OA12431A - Oxidized protein, their biological activity, and therapeutic and diagnostic measures, which are derived from the active mechanism, from the use of these proteins or from the inhibition thereof. - Google Patents

Abstract

Medicaments (A and B) for use in humans or animals are new. (A) contains an agent (I) which inhibits the binding of oxidized proteins to CD36 or the functions of CD36 induced by oxidized proteins. (B) contains at least one oxidized protein (II) or oxidized peptide (or a structural analog or mimetic) : An independent claim is included for the preparation of oxidized proteins/peptides, involving reaction with hypochlorous acid or peroxynitrites. ACTIVITY : Anticoagulant; Thrombolytic; Antiinflammatory; Immunosuppressive; Antiarteriosclerotic; Hypotensive; Nephrotropic; Vasotropic; Cardiant; Cerebroprotective; Antidiabetic; Vulnerary; Antibacterial; Anti-HIV; Cytostatic; Antirheumatic; Antiarthritic; Antiulcer; Dermatological. MECHANISM OF ACTION : CD36 antagonist; CD36 agonist.

Description

012431

Oxidized Proteins, their biological activity, and therapeutic and diagnostic measures, which are derived from the active mechanism, from the use of these proteins and from the inhibition thereof 5

Description

The présent invention relates to substances that inhibit the binding of oxidized proteins toCD36 or that inhibit fonctions of CD36 induced by the interaction of CD36 with oxidized 10 proteins, and their use as a médicament for humans and animais.

Oxidative modification of proteins is regarded as a critical step for the pathogenesis of variousdiseases, which range from atherosclerosis (arteriosclerosis) and neurodegenerative diseasesto the process of aging itself (Holvoet and Collen, 1997 Curr Opin Lipidol, Witztum and 15 Steinberg 1991, J Clin Invest 88, 1785-92, Smith MA et al., 1996, Nature, Oxidative damagein Alzheimers, Stadtmann ER, Protein Oxidation and Aging, 1992, Science 257: 1220-24).High LDL concentrations in the blood are considered to be the major risk factor for thedevelopment of arteriosclerotic vesseî diseases (Brown MS, Goldstein IL, 1986, Science 232:34-37). 20

However, today there is overwhelming evidence to believe that not LDL itself but its oxidizedform is the décisive trigger for changes leading to diseases that lead to arteriosclerosis(Steinberg D, Circulation 95: 1062-71, 1997). U.S. Patent No. 5,756,067 discloses thatmeasurement of cholesterin, triglycérides, and lipoproteins, as risk markers for developing 25 · arteriosclerosis is not sufficient/ because approximately half of ail heart diseases based onarteriosclerosis is présent in patients who show normal plasma triglycéride and normalcholesterin values, and because arteriosclerosis can also be demonstrated angiographically inpatients with normal lipid values. Therefore, processes that hâve not yet been published mustplay a causal rôle in the development of arteriosclerosis. 30

Oxidation of lipids in LDL, eithet ïn vitro, e.g. by copper induced oxidation, or in vivo, leads to the formation of reactive aldéhydes (WO 98/59248). Uptake of oxidized LDL (oxLDL) by macrophages leads to the formation of so-called foam cells, a process which is regarded as initial step in the development of arteriosclerosis (WO 98/59248). 012431 r

Oxidation of the lipid portion of LDL is regarded as responsible for this process. Therefore,oxLDL is induced by almost ail scientists in order to investigate the formation ofarteriosclerosis, by the addition of CuSOî or malonedialdehyde, an end product in lipid 5 peroxidation. The concentration of oxLDL in plasma of patients with coronary primary heartdisease or transplantation associated coronary heart disease corresponds closely to theprogression of the disease, while in healthy control persons no elevated oxLDL levels caa bemeasured (Holvoet et al., Circulation 98: 1487-94, 1998). Also chrome kidney diseases andtransplant rejections are associated with high oxLDL levels (Holvoet, Collen Thromb 10 Haemost 76(5): 663-9, 1996 + ATVB 18(1) 100-7,1998).

