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(12) United States Patent (10) Patent No.: US 6,838,452 B2 Harats Et Al USOO68384.52B2 (12) United States Patent (10) Patent No.: US 6,838,452 B2 Harats et al. (45) Date of Patent: Jan. 4, 2005 (54) METHODS EMPLOYING AND (58) Field of Search ................................. 558/169, 170, COMPOSITIONS CONTAINING DEFINED 558/172; 514/114 OXIDIZED PHOSPHOLIPIDS FOR PREVENTION AND TREATMENT OF (56) References Cited ATHEROSCLEROSIS U.S. PATENT DOCUMENTS (75) Inventors: Dror Harats, Ramat Gan (IL); Jacob 5,061,626 A * 10/1991 Baldo et al. ................ 435/174 George, Petah Tikva (IL); Gideon OTHER PUBLICATIONS Halperin, Jerusalem (IL) Macpherson et al., 1992, CAS: 117:68162.* (73) Assignee: Vascular Biogenics Ltd., Or Yehuda Wang et al., 1990, CAS: 114:40661.* (IL) Karasawa et al., 1991, CAS:116:39343.* Small et al., 1989, CAS: 111:192750.* (*) Notice: Subject to any disclaimer, the term of this Nitta et al., 1984, CAS: 102:4277.* patent is extended or adjusted under 35 Berchtold et al., 1981, CAS: 95:42295.* U.S.C. 154(b) by 0 days. Cooney et al., 1990, CAS: 113:170033.* * cited by examiner (21) Appl. No.: 10/445,347 Primary Examiner Rita Desai (22) Filed: May 27, 2003 Assistant Examiner Rei Tsang Shiao (65) Prior Publication Data (74) Attorney, Agent, or Firm-G.E. Ehrlich (1995) Ltd. (57) ABSTRACT US 2003/0225035 A1 Dec. 4, 2003 Novel synthetic forms of etherified oxidized phospholipids Related U.S. Application Data and methods of utilizing Same for preventing and treating atherOSclerosis and other related disorders, as well as inflam (63) Continuation-in-part of application No. PCT/IL01/01080, matory disorders, immune mediated diseases, autoimmune filed on Nov. 22, 2001. (60) Provisional application No. 60/252,574, filed on Nov. 24, diseases and proliferative disorders, are provided. In 2000. addition, methods of synthesizing etherified and esterified oxidized phospholipids and of using Same for preventing (51) Int. Cl. ........................... A61K 31/66; CO7F 9/02; and treating atherosclerosis and other related disorders are A61P 9/00 also provided. (52) U.S. Cl. ....................... 514/114; 558/169; 558/170; 558/172 11 Claims, 9 Drawing Sheets U.S. Patent Jan. 4, 2005 Sheet 1 of 9 US 6,838,452 B2 1. CH=CH-CH.) SO-CH, H.O.C.", H.O.C.H. 2. KOH, benzene Hoh anale Hoh assaulture CH, OH CH-O-Tryt" Compound I Compound II CH-O-CH CH-O-Chi Ho (CH)-CH=CH, CHOH/HC-memb- Ho (CH)-CH=CH, CH-O-Tryt' CH-OH Compound III Compound IV 1. Br CHCH-O-POCl, CH-O-Chi 2. N(CH.), -> HC-O-(CH)-CH=CH, OH CH-O-P-OCHCH.N(CH), O Compound V 1. HCOOH+ HO, 2. NaOH CH-O-CH 3. KIO, + HPO, 2 is 33 someonmasama- H-O-(CH)--0 | 9 | CH-O-P-O-CHCH-N(CH), Ot O) Compound VI ALLE "Tryi -C-O) O) Fig. 1 U.S. Patent Jan. 4, 2005 Sheet 2 of 9 US 6,838,452 B2 O 1-Hexadecanoyl-sn-3- H-occul, glycerophosphocholine HC-OH Q Compound I CH-O-P-O-CHCH-N(CH), O CH=CH-(CH)-COOH DCC - DMP CH-O-C-Chi W 1-Hexadecanoyl-2-(5-hexenoyl) HC-O-C-(CH)-CH=CH, sn-3-glycerophosphocholine p. • CH-O-P-O-CHCH-N (CH), Compound II O 1.HCOOH+HO, 2.NHOH 3.NaIO + HPO, O POVPC CH-O-C-CH 1-Hexadecanoyl-2-(5-oxopentanoyl)- H sn-3-glycerophosphocholine tio -(CH)=0p 4. choochon (CH), Compound TV Fig. 2 U.S. Patent Jan. 4, 2005 Sheet 3 of 9 US 6,838,452 B2 - 250 E 3 200 15O 1 OO 5 O O Control PPD ALLE(L+D) Fig. 3 3 V X 5 100 G) - O f Control ALLE(L+D) 10pg ALLE(L+D) 1mg Fig. 4 U.S. Patent Jan. 4, 2005 Sheet 4 of 9 US 6,838,452 B2 160 140 120 1 OO 2 g Control NT 10 ug Fig. 5 U.S. Patent Jan. 4, 2005 Sheet 5 of 9 US 6,838,452 B2 450 E 400 16.4% 350 103. loss loss 300 P-0.001O P-0.001 P=0.002 P=0.001 250 200 150 1OO 5 O O Time 0 Control L-ALLE D-ALE POVPC Fig. 7 U.S. Patent Jan. 4, 2005 Sheet 6 of 9 US 6,838,452 B2 5 10 15 20 25 30 35 40 45 Fraction number Fig. 8 150 100 50 5 10 15 20 25 30 35 40 45 Fraction number Fig. 9 U.S. Patent Jan. 4, 2005 Sheet 7 of 9 US 6,838,452 B2 Figure 10 OH O Compound I SH s e / CHO(CH2)5CH S d Nchochsch, OYN/ O O Ry OCH OCHCH HCO HaCHCO Compound IIa Compound Ib U.S. Patent Jan. 4, 2005 Sheet 8 of 9 US 6,838,452 B2 Figure 11 Aortic Sinus Lesion (um?) 250000 200000 -3.3% 50000 00000 S0000 O P 9S C -20i U.S. Patent Jan. 4, 2005 Sheet 9 of 9 US 6,838,452 B2 IOZ-IO GITTV d. ZI9InãIA ZI-TI “IGITxo(8139e I0?