudication. For example, atherosclerosis of the coronary arteries commonly causes coronary artery disease, myocardial infarction, coronary thrombosis, and angina pectoris. Atherosclerosis of the arteries supplying the central nervous system frequently provokes strokes and transient cerebral ischemia. In the peripheral circulation, atherosclerosis causes intermittent claudication and gangrene and can jeopardize limb viability. Atherosclerosis of an artery of the splanchnic circulation can cause mesenteric ischemia. Atherosclerosis can also affect the kidneys directly (e.g., renal artery stenosis). It is intended that persons who have previously experienced one or more non-fatal atherosclerotic disease events are those for whom the potential for recurrence of such an event exists. [55] Acute coronary syndrome represents a form of acute destabilization of atherosclerotic plaques often caused by plaque rupture that results in acute myocardial ischemia. Acute myocardial ischemia is chest pain due to insufficient blood supply to the heart muscle that results from coronary artery disease (also called coronary heart disease). Patients who have symptoms of acute coronary syndrome may or may not exhibit an ST elevation (also referred to as an ST displacement) by electrocardiogram (ECG or EKG), which is diagnostic of damage to the cardiac muscle or strain on the ventricles. Most patients who exhibit ST- segment elevation in an ECG ultimately develop a Q-wave acute myocardial infarction (i.e., heart attack). Patients who have ischemic discomfort without ST-segment elevation are generally diagnosed as having unstable angina or a non-ST-segment elevation myocardial infarction (the latter of which can lead to a non-Q-wave myocardial infarction). Acute coronary syndrome thus encompasses the spectrum of clinical conditions ranging from unstable angina to non-Q-wave myocardial infarction and Q-wave myocardial infarction.

[56] The method of this invention particularly serves to slow new atherosclerotic lesion or plaque formation, and to slow progression, including stopping progression, of existing lesions or plaques, as well as to cause regression of existing lesions or plaques. In addition, this intervention may accelerate healing of unstable or ruptured plaque.

[57] The methods of the invention contemplate methods for slowing the progression, including stopping progression, of atherosclerosis, including slowing atherosclerotic plaque progression, comprising administering a therapeutically effective amount of GW2580 or a derivative thereof to a patient in need of such treatment. This method also includes slowing progression, including stopping progression, of atherosclerotic plaques existing at the time the instant treatment is begun (i.e., "existing atherosclerotic plaques"), as well as halting or stopping formation of new atherosclerotic plaques in patients with atherosclerosis. [58] The methods disclosed herein also encompass methods for regression of atherosclerosis, including regression of atherosclerotic plaques existing at the time the instant treatment is begun, comprising administering a therapeutically effective amount of GW2580 or a derivative thereof to a patient in need of such treatment. [59] In addition, the methods disclosed herein also encompass methods for slowing atherosclerotic plaque progression and/or accelerating plaque healing so as to reduce the risk of atherosclerotic plaque rupture comprising administering a prophylactically effective amount of GW2580 or a derivative thereof to a patient in need of such treatment, e.g. acute syndrome associated with impending plaque rupture. Rupture as used herein refers to rupture of an atherosclerotic plaque often at the site of a thin fibrous cap, which potentially leads to thrombus formation and an acute event. A further aspect of this invention involves a method for preventing or reducing the risk of developing atherosclerosis, comprising administering a prophylactically effective amount of the compounds described herein to a patient in need of such treatment.

[60] Subject suitable for treatment according to the methods of the invention include those who a medical practitioner has diagnosed as having one or more symptoms of atherosclerosis, and particularly those patients who have had or are at risk of an atherosclerotic disease event. Diagnosis may be done by any suitable means. Methods for diagnosing atherosclerosis by measuring systemic inflammatory markers are described, for example, in U.S. Pat. No. 6,040,147, hereby incorporated by reference. Diagnosis and monitoring may employ an electrocardiogram, chest X-ray, cardiac catheterization, ultrasound (for the measurement of vessel wall thickness), or measurement of blood levels of CPK, CPK- MB, myoglobin, troponin, homocysteine, or C-reactive protein.

[61] One in the art will understand that a patient at risk of development of an atherosclerotic disease event may have been subjected to the same tests (electrocardiogram, chest X-ray, etc.) or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., family history, hypertension, diabetes mellitus, high cholesterol levels, smoking, obesity, etc.).

[62] Usually, atherosclerosis does not produce symptoms until it narrows the interior of an artery by more than 70%. Symptoms depend on location of the narrowing or blockage, which can occur almost anywhere in the body. Symptoms occur because as atherosclerosis narrows an artery more and more, tissues supplied by the artery may not receive enough blood and oxygen. The first symptom of a narrowing artery may be pain or cramps at times when blood flow cannot keep up with the tissues' need for oxygen. Typically, symptoms develop gradually as the atheroma slowly narrows an artery. However, sometimes the first symptoms occur suddenly because the blockage occurs suddenly — for example, when a blood clot lodges in an artery narrowed by an atheroma, causing a heart attack or stroke.

[63] In some embodiments, an effective amount of GW2580 or a derivative thereof reduces the area of an atherosclerotic lesion in an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more than 50%, compared to the area of atherosclerotic lesion in an individual not treated with GW2580. [64] GW2580 suitable for administration includes the structure (GW2580, SPG-1 ):

[65] where R₁ and R₂ are independently selected from H, from a protecting group, e.g. Boc; a cleavable moiety useful in prodrug formulation, including a cleavable oligopeptide, an ester linkage, a cleavable carbohydrate, and the like; and a polymeric carrier, e.g. PEG, PLGA, and the like. R₃ is selected from CH₃, and a polymeric carrier, e.g. PEG, PLGA, and the like. In some embodiments, the inhibitor is GW2580 or a prodrug derivative therefore. Such derivatives may be ester derivatives, where the attached ester substituents include one or more amino or carboxylate groups.

[66] Selection of the therapeutically effective dose can be determined (e.g., via clinical trials) by a skilled artisan, such as a clinician or a physician, based upon the consideration of several factors which will be known to one of ordinary skill in the art. Such factors include, for example, the particular form of GW2580, and the compound's pharmacokinetic parameters such as bioavailability, metabolism, half-life, and the like, which is established during the development procedures typically employed in obtaining regulatory approval of a pharmaceutical compound. Further factors in considering the dose include the disease or condition to be treated, the benefit to be achieved in a subject, the subject's body mass, the subject's immune status, the route of administration, whether administration of the compound or combination therapeutic agent is acute or chronic, concomitant medications, and other factors known by the skilled artisan to affect the efficacy of administered pharmaceutical agents.