Also oxidations of the protein portion of LDL can lead to physiological/pathophysiologicalmodifications. Thus, delipidated HOCl-oxLDL induces oxidative burst in macrophages(Nguyen-Khoa et al., BBRC 263: 804-9, 1999) and HOCl-oxLDL leads to thrombocyte 15 aggregation (Volf et al., ATVB 20(8): 2011-18,2000).

Reactive oxidants can be released from the body by phagocytes and play a décisive rôle in thedefense against pathogenic agents, tumor monitoring and ail inflammatory processes (Babior,NEJMed 298: 659-663, 1978, Weiss J, NEJMed 320: 365-376, 1989). Besides "professional” 20 phagocytes, like granulocytes and monocytes, other cells like e.g. endothélial cells or smoothmuscle cells also produce and release reactive oxidants.

Among these oxidative substances are O2, superoxide, hydrogen peroxide, peroxynitrite, QH-radicals, hypochloric acid HOC1, Cl2-gas and chloramine. It remains unclear which processes 25 in detail contribute to the development of these oxidative substances. Céruloplasmine, 15-lipooxygenase, myeloperoxidase (MPO) and nitric oxide synthase (NO-S) were found inarteriosclerotical lésions in animais and humans and may contribute to oxidation of LDL(Carr et al., ATVB 20:1716-23, 2000). A possible oxidation pathway involves MPO. MPO, aheme-protein enzyme can halogenate and peroxidate (Carr et al.,). The best-described product 30 of myeloperoxidase is hypochloric acid HOC1', Cl' + H2O2 + H+ -> HOC1 + H2O.Hypochlorite-modified proteins, in particular HOCl-oxLDL, are found in arterioscleroticallésions (Hazell et al., 1996). HOC1 modification of proteins also plays a rôle in other diseases,e.g. inflammatory diseases of the joints (Davies et al., Free-Radical-Biol-Med 15(6): 637-43, . 1993), coagulation disorders (oxidation of thrombomodulin) (Glaser et al., J Clin Invest 3 01 243 1 { 90(6): 2565-73, 1992), tissue destruction mediated by granulocytes in inflammatory reactionsin general (Schraufstatter et al., J Clin Invest 85(2): 554-62, 1990), ischemia, reperfusiondamage (Samanta et al., Fee Radie Res Commun 7(2): 73-82, 1989), glomerulonephritis(Shah et al., J Clin Invest 79(1): 25-31, 1987), and immune régulation (NK-cell-apoptosis) 5 (Hansson et al., J Immunol 156(1): 42-7, 1996). CD36, also named glycoprotein Illb (GPIIIb) or glycoprotein IV (GB IV), is a majorglycoprotein of platelets, endothélial cells, monocytes, erythroblasts, épithélial cells, andsome tumor cell lines such as melanoma cells and osteosarcoma cells (Asch et al J. Clin. 10 Invest. 79:1054-1061 (1987), Knowles et al, J. Immunol. 132, 2170-2173 (1984), Kieffer etal, Biochem. J. 262:835-842 (1989)). CD36 belongs to the family of class B scavengerreceptors. Members of the family also include the intégral lysosomal membrane proteinLIMP-II (lysosomal intégral membrane protein II, Vega et al, 1991), the CLA-1 (CD36 andLIMP-II analogous, Calvo and Vega 1993), the FAT protein of adipocyte membrane 15 (Abumrad et al, 1993), PAS IV from breast épithélial cells (Greenwalt et al, 1990 and 1992),and SR-B1 (Acton et al, (1994). FAT protein of adipocytes is involved in binding and transportation of long chain fatty acids.PAS IV protein is an intégral membrane protein of lactating breast épithélial cells, and is 20 concentrated in apical plasmalemma. With sécrétion of triglycérides, it reaches the milk and isfound in the milk fat globule membrane (MFGM) fraction. The sequence of PAS IV is almostidentical to CD36, but there are différences in glycosylation. SR-B1 is a scavenger receptor for LDL (Acton et al, 1994). CD36 consists of a single heavy 25 glycosylated polypeptide chain with an apparent molecular weight of 88,000 in reduced andnon-reduced condition, and has an isoelectric range between 4.4 and 6.3 (McGregor et al,1980, Clementson 1987). The reason for not being able to détermine a clearly definedisoelectric point is the variable content of sialylic acid (McGregor et al, 1981). Thecarbohydrate portion of 26 % and a strong hydrophobicity provides CD36 with a high 30 résistance towards dégradation by proteases as long as the protein is located in the membrane (Greenwalt et al, 1992). This explains the observation that the protein is protected from attacks in régions in which inflammatory processes take place. CD36 has N- and O-linked

glycosidic modifications. The amino acid sequence for CD36 derived from placenta cDNA 012431 (Oquendo et al., 1989) shows multiple hydrophobie régions and two presumablytransmembrane régions.