-IOPITTVSAHA -9JNIe?908-13 |01-T US 6,838,452 B2 1 2 METHODS EMPLOYING AND When the involved arteries block the blood flow to the heart, COMPOSITIONS CONTAINING DEFINED a person is afflicted with a heart attack; when the brain OXIDIZED PHOSPHOLIPIDS FOR arteries occlude, the perSon experiences a stroke. When PREVENTION AND TREATMENT OF arteries to the limbs narrow, the result is Severe pain, ATHEROSCLEROSIS decreased physical mobility and possibly the need for ampu tation. This application is a continuation-in-part of PCT/IL01/ Oxidized LDL has been implicated in the pathogenesis of 01080, filed Nov. 22, 2001, which claims the benefit of atherOSclerosis and atherothrombosis, by its action on mono priority from U.S. Provisional Patent Application No. cytes and Smooth muscle cells, and by inducing endothelial 60/252,574, filed Nov. 24, 2000. 1O cell apoptosis, impairing anticoagulant balance in the endot helium. Oxidized LDL also inhibits anti-antherogenic HDL FIELD AND BACKGROUND OF THE associated breakdown of oxidized phospholipids (Mertens, INVENTION A and Holvoet, P, FASEBJ October 2001; 15(12):2073–84). The present invention relates to defined, oxidized LDL This association is also Supported by many Studies demon (OXLDL) components for prevention and treatment of ath 15 Strating the presence of oxidized LDL in the plaques in erosclerosis and related diseases and disorders, as well as various animal models of atherogenesis, the retardation of other inflammatory, immune mediated, autoimmune and atherogenesis through inhibition of oxidation by pharmaco proliferative diseases and disorders and, more particularly, logical and/or genetic manipulations, and the promising to methods and compositions employing oxidized phospho results of intervention trials with anti-oxidant Vitamins (see, lipids effective in inducing mucosal tolerance and inhibiting for example, Witztum J and Steinberg, D, Trends Cardiovasc inflammatory processes. Med 2001 April–May; 11(3–4):93-102 for a review of current literature). Indeed, oxidized LDL and malondialde Cardiovascular disease is a major health risk throughout hyde (MDA)-modified LDL have been recently proposed as the industrialized World. AtherOSclerosis, the most prevalent accurate blood markers for 1 and 2" stages of coronary of cardiovascular diseases, is the principal cause of heart 25 attack, Stroke, and gangrene of the extremities, and as Such, artery disease (U.S. Pat. No. 6,309,888 to Holvoet et al. and the principle cause of death in the United States. Athero U.S. Pat. No. 6,255,070 to Witztum, et al.). Sclerosis is a complex disease involving many cell types and Reduction of LDL oxidation and activity has been the molecular factors (for a detailed review, see Ross, 1993, target of a number of Suggested clinical applications for Nature 362: 801-809). The process, which occurs in treatment and prevention of cardiovascular disease. Bucala, response to insults to the endothelium and Smooth muscle et al. (U.S. Pat. No. 5,869,534) discloses methods for the cells (SMCs) of the wall of the artery, consists of the modulation of lipid peroxidation by reducing advanced formation of fibrofatty and fibrous lesions or plaques, pre glycosylation end product, lipid characteristic of age-, ceded and accompanied by inflammation. The advanced disease- and diabetes-related foam cell formation. Tang et lesions of atherosclerosis may occlude the artery concerned, 35 al., at Incyte Pharmaceuticals, Inc. (U.S. Pat. No. 5,945,308) and result from an excessive inflammatory-fibroproliferative have disclosed the identification and proposed clinical appli response to numerous different forms of insult. For example, cation of a Human Oxidized LDL Receptor in the treatment Shear Stresses are thought to be responsible for the frequent of cardiovascular and autoimmune diseases and cancer. occurrence of atherOSclerotic plaques in regions of the AtherOSclerosis and Autoimmune Disease circulatory System where turbulent blood flow occurs, Such 40 Because of the presumed role of the excessive as branch points and irregular Structures. inflammatory-fibroproliferative response in atherOSclerosis The first observable event in the formation of an athero and ischemia, a growing number of researchers have Sclerotic plaque occurs when inflammatory cells Such as attempted to define an autoimmune component of vascular monocyte-derived macrophages adhere to the vascular injury. In autoimmune diseases the immune System recog endothelial layer and transmigrate through to the Sub 45 nizes and attacks normally non-antigenic body components endothelial space. Elevated plasma LDL levels lead to lipid (autoantigens), in addition to attacking invading foreign engorgement of the vessel walls, with adjacent endothelial antigens. The autoimmune diseases are classified as auto- (or cells producing oxidized low density lipoprotein (LDL). In Self-) antibody mediated or cell mediated diseases.
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