[67] In some embodiments, the total pharmaceutically effective amount of GW2580 administered orally per dose will be in the range of about 1 μg/kg/day to about 500 mg/kg/day, including about 10 μg/kg/day to about 200 mg/kg/day, such as, about 40 μg/kg/day to about 100 mg/kg/day, of subject body weight, although, this will be subject to a great deal of therapeutic discretion. In some embodiments, the GW2580 therapy of the invention may be administered to the patient in the form of a single or twice daily administration of an immediate release formulation of GW2580.

[68] Administration of the pharmaceutical compositions of the invention includes, but is not limited to, oral, intravenous infusion, subcutaneous injection, intramuscular, topical, depo injection, implantation, time-release mode, intracavitary, intranasal, inhalation, intralesional, intraocular, immediate release, and controlled release. The pharmaceutical compositions of the invention also may be introduced parenterally, transmucosally (e.g., orally), nasally, rectally, intravaginally, sublingually, submucosally, or transdermally. In some embodiments, administration is parenteral, i.e., not through the alimentary canal but rather through some other route via, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, intraorbital, intracapsular, intraspinal, intrastemal, intra-arterial, or intradermal administration. In some embodiments, the administering of GW2580 is by other than direct administration to the pericardial space. In other embodiments, the administering of GW2580 is by systemic administration. In some of these embodiments, GW2580 is systemically administered by subcutaneous bolus injection.

[69] The present invention further provides methods for treating a subject having atherosclerosis using a pharmaceutical composition of GW2580 or derivative thereof, and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic pharmaceutical compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidylethanolamine (DOPE), and liposomes. Such pharmaceutical compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[70] The pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[71] Pharmaceutical compositions adapted for oral administration may be provided, for example, as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids); as edible foams or whips; or as emulsions. Tablets or hard gelatine capsules may comprise, for example, lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatine capsules may comprise, for example, vegetable oils, waxes, fats, semi-solid, or liquid polyols, etc. Solutions and syrups may comprise, for example, water, polyols and sugars. [72] An active agent intended for oral administration may be coated with or admixed with a material (e.g., glyceryl monostearate or glyceryl distearate) that delays disintegration or affects absorption of the active agent in the gastrointestinal tract. Thus, for example, the sustained release of an active agent may be achieved over many hours and, if necessary, the active agent can be protected from being degraded within the gastrointestinal tract. Taking advantage of the various pH and enzymatic conditions along the gastrointestinal tract, pharmaceutical compositions for oral administration may be formulated to facilitate release of an active agent at a particular gastrointestinal location.

[73] In yet another embodiment, GW2580 may be administered using long-acting GW2580 formulations that either delay the clearance of GW2580 from the site or cause a slow release of GW2580 from, e.g., an injection or administration site. The long-acting formulation that prolongs GW2580 plasma clearance may be in the form of GW2580 complexed, or covalently conjugated (by reversible or irreversible bonding) to a macromolecule such as a water-soluble polymer selected from PEG and polypropylene glycol homopolymers and polyoxyethylene polyols, i.e., those that are soluble in water at room temperature. See, e.g., U.S. Patent No. 5,824,642, hereby expressly incorporated by reference in its entirety. Alternatively, the GW2580 may be complexed or bound to a polymer to increase its circulatory half-life. Examples of polyethylene polyols and polyoxyethylene polyols useful for this purpose include polyoxyethylene glycerol, polyethylene glycol, polyoxyethylene sorbitol, polyoxyethylene glucose, or the like. The glycerol backbone of polyoxyethylene glycerol is the same backbone occurring in, for example, animals and humans in mono-, di-, and triglycerides. The polymer need not have any particular molecular weight. In some embodiments, the molecular weight is between about 3500 and 100,000, or between 5000 and 40,000. In some embodiments, the PEG homopolymer is unsubstituted, but it may also be substituted at one end with an alkyl group. An exemplary alkyl group is a C1-C4 alkyl group, e.g., a methyl group. In some embodiments, the polymer is an unsubstituted homopolymer of PEG, a monomethyl- substituted homopolymer of PEG (mPEG), or polyoxyethylene glycerol (POG) and has a molecular weight of about 5000 to 40,000.

[74] In another aspect, the GW2580 regimen of the invention can be modified to include the use of additional agents for treating atherosclerosis or an atherosclerotic disease event, e.g., stroke, heart attack, heart disease including congestive heart failure, peripheral artery occlusive disease, and the like.

[75] One or more additional active agents, for example but not limited to anti- atherosclerotic agents, may be used in combination with the GW2580 therapy of this invention in a single dosage formulation, or may be administered to the patient in a separate dosage formulation, which allows for concurrent or sequential administration of the active agents. The additional active agent or agents can be lipid altering compounds such as HMG-CoA reductase inhibitors, or agents having other pharmaceutical activities, or agents that have both lipid-altering effects and other pharmaceutical activities. Examples of HMG-CoA reductase inhibitors useful for this purpose include statins in their lactonized or dihydroxy open acid forms and pharmaceutically acceptable salts and esters thereof, including but not limited to lovastatin (see U.S. Pat. No. 4,342,767) (ALTOPREV™); simvastatin (see U.S. Pat. No. 4,444,784) (ZOCOR™); dihydroxy open-acid simvastatin, particularly the ammonium or calcium salts thereof; pravastatin (PRAVACHOL™), particularly the sodium salt thereof (see U.S. Pat. No. 4,346,227); fluvastatin (LESCOL™), particularly the sodium salt thereof (see U.S. Pat. No. 5,354,772); atorvastatin (LIPITOR™), particularly the calcium salt thereof (see U.S. Pat. No. 5,273,995); nisvastatin (PITAVA™), also referred to as NK-104 (see PCT international publication number WO 97/23200); and rosuvastatin (CRESTOR™) (also known as ZD4522, see U.S. Pat. No. 5,260,440).