Certain fonctions hâve been postulated for CD36. It has been described as a receptor forcollagen (Tandon et al., 1989). Purified CD36 binds to fibrils of collagen type I, and Fabfragments of a polyclonal antibody directed against CD36 inhibit collagen-inducedaggregation.

However, analysis of platelets, which lack CD36, show that CD36 is not strictly required foractivation of platelets by collagen. Our joint experiments with colleagues of the group of J. J.Sixma (Utrecht) showed no différence in adhesion of CD36-deficient platelets and controlplatelets to bovine or human collagen type I or ΙΠ in a static System or under the influence oflow, intermediate, and high shear rates in a perfusion chamber when using heparin blood andphysiological Ca2+ concentrations (Saelman et al., 1994). CD36 déficient platelets aggregate normal with hom collagen, a mixture of equine type I andtype III collagen, and with purified bovine and human collagen type I and III (Kehrel et al.,1991 and 1993). The sécrétion of α-granulae and dense granulae induced by type I or ΙΠcollagen is not different in CD36 déficient platelets and control platelets (Kehrel et al., 1993).Daniel and coworkers showed that the signal transduction after activation with collagen type Iin CD36 déficient platelets and control platelets is equal (Daniel et al., 1993). CD36 is a receptor for thrombospondin-1 (TSP-1) (Asch et al., 1987, McGregor et al., 1989).On resting, thrombocyte CD36 threonine (92) is phosphorylated. Déphosphorylation allowsthe binding of thrombospondin (Asch, Science 1993). Purified CD36 binds specifically tothrombospondin. This binding is Ca2+-dependent and cannot be inhibited by RGE peptides.The monoclonal antibody 0KM5, which is directed against CD36, inhibits binding ofthrombospondin to platelets activated by thrombin (Asch et al., 1989). Leung and coworkersreported that two peptide régions on CD36 influence binding of thrombospondin. Peptide139-155 enhances platelet aggregation in platelet-rich plasma, which has been induced byADP or collagen. However, peptide 93-110 partly inhibits collagen-induced aggregation, andalso blocks binding of CD36 to immobilized thrombospondin. This peptide is not able to bindthrombospondin on its own, but can in the presence of peptide 139-155 (Leung et al., 1992,Pearce et al., 1993). The sequence SVTCG of thrombospondin binds to CD36 with high 5 012431 ( affinity (Li et al., 1993). Silverstein et al., (1992) demonstrated the relevance of CD36 forthrombospondin binding by experiments with “sense” and “antisense” transfected melanomacells. The binding site for thrombospondin on CD36 is between amino acids 93-120 (Frieda etal., 1995). 5 CD36 is also described as a binding mediator between platelets, endothélial cells, monocytes,or C32 melanoma cells on the one hand and érythrocytes infected with malaria parasitePlasmodium falciparum on the other hand (Bamwell et al., 1989). Binding of infectedérythrocytes to Caterpillar endothélial cells, called séquestration, is of décisive significance for 10 the often deadly end of malaria tropica, if séquestration takes place in the brain (célébraimalaria). Infected érythrocytes bind to immobilized purified CD36 (Ockenhouse et al., 1989).COS cells in which cDNA for CD36 is expressed are capable of binding to malaria-infectedérythrocytes (Oquendo et al., 1989). With the help of anti-idiotype antibodies against themonoclonal anti-CD36 antibody OKM5, a binding partner on infected érythrocytes, the 15 sequestrine was found (Ockenhouse et al., 1991). Contact with infected érythrocytes activâtesplatelets and monocytes (Ockenhouse et al., 1989). CD36-deficient platelets do not show anybinding capability for infected érythrocytes in the hands of Tandon et al., (1991). In contrast,we hâve observed that binding is only disrupted in the presence of EDTA. In the presence ofCa2+ and Mg2+ we could clearly observe rosetting of Plasmodium falciparum-infected 20 érythrocytes and CD36-deficient platelets (Kronenberg et al., 1992). Besides CD36, furtherbinding mediators for malaria infected érythrocytes hâve been described and among these arethrombospondin, which needs bivalent cations for this fonction (Berendt et al., 1989, Robertset al., 1985). Thrombospondin might be able to médiate the binding of CD36-deficientplatelets to infected érythrocytes, which was observed by us. Binding of Plasmodium 25 falciparum-ïafeçted érythrocytes to CD36 is inhibited by CD36 peptides 145-171 and 156-184 (Baruch et al., 1999). Also, a rôle of CD36 in signal transduction is often discussed(Shattil and Brugge 1991). In immunoprécipitation of CD36 from resting platelets, tyrosinekinases of the src gene family pp60fy”, pp62yes and pp54/58Iyn are coprecipitated, whichindicates a close association with CD36 (Huang et al., 1991). The meaning of this discovery is 30 still unclear. Some antibodies against CD36 activate platelets and monocytes (Aiken et al.,1990, Schüepp et al., 1991). IgM antibody NLO7 activâtes platelets with the help of thecomplément System (Alessio et al., 1993). As a forther fonction of CD36, its rôle inthrombotic thrombocytopénie purpura (TTP) is described (Lian et al., 1991). A protein (p37),which is présent in plasma of TTP patients, agglutinâtes platelets, mediated by CD36. The 01243 1 6 meaning of this finding is still unknown. Peptide VTCG from thrombospondin inhibits thephosphatidylinositol (3,4) bisphosphate synthesis in platelets activated with thrombin. CD36is one of the thrombospondin receptors, which médiate the later activation of PI-3-kinase andphospholipase C (Trumel, Payrastre, Mazarguil, Chap, Plantavid, personal communication). 5 CD36 is involved in the transport of long chain fatty acids in muscle tissue cells.