[76] Additional active agents which may be employed in combination with GW2580 include but are not limited to 5-lipoxygenase inhibitors, HMG-CoA synthase inhibitors; cholesterol ester transfer protein (CETP) inhibitors, for example JTT-705 and CP529.414; squalene epoxidase inhibitors; squalene synthetase inhibitors (also known as squalene synthase inhibitors); acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors including selective inhibitors of ACAT-1 or ACAT-2 as well as dual inhibitors of ACAT1 and -2; microsomal triglyceride transfer protein (MTP) inhibitors; probucol; niacin; bile acid sequestrants; LDL (low density lipoprotein) receptor inducers; platelet aggregation inhibitors, for example glycoprotein llb/llla fibrinogen receptor antagonists and aspirin; human peroxisome proliferator activated receptor gamma (PPARy) agonists including the compounds commonly referred to as glitazones for example troglitazonE (REZULIN™), pioglitazone (ACTOS™) and rosiglitazone (AVANDIA™), including those compounds included within the structural class known as thiazolidinediones as well as those PPARy agonists outside the thiazolidinedione structural class; PPARα agonists such as clofibrate (ATROMID-S™), fenofibrate including micronized fenofibrate, and gemfibrozil; PPAR dual α/γ agonists such as 5-[(2,4-dioxo-5- thiazolidinyl)methyl]-2-methoxy-N-[[4-(trifluoromethyl)ph- enyl]methyl]-benzamide, known as KRP-297; vitamin B₆ (also known as pyridoxine) and the pharmaceutically acceptable salts thereof such as the HCI salt; vitamin Bi₂ (also known as cyanocobalamin); folic acid or a pharmaceutically acceptable salt or ester thereof such as the sodium salt and the methylglucamine salt; anti-oxidant vitamins such as vitamin C and E and β-carotene; β- blockers; angiotensin Il antagonists such as losartan (COZAAR™); angiotensin converting enzyme inhibitors such as enalapril (VASOTEC™) and CAPTOPRIL™; calcium channel blockers such as nifedipine (ADALAT™, PROCARDIA™) and diltiazem (CARDIZEM™, DILACOR™, TIAZAC™); endothelin antagonists such as bosentan, tezosentan, sitaxsentan, enrasentan and ambrisentan; agents that enhance ABC1 gene expression; FXR and LXR ligands including both inhibitors and agonists; bisphosphonate compounds such as alendronate sodium; and cyclooxygenase-2 inhibitors such as rofecoxib (VIOXX™) and celecoxib (CELEBREX™).

[77] Cholesterol absorption inhibitors can also be used in combination with GW2580 of the present invention. Such compounds block the movement of cholesterol from the intestinal lumen into enterocytes of the small intestinal wall, thus reducing serum cholesterol levels. Examples of cholesterol absorption inhibitors are described in U.S. Pat. Nos. 5,846,966, 5,631 ,365, 5,767,115, 6,133,001 , 5,886,171 , 5,856,473, 5,756,470, 5,739,321 , 5,919,672, and in PCT application Nos. WO 00/63703, WO 00/60107, WO 00/38725, WO 00/34240, WO 00/20623, WO 97/45406, WO 97/16424, WO 97/16455, and WO 95/08532. The most notable cholesterol absorption inhibitor is ezetimibe (ZETIA™), also known as 1-(4-fluorophenyl)-3(R)- [3(S)-(4-fl- uorophenyl)-3-hydroxypropyl)]-4(S)-(4-hydroxyphenyl)-2-azetidinone, described in U.S. Pat. Nos. 5,767,115 and 5,846,966.

[78] GW2580 may also be administered in conjunction with one or more additional agents such as anti-inflammatory agents (e.g., non-steroidal anti-inflammatory drugs (NSAIDs; e.g., detoprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate, mefenamic acid, meloxicam, nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib, rofecoxib, aspirin, choline salicylate, salsalte, and sodium and magnesium salicylate) and steroids (e.g., cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone)), antibacterial agents (e.g., azithromycin, clarithromycin, erythromycin, roxythromycin, gatifloxacin, levofloxacin, amoxicillin, or metronidazole), platelet aggregation inhibitors (e.g., abciximab, aspirin, cilostazol, clopidogrel, dipyridamole, eptifibatide, ticlopidine, or tirofiban), anticoagulants (e.g., dalteparin, danaparoid, enoxaparin, heparin, tinzaparin, or warfarin), or antipyretics (e.g., acetaminophen).

[79] Surgical treatment is also envisioned as a combination therapy in conjunction with administration of GW2580. For example, balloon angioplasty can open up narrowed vessels and promote an unproved blood supply. In addition, a metallic stenting element can be inserted and used to permanently maintain the walls of the vessel treated in its extended opened state. Vascular stents are small mesh tubes made of stainless steel or other metals and are used to prop open the weak inner walls of atherosclerotic arteries. They are often used in conjunction with balloon angioplasty to prevent restenosis after the clogged arteries are treated. The blood supply to the heart muscle can also be restored through a vein graft bypass. Large atheromatous and calcified arterial obstructions can be removed by endarterectomy, and woven plastic tube grafts can replace entire segments of diseased peripheral vessels. [80] These secondary therapeutic agents may be administered within 14 days, 7 days, 1 day, 12 hours, or 1 hour of administration of GW2580, or simultaneously therewith. The additional therapeutic agents may be present in the same or different pharmaceutical compositions as GW2580. When present in different pharmaceutical compositions, different routes of administration may be used. For example, a second agent may be administered orally, while GW2580 may be administered by intravenous, intramuscular, or subcutaneous injection.

[81] Kits with unit doses of the subject compounds, usually in oral or injectable doses, are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the GW2580 in treating atherosclerosis.

[82] The present invention may be better understood by reference to the following non- limiting Examples, which are provided only as exemplary of the invention. The following examples are presented to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broader scope of the invention.

EXPERIMENTAL

[83] Briefly, human LDL was incubated with a human endothelial cell/smooth muscle cell coculture. This LDL induces the production of factors, including M-CSF and MCP-1 , that mediate monocyte migration. Subsequently, labeled human monocytes were added to the culture in addition to various doses of GW2580. The monocytes were and allowed to migrate into the coculture. Migrated monocytes were then quantitated microscopically. Human- derived HDL (which inhibits the production of M-CSF and MCP-1 ) and the DMSO carrier were used as positive and negative controls in this experiment. We also tested the ability of GW2580 to decrease monocyte migration in response to direct MCP-1 addition to the culture media. In both cases, GW2580 (in a dose dependant manner) was able to decrease monocyte migration as well as the control HDL.

[84] We also conducted an in vivo animal study using LDL receptor null mice, an established model for human atherosclerosis. GW2580 was dosed orally at 80 mg/kg twice a day for 8 weeks, which resulted in about a 50% reduction in atherosclerotic lesion size with no decrease in lipid levels, body weight or bone density. Plasma paraoxonase activity, a marker of HDL function and anti-oxidant function was significantly increased with treatment. [85] Mechanisms underlying the resistance to atherosclerosis of mice deficient in macrophage colony stimulating factor (M-CSF) were examined. Based on these results, we studied the effects of pharmacological inhibition of M-CSF signaling on atherogenesis using the M-CSF receptor kinase inhibitor GW2580. Lesions of M-CSF^"'^' mice were examined for apoptosis, proliferation, chemotaxis and inflammation. The lesions had increased numbers of apoptotic macrophages but not of proliferating cells. In vitro studies indicated that M-CSF is chemotactic for monocytes. Bone marrow transplantation studies suggested that vascular cell-derived M-CSF is responsible for the effect on atherosclerosis. M-CSF's effect on lesion development was dose dependant, since heterozygous mice had significantly reduced lesions. This suggested that pharmacological inhibition of M-CSF might achieve similar results. We observed that pharmacological inhibition of M-CSF decreased lesion size, arterial and hepatic expression of adhesion factors (ICAM-1 ), macrophage markers (F4/80) and inflammatory cytokines (II-6, 11-1 beta) along with macrophage matrix degradation enzymes (MMP-9), indicating that GW2580 can suppress inflammatory aspects of early, intermediate and advanced atherosclerotic lesions. Vascular cell-derived M-CSF is primarily responsible for lesion formation and mechanisms for M-CSF's effect on atherosclerosis include monocyte recruitment and macrophage survival. GW2580 suppresses inflammation and lesion formation by inhibiting M-CSF signaling within the artery wall.

[86] Based on this improved understanding of atherogenesis in genetically homogenous M- CSF heterozygous mice, in particular the finding that M-CSF exhibited a dose-dependant effect on atherosclerosis, we examined the ability of the selective M-CSF receptor kinase inhibitor GW2580(15) to specifically suppress inflammation and lesion formation in LDLR^''^' mice. Our results indicate that GW2580 is a potent inhibitor of atherosclerosis at concentrations that do not result in the deleterious physiological problems observed with complete inhibition of M-CSF signaling.

Results:

[87] Dose-dependant effects of an M-CSF deficiency in a defined genetic background Interaction of M-CSF Mutation and Genetic Background. To study the mechanism by which an M-CSF deficiency contributes to atherosclerosis, we sought to place the M-CSF null mutation on an inbred genetic background. For this, we repeatedly crossed the mutation, originally on a mixed C57BL/6JxC3HeB/FeJ background, to inbred C57BL/6J mice. We felt this was important since C3H mice are highly resistant to atherosclerosis and nearly a dozen major loci contribute to the differences in susceptibility between C57BL/6 and C3H mice on an apoE null background. After 10 generations of backcrossing, we intercrossed heterozygous mice. Over 120 mice from this cross were genotyped, resulting in a ratio that was greatly skewed from the expected 1 :2:1 ratio, such that approximately equal numbers of ^+/+ and ^+/" mice were observed and only two of the mice were of the ^"'^" genotype. Thus, both the heterozygous and homozygous null genotypes were associated with dramatically reduced viability. Even on a mixed genetic background, M-CSF^"'^" mice had reduced size and weight at birth as well as osteopetrosis and a lack of tooth eruption. Therefore, we chose to use primarily heterozygous mice in this study. Both male and female mice were examined. [88] Atherosclerotic Lesions. Examination of the aortic root by immunostaining clearly revealed a dose-dependant decrease in macrophage content and lesion size in female M-CSF deficient mice, lntimal lesion macrophage content decreased 50% (Fig. 1 ) in female M-CSF⁺'^" LDLR^''^" mice as compared to M-CSF^+/+ LDLR^"Λ mice. Movat staining showed that about 45% of lesion area consisted of matrix components including collagen and proteoglycans (Table I). M-CSF⁺'^' LDLR^"'^" female mice showed a 40% decrease in proximal aorta atherosclerosis (Fig. 2). This decrease was not observed in the thoracic and abdominal aorta of female M-CSF⁺'^" mice, as measured by en face staining of lipids (Fig. 7). We also examined other characteristics of those mice and observed no differences in plasma lipids, circulating monocytes (Table I), overall number of cells per unit of lesion area or in the number of proliferating cells (Figure 8), fibrous caps or prominent lipid cores per aortic root section (Table II).

Table

Values were expressed as mean +/- SEM

Table Il

Values were expressed as mean *l- SEM

[89] M-CSF Derived from Monocyte/Macrophages does not Influence Lesion Development. As discussed above, monocytes/macrophages are capable of abundant M-CSF expression. In order to test whether monocyte/macrophage-derived M-CSF contributed to lesion development, we conducted bone marrow transplantation experiments. We transplanted bone marrow from M-CSF^+/+, ^+/" and ^"'^" donors into lethally irradiated LDLR^"'^" recipients and placed the animals on a 12 week Western diet. No significant differences were detected in aortic root atherosclerosis (Figure 2), plasma M-CSF levels or circulating monocyte counts (Table II) between animals receiving M-CSF ^+/+, ^+/' or ^"'^" bone marrow.