Overexpression of CD36 in muscle cells of transgenic mice led to enhanced cellular uptake offatty acids, increased fatty acid oxidation by contractile muscles, and reduced the 10 concentration of triglycérides and free fatty acids in the plasma. The mice had reduced bodyweight, in particular reduced body fat in comparison to control mice.

The lack of CD36 in humans leads to the loss of uptake of long chain fatty acids, which arethe main energy source for the heart muscle, in heart muscle cells, and consequently to 15 elevated appearance of innate hypertrophie cardiomyopathies (Fuse et al., 1998). Also, CD36- defîcient mice show a defect in the transport of fatty acids in cells and a disturbed lipoproteinmetabolism (Febrraio et al., 1999). CD36 médiates the arachidonic acid-mediated platelet aggregation (Dutta-Roy et al., 1996) 20 and binds to negatively charged phospholipids in cell membranes (Ryeom et al., 1996). Inparticular, phosphatidylserine (PS) and phosphatidylinositol (PI) are specifically bound byCD36 with high affïnity (Rigotti et al., 1995). Because apoptotic cells expressphosphatidylserines on their surface, contact with phagocytotic cells can be mediated byCD36 (Fadok et al., 1998, Alberts et al., 1998). Phosphatidylserine presumably binds to the 25 CD36 sequence 162-183 (Yamaguchi et al., 2000). CD36 binds to cholesteryl hemisuccinateand can be easily purified by this reaction (Kronenberg et al., 1998).

The main function of CD36 might be its rôle as receptor for oxidized LDL. This rôle was firstdescribed by Endemann et al., 1993. Transfection experiments with a cDNA clone, which 30 codes for CD36 in the human macrophages-like-THP-cell line, led to a newly identified binding capability of the cell for Cu2+-oxidized LDL. The monoclonal CD36-specific antibody 0KM5 inhibits binding of Cu2+-oxLDL to platelets by 54 %. The binding site for