[90] We also performed the reciprocal experiments. Thus, we transplanted M-CSF^+/+ bone marrow into lethally irradiated female M-CSF⁺'^" LDLR^"'^" mice and placed them on the same diet regimen as before. These mice exhibited a 50% decrease in aortic root atherosclerosis (Fig. 2C) and a 35% decrease in plasma M-CSF levels but no decrease in circulating monocyte counts (Table III). We conclude that the levels of artery wall-derived M-CSF are most important in mediating the effect of M-CSF on atherosclerosis and that even a total absence of macrophage-derived M-CSF has little to no impact on atherosclerosis. Table

Values were expressed as mean +/- SEM

ND Not determined

NA- Not aplicable

*P<00009 Plasma M-CSF levels are a combination of values from male and female M-CSF +/- LDLR -/- recipients

[91] Mechanisms contributing to lesion development: inflammatory gene expression. In M-

CSF^+/" LDLR^"'^" mice we detected a 7-fold decrease in M-CSF expression and a 3-fold decrease in SR-A expression (Fig. 3). One would expect a 2-fold decrease in M-CSF expression when examining heterozygous mice but since macrophages express this cytokine themselves, the large decrease we have observed in aortic M-CSF expression probably reflects the decreased macrophage content (Fig. 1 ) and atherosclerosis (Fig. 2) of M-CSF⁺'^" LDLR^"'^" mice. SRA is known to be up-regulated by M-CSF. No significant changes in CD36, KC, and MCP-1 expression were detected.

[92] Effect of M-CSF on Monocyte Migration. We examined the possible role of M-CSF in monocyte recruitment using a monocyte migration assay and a peritoneal macrophage recruitment assay (Figure 9). Addition of an M-CSF neutralizing antibody to an in vitro assay consisting of cocultures of endothelial and smooth muscle cells significantly decreased monocyte migration in response to oxidized lipids while control antiserum had no effect. Consistent with this, peritoneal cavity monocyte recruitment in response to thioglycolate also exhibited a significant decrease in M-CSF⁺'^" LDLR^"'^" animals. These experiments support the idea that M-CSF has a chemotactic role in the artery wall and that haploinsufficiency of M- CSF levels in M-CSF⁺'^" animals can alter monocyte migration in the context of atherosclerosis.

[93] Circulating Monocytes. We did not detect a difference in circulating levels of CD115+ (M-CSF receptor) monocytes or Ly6C+ granulocytes in M-CSF⁺'^" mice on an inbred C57BL/6J background (Figure 9). Nor did we detect a difference in circulating levels of the inflammatory monocyte subset of CD115+/Ly6C-high cells or the more resident CD115+/Ly6C-low monocyte subset.

[94] M-CSF Influences Macrophage Apoptosis in Lesions. The decreased macrophage content we observed in atherosclerotic lesions from M-CSF deficient mice raised the possibility of an effect on macrophage apoptosis. We quantitated apoptosis in lesions by measuring DNA fragmentation using TUNEL staining. The number of apoptotic nuclei varied from 0 to 22 per section in each group and overlapped almost exclusively with lesion areas positive for the macrophage marker MOMA-2 (Fig. 4A-B; Figure 12). Female M-CSF⁺'^' lesion sections did not show an increase in apoptotic cell content relative to lesion. However, after normalizing for the number of apoptotic cells by the percentage of MOMA-2 positive lesion area, we observed approximately two-fold increased apoptosis in M-CSF⁺'^' LDLR^"'^" mouse lesions (p=0.014) (Fig. 4D).

[95] Pharmacologic Inhibition of M-CSF Signaling Decreases Atherosclerosis. Based on the clear dose-dependant inhibitory effect of an M-CSF reduction on lesion formation, we decided to test the effect of blocking M-CSF signaling by inhibiting the intrinsic kinase activity of the M-CSF receptor with a highly selective, orally bioavailable, small molecule kinase inhibitor GW2580. We initially detected the ability of this compound to inhibit LDL or MCP-1 mediated monocyte migration in a dose dependant manner using an artery wall coculture model (Fig. 10). This suggested that there was potential for the compound to be effective in inhibiting atherogensis in vivo. The inhibitor was dosed orally twice a day at 80 mg/kg into LDLR^"'^" female mice for 8 weeks. This dose was chosen based on previous work indicating that free drug levels in the plasma would remain within the IC₅₀ range for suppression of monocyte proliferation while maintaining 100-fold selectivity for kinase inhibition. Lesion size was reduced -40% (p=0.019) and relative plasma PON activity was elevated 28% (p<0.05) (Fig. 5). Using the same coculture model mentioned previously, we observed that HDL from the group treated with GW2580 retained the protective capacity to inhibit LDL mediated monocyte migration while HDL from control mice lost this ability (Fig. 10). No changes in plasma lipid levels were detected (Fig. 11 ).

[96] To better understand the anti-inflammatory effects of GW2580, inflammatory gene expression was determined in the atherosclerotic aorta and the liver. As in the M-CSF⁺'^" mouse lesions, 70% to 80% reductions were observed in the tissue macrophage specific marker F4/80 (p=0.004), the pan-monocyte/macrophage marker CSF1-R (the M-CSF receptor) (p=0.0003), and the scavenger receptor SRA (p=0.006) (Fig. 6A). Matrix metallopeptidase-9 (MMP-9) expression, an enzyme secreted by macrophages that can degrade the matrix and fibrous cap causing lesion destabilization, declined -90% (p=1x10⁴). ICAM-1 and E-Selectin, both factors expressed by the endothelium early in plaque formation and involved in the firm adhesion of monocytes to the artery wall prior to their entry showed 70% to 80% reduced expression in the M-CSF receptor inhibitor treated group. Urokinase plasminogen activator receptor (uPAR), a direct target of M-CSF signaling was significantly down-regulated as well (Fig. 6B). In contrast to the aorta, hepatic CSF-1 R expression tripled (p=0.01 ) while F4/80 levels declined 50% (Fig. 6C), suggesting that while the overall macrophage content of liver decreased, there appears to be a feedback loop linking the inhibition of M-CSF signaling with increased expression of M-CSF receptor in the remaining tissue macrophages. The expression of other key inflammatory mediators and markers of inflammatory macrophages, such as IL-6 and IL-1 beta, decreased by 50%-80% in the liver. Hepatic M-CSF expression itself declined 50% (P=O.0484).

[97] Genetic deficiencies of M-CSF or its receptor are known to affect body composition, particularly by increasing bone density and decreasing adiposity. We wanted to determine if pharmacologic inhibition of this pathway would have similar effects. No significant changes in body weight or body composition as measured by NMR were noted except for a small increase in percent body fat from 19.1% to 22.5% (p=0.014). No changes in bone density as determined by DEXA were noted in the femur or in across the entire body in bone mineral content, bone mineral density or total bone area. We also found no changes in food intake or blood monocyte counts in mice treated with the inhibitor.

[98] Effect of M-CSF on Cell Proliferation. Because M-CSF promotes macrophage proliferation and because it is known that macrophages proliferate within lesions, we quantitated the number of Ki67+ cell nuclei in lesions from M-CSF deficient animals by immunohistochemistry. Ki67 is found in the nucleus in all phases of the cell cycle except G₀, serving as a marker of proliferation. Between 400 and 1000 nuclei from each section, derived from four to six animals per group per sex were examined. We detected an average proliferation rate of 2.38^+/" 0.76% for M-CSF^+/+ female lesion sections (ranging from 0% to 5% proliferating cells/section) and an average rate of 4.27⁺'^"0.73% for M-CSF⁺'^' female lesion sections (ranging from 0 to 8% proliferating cells/section). Our results using Ki67 are similar in magnitude to those previously observed using a BrdU labeling approach. No differences in Ki67+ cells were observed in atherosclerotic lesions between ^+/+ and ^+/" mice of either sex, suggesting that cell proliferation is not significantly affected by the M-CSF deficiency (Fig. 8). Ki67+ cells did appear to cluster near the medial side of lesions as highlighted by arrows. [99] We have confirmed an effect of M-CSF on atherosclerosis using healthy and genetically homogenous M-CSF⁺'^" LDLR^''^" mice. This effect was dependent on decreased artery wall-derived M-CSF, rather than monocyte/macrophage-derived M-CSF, and resulted in decreased lesional macrophage content and increased lesional macrophage apoptosis. Studies with an artery wall model of monocyte migration, along with other histological measurements, suggested that the decreased atherosclerosis in M-CSF deficient mice is due to both decreased monocyte migration into the artery wall and decreased macrophage survival within the artery wall. The fact that the effect of M-CSF on atherosclerosis is clearly dose-dependant also raised the possibility of therapeutic intervention. To this end, we tested the effect of a specific M-CSF receptor kinase inhibitor (GW2580) on atherosclerosis. [100] Studies of M-CSF^"'^" ApoE^"'^" and M-CSF^"'^" LDLR^"'^" animals have revealed dramatic decreases in aortic root atherosclerosis. These studies were conducted using mice on a mixed background that had significant decreases in circulating monocytes and were generally unhealthy, with striking osteopetrosis. The current study used M-CSF⁺'^' LDLR^"'^" animals backcrossed for ten generations to the C57BL/6J background to demonstrate an approximate two-fold decrease in female aortic root lesions. Though not reaching significance, aortic root lesions in male M-CSF⁺'^" mice trended strongly toward a similar decrease in lesion size while showing an actual decrease in lesions when measured by an en face approach. Sex-specific and region-specific differences in lesion susceptibility are frequently observed in mouse atherosclerotic lesion studies. These data demonstrate that decreasing M-CSF levels results in decreased atherosclerosis but that the interplay of complex factors outlined above are responsible for the variations observed between males and females. The differences in lesion size observed in previous studies may well be attributable to the mixed genetic background, since the C3H background imparts a powerful resistance to atherosclerosis.

[101] Accompanying the decreased atherosclerosis was decreased arterial expression of a panel of inflammatory genes (Fig. 3). M-CSF expression was substantially decreased in M- CSF⁺'^" mice, but by far more than 50% that would be expected from the decreased female MOMA-2 staining. Macrophages themselves can express M-CSF and a significant decrease in macrophage content was noted in M-CSF ^+/" lesions. Also, decreased expression of SR-A, a macrophage scavenger receptor up regulated in response to M-CSF, was noted.

[102] We distinguish the effects of a macrophage specific M-CSF deficiency on atherosclerosis, as opposed to an artery wall specific deficiency on atherosclerosis, using bone marrow transplantations. These experiments demonstrate that artery wall derived M- CSF is the primary source of circulating M-CSF and is the key source of M-CSF as it relates to atherosclerosis. Infusion of a neutralizing antibody to the M-CSF receptor in ApoE^"'^" mice resulted in significantly decreased atherosclerotic lesions despite no significant differences in circulating monocytes. Reducing signaling through the M-CSF receptor (primarily expressed on mononuclear phagocytes) by decreasing artery wall derived-M-CSF or blocking M-CSF receptor ligand binding results in decreased atherosclerosis.

[103] We have shown a specific role for M-CSF in monocyte migration in the context of an actual model of the artery wall by neutralizing oxidized LDL stimulated endothelial and smooth muscle cell derived M-CSF. Our results show that M-CSF also has a significant role in this process and it is possible that M-CSF and MCP-1 signaling pathways interact in the monocyte or that M-CSF itself can induce endothelial cell derived MCP-1. We also showed that M- CSF⁺'^" LDLR^"'^" animals have significantly fewer thioglycolate elicited peritoneal cells, a population of cells rich in macrophages, suggesting an in vivo role for M-CSF in macrophage migration.

[104] The peripheral leukocyte surface markers CD115(M-CSF receptor) and Ly6C have been used to define two different populations of monocytes. CD115+/Ly6C-high expressing cells represent monocytes that are prone to migrate to sites of inflammation, including atherosclerotic lesions. CD115+/Ly6C-low monocytes, on the other hand, tend to become resident tissue macrophages rather than migrating in response to inflammatory stimuli. We observed no change in the frequencies of either sub- population of monocytes or in the overall frequency of CD115+ circulating monocytes in M-CSF⁺'^" LDLR^"'^' mice as compared to M- CSF^+/+ LDLR^"'^" mice, suggesting that M-CSF does not alone strongly regulate these aspects of monocyte development. The data also suggest that the decreased atherosclerosis observed in M-CSF deficient mice is not attributable to fewer circulating monocytes. [105] Small molecule kinase inhibitors frequently do not inhibit 100% of their target's function. This suggests that a heterozygous deficiency of a target gene represents a more realistic model of the effects of an inhibitor and the mechanism underlying such effects. We obtained a better understanding of what effect a 50% reduction of M-CSF has on suppressing atherogenesis using heterozygous mice. That understanding led us to test GW2580, a compound that has demonstrated to have at least 100-fold greater inhibitory capacity for the M-CSF receptor kinase relative to an array of related kinases such as the PDGF and VEGF receptor tyrosine kinases. GW2580 was administered at a dose required to maintain free plasma levels (unbound to albumin) of the inhibitor in the range of 100 nM, near the IC₅₀ for inhibiting monocyte growth with the assumption that albumin associated compound having very limited bioactivity. At the dose used, GW2580 suppressed atherogenesis to about the same extent observed in M-CSF heterozygous mice (Figure 2A). Although other kinase inhibitors including imatinib and SU11248 can inhibit M-CSF receptor kinase activity, they also inhibit several other kinases and are in no way selective for that target.

[106] We also observed that GW2580 could suppress monocyte migration in an artery wall coculture model and that HDL from treated LDLR^"'^' mice retained the ability to block LDL mediated monocyte migration while HDL from control LDLR^"'^" mice lost this protective capacity (Fig. 10). This phenomenon presumably occurs as a result of elevated inflammatory state engendered by a high fat diet in atherosclerotic mice and is associated with the displacement of PON1 and platelet-activating factor acetyl hydrolase (PAF-AH) enzymes from HDL particles. Both PON 1 and PAF-AH have the ability to inactivate oxidized lipids. The ability to suppress lesion formation, improve the anti-oxidant capacity of HDL, as well as reduce the overall inflammatory state while not altering lipid levels, body composition or bone density demonstrates that pharmacologic inhibition of M-CSF signaling can selectively suppress inflammation and lesion formation, properties that are desirable when used in human CAD. These results also show that such an inhibitor would work in a synergistic or complementary manner with lipid lowering therapies such as statins or HDL elevating therapies such as niacin. Furthermore, the ability of GW2580 and M-CSF/M-CSF receptor kinase inhibitors to decrease monocyte migration to sites of inflammation suggests their possible use in other disease states such as restenosis.

Materials and Methods

[107] Animal Husbandry. M-CSF heterozygous mice on a C57BL/6JxC3HeB/FeJ mixed genetic background were purchased from the Jackson Laboratory and backcrossed for ten generations to C57BL/6J mice. M-CSF heterozygous mice on an inbred genetic background were then crossed to LDLR^"'^" mice on a C57BL/6J background to produce double heterozygous mice that were again crossed to LDLR^"'^' mice. M-CSF⁺'^' LDLR^"'^" mice were selected and intercrossed to produce M-CSF^+/+, ^+/" and ^"'^" mice on an LDLR null background. However, only two M-CSF^"'^' LDLR^"'^" mice on the inbred C57BL/6J background were ever observed at weaning and, therefore, only M-CSF^+/+ and ^+/" inbred animals were used for subsequent experiments. At approximately 10 weeks of age the mice were placed on an ad libitum Western type diet containing 42% fat, 0.15% cholesterol, and 19.5% casein without sodium cholate (TD 88137, Teklad, Madison, Wl) for 13 weeks before sacrifice. [108] Genotyping. In order to produce M-CSF^"'^" mice as donors for bone marrow transplantation, we transferred the M-CSF null mutation onto a mixed genetic background that still maintained a b H2 haplotype, allowing tissue transplant compatibility with the C57BL/6J strain. M-CSF⁺'^" animals on the C57BL/6J background were crossed to the inbred 129T2/SvEMsJ strain (Jackson Laboratory) to create mice that preserved the b H2 haplotype on a mixed genetic background. M-CSF⁺'^" animals were selected and intercrossed to produce M-CSF^+/+, M-CSF⁺'^" and M-CSF^"'^" animals on the mixed background. M-CSF^+/+ and M-CSF⁺'^" animals were maintained on a chow diet but M-CSF^"'^", which lack teeth, were maintained on a powdered chow diet. The region containing the single base pair insertion creating the M-CSF null allele was amplified by fluorescent PCR using the following primers: 5'-CGC ATG GTC TCA TCT ATT ATG TCT TG-3' and 5'-CTG CTC CTC ATA GTC CTT GG-3'. The products (153 bp for the normal allele and 154 bp for the mutant allele) were resolved on an ABI Genotyper.

[109] M-CSF Receptor Kinase Inhibitor Synthesis. GW2580 was synthesized as previously described. Purity was greater than 98.6% as determined by HPLC analysis. Further characterization and validation of the compound structure was done by NMR and mass spectrometry as well as elemental analysis to rule out contaminating elements. GW2580 was added to a carrier solution of 0.5% hydroxymethycellulose/0.1 % Tween 20 prior to oral administration of 80mg/kg twice a day for 8 weeks to 8-week-old LDLR ^"'^" female mice purchased from the Jackson Laboratory.

[110] Plasma Lipid, Glucose, and M-CSF Levels. Animals were fasted overnight before being bled from the retroorbital sinus. Plasma was collected and used to determine total cholesterol and HDL cholesterol levels. Plasma paraoxonase activity and lipids were determined as described previously. Plasma glucose was determined in triplicate using a commercial kit (#315-100; Sigma). Plasma collected at time of sacrifice was used to measure circulating levels of M-CSF by ELISA (R&D Systems).

[111] Quantitation of Atherosclerosis, Plaque Composition and Immunohistochemistry. Methods for the quantitation of atherosclerotic lesions in the aortic root were as previously reported. Briefly, the heart and proximal aorta were excised and embedded in OCT compound (Tissue-Tek) before freezing. Serial 10 μm thick cryosections from the middle portion of the ventricle to the aortic arch were collected and mounted on poly~lysine-coated slides. In the region from the appearance to the disappearance of the aortic valves, every other section was collected. In all other regions, every fifth section was collected. Sections were stained with oil red O and hematoxylin, counterstained with fast green, and lesion areas quantitated by light microscopy. Additionally, the ascending and descending aorta (to the diaphragm) was dissected, cleaned of connective tissue and fixed. The aortas were then pinned out en face and stained with Oil red O as in Tangirala et al. Lesion surface area and total aortic surface area were measured using Image Pro Plus (Media Cybernetics). Additional cryosections from the heart and proximal aorta were stained for plaque composition by a Movat pentachrome stain, macrophages (rat anti-mouse MOMA-2, BD Biosciences) and proliferating cells (rabbit- anti mouse Ki67, Abeam). Three to four sections from four to six animals per group were analyzed. Sections were dried at room temperature, fixed in 1 % PFA (for MOMA-2) or acetone (for Ki67). Sections were then blocked in 5% serum appropriate for the biotinylated secondary antibody used (rabbit anti-rat or horse anti-goat). Staining was detected using the Alkaline Phosphatase Standard ABC Kit (Vector Labs) and Vector Red as a substrate that fluoresces in a spectrum similar to Rhodamine. Sections were counterstained with DAPI and imaged on a Zeiss Axioskop 2 florescent microscope. Omission of the primary antibody was included as one control to determine staining specificity.

[112] Peritoneal Macrophage and Monocyte Migration Assays. Mice were injected intraperitoneal^ with 1 mL of 3% brewers yeast thioglycolate four days before sacrifice and cells were isolated by flushing the peritoneal cavity with cold PBS. Red blood cells were lysed and peritoneal cells counted with a hemocytometer. Monocyte migration was determined using a human artery wall co-culture model. Briefly, an aliquot of human LDL or HDL cryopreserved in sucrose from a normal donor (standard LDL) was incubated with an endothelial cell/smooth muscle cell coculture to which GW2580 (dissolved in DMSO) or neutralizing monoclonal antibodies to human M-CSF, MCP-1 , and G-CSF were added. All antibodies were monoclonal mouse anti-Human (R&D Systems). Subsequently, di-l labeled human monocytes were added and allowed to migrate into the coculture. Migrated monocytes were then quantitated microscopically and expressed as the mean of 9 high-power fields counted in triplicate. Pre-immune serum, an irrelevant antibody and anti-granulocyte colony stimulating factor (G-CSF) antibodies, were used as negative controls. Human-derived HDL and an anti-macrophage chemotatic protein 1 (MCP-1 ) were included as controls for decreasing monocyte migration.

[113] Apoptosis Quantitation. The ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon), utilizing a terminal deoxynucleotidyl transferase (TdT) based technique, was used to quantitate apoptosis within atherosclerotic lesions of the aortic root. Five to six 5μm cryosections sections from six to eight animals of each genotype and sex were selected and dried overnight at room temperature before using the assay. Lesions were examined at 20Ox magnification for apoptotic nuclei and over 150 TUNEL+ nuclei for each genotype were detected. Positive nuclei were confirmed by colocalization with nuclear DAPI staining at 40Ox magnification.

[114] Bone Marrow Transplantation Bone marrow was flushed from the femurs of M-CSF^+/+ and M-CSF⁺'^' mice on the C57BL/6J background and from the femurs of M-CSF^+/+ and M- CSF^"'^" mice on a mixed C57BL/6J x 129T2/SvEMs J background. Eight to ten week old M- CSF^+/+ LDLR^"'^' and M-CSF⁺'^" LDLR^"'^" were lethally irradiated and injected via the tail vein with 10⁷ bone marrow cells as previously described. Five weeks after transplantation the animals were placed on a Western-type diet for 12 weeks at which time blood was collected for automated differential monocyte counting and plasma lipid levels determined. Leukocyte DNA was isolated to confirm the presence of the M-CSF null allele or the presence of the SRY gene by PCR as appropriate.

[115] Flow Cytometry Blood from four to five mice of each genotype was collected from the retroorbital sinus followed by red blood cells lysis. The remaining leukocytes were incubated with an anti- mouse CD16/CD32 Fc gamma lll/ll receptor 2.462 blocking antibody (BD Pharmingen) prior to double staining with a PE labeled anti-mouse CD115 antibody (eBioscience) and a FITC labeled anti-mouse Ly6C antibody (BD Pharmingen). Both unstained singly stained leukocyte control cells were used to determine the appropriate gating parameters. 100,000 cells were counted using a BD FACS scanner running CellQuestsoftware.

[116] Quantitation of Inflammatory Gene Expression The ascending and descending aorta was isolated from animals at the time of sacrifice. Connective tissue and adventitia were dissected away from the aorta prior to total RNA isolation using an RNEASY kit (Qiagen). First strand cDNA synthesis was performed using an iScript cDNA Synthesis Kit (Biorad). Realtime quantitative PCR was performed on an Applied Biosystems 7700 unit using Platinum SYBR Green qPCR Supermix UDG (Invitrogen). Samples were analyzed in duplicate and normalized to beta(2)-microglobulin expression. [117] Statistical Tests Data were expressed as mean ^+/" SEM. Statistical analyses were performed using the non-parametric Mann Whitney test (Statview) for all comparisons. Lesion data were displayed as a box plot with the whiskers representing the maximum and minimum data points, the central line representing the overall median, the bottom of the box representing the median of the first quartile of the data set and the top of the box representing the median third quartile of the data set. Open circles represent outliers.

Example 2

Modification and Improvement of SPG-1 (GW-2580)

[118] Many therapeutic compounds exhibit significant lipophilic properties that are important in allowing them to permeate cell membranes. However, these lipophilic properties may result in poor oral bioavailability, poor solubility in aqueous solutions, and a high affinity for binding plasma proteins. In some cases, solubility is a factor of pH in buffered solutions or can vary between the free base and salt form of a compound, and thus the pH and buffer are modified to optimize delivery. Compound absorption is also increased by milling the material into micronised particles of around about 5 to about 25 μm in diameter, e.g. around about 10 μm in diameter.

[119] GW2580 can also be formulated in a combination with lactic and glycolic acid (PLGA) to provide microspheres with improved solubility and sustained drug release. [120] GW2580 is conjugated to improve pharmacodynamic properties. A synthetic scheme to conjugate monomethoxy-polyethylene glycol-polylactic acid is shown below:

[121] GW2580 can also be modified by conjugation of an esterase, protease, phosphatase cleavable moiety to the drug, e.g. as shown below, where R is an ester, cleavable oligopeptide, cleavable polysaccharide, and the like. The compound is activated to the active compound by cleavage of the group.

Claims

What is claimed is:

1. A method of treating inflammatory cardiovascular disease, the method comprising: administering an effective dose of a cFMS inhibitor.

2. The method of Claim 1 , wherein the inflammatory cardiovascular disease is atherosclerosis.

3. The method of Claim 1 , wherein the cFMS inhibitor has the structure:

where R₁ and R₂ are independently selected from H, from a protecting group; a cleavable moiety; and a polymeric carrier;

R₃ is selected from CH₃, and a polymeric carrier.

4. The method of Claim 3 wherein the inhibitor is