Cu2+-oxLDL lies in the région ofthe CD36 sequence 155-183 (Puente et al., 1996). Nicholson et al. suggested that Cu2+-oxLDL presumably binds to CD36 by its lipid portion (Nicholson et 7 01243 1 al., 1995). Macrophages from blood donors, which are déficient for CD36 on monocytes,hâve signifîcantly reduced (-40%) uptake of oxLDL in comparison to Controls (Nozaki et al.,1995). 5 Vitamin E (alpha-tocopherol) inhibits the uptake of Cu2+-oxLDL in smooth muscle cells ofthe aorta by inhibiting CD36 (Ricciarelli et al., 2000). The binding of oxLDL to murine CD36is partly prevented by oxidized phospholipids, which are associated with the lipid and proteinportion (Boullier et al., 2000). It has recently been found that CD36 is not only the receptorfor Cu2+-oxLDL, but also for NO2-LDL, and that CD36 is responsible for NCh-LDL-mediated 10 foam cell formation (Podrez et al., 2000).

This binding is competitively inhibited by oxidation products of the lipid l-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine. Therefore, the authors speculate that themyeloperoxidase-catalyzed peroxidation of lipids is required to mark phospholipid containing 15 targets for phagocytosis by CD36 containing cells. In accord with its rôle as receptor foroxLDL, CD36 is found on macrophages loaded with lipids in arteriosclerotic plaques (Nakataet al., 1999), and smooth muscle cells may develop to foam cells by expression of CD36 invivo (Ohya et al., 2000). 20 Lack of CD36 seems to be a protection against arteriosclerosis. Therefore, mice that lackApoE protein develop arteriosclerotic plaques under a corresponding diet. If the CD36 gene inthese mice is also knocked out then, under the same conditions as their CD36 containingcontrol relatives, the mice develop 76 % less arteriosclerotic lésions in the aortic tree under afat rich diet, and 45 % less plaques in the aortic sinus under normal diet. 25

Macrophages of CD36- and ApoE-double knock-out mice intemalize less than 40% of copperoxidized LDL and NO2-LDL (Febbraio et al., 2000).

Blocking thrombotic and arteriosclerotic fonctions of CD36, while simultaneously not 30 affecting the important CD36-mediated uptake of long chain fatty acids in the cell, would be an important step in the fight against vessel diseases.

This problem is solved by the présent invention. In the context of the présent invention it has been found that cell fonctions that oxLDL triggers through CD36 can also be triggered by δ 01243 1 ( other substances. Surprisingly, these other substances are oxidized proteins, which do notneed to hâve a lipid portion. In the body, proteins are oxidized for defense against infection,in arteriosclerotic plaques, or in acute or chronic inflammations.

In the following disclosure, some reactions are mentioned by way of example that are not5 only triggered by oxLDL, but are also triggered by other oxidized proteins and CD36: (1) 10 (2) (3) 15 20 (4) 25 (5) (6) 30 (7) (8)

Activation of thrombocytes -» on the one hand, thrombosis, heart attack, and stroke, but on the other hand,also hemostasis

Damage of endothélial cells -> ischemia, inflammatoiy reactions, edema formation, and disturbance ofprostacyclin release

Activation of leucocytes, e.g.: a) elevated adhesion of leucocytes to endothélial cells; b) support of transmigration of leucocytes through endothélium and epithelia; c) homing of leucocytes in arteriosclerotic plaques; d) priming and triggering of oxidative burst in phagocytes; and e) tissue factor expression on monocytes, in particular increase of the reactiontriggered by lipopolysaccharide (LPS). —» damages by inflammatory reaction, thrombosis etc.

Activation of smooth muscle cells (SMCs): a) prolifération of SMCs, and b) intimai swelling in arteriosclerotic plaques. —» reocclusion after bypass, stent, PTCA; development of arteriosclerosis

Stimulation of renin release from juxta-glomerulic cells-» renin dépendent high blood pressure in kidney diseases.

Formation of foam cells by stimulation of the uptake of coincubated LDL bymacropinocytosis. -» development/increase of arteriosclerosis

Apoptosis of vessel cells in the center of arteriosclerotic lésions. -> necrosis, plaque rupture

Stimulation of the expression of matrix metalloproteinases in endothélial cells. -» stimulation of rupture of arteriosclerotic plaques. 9 012431 /

Additionally, it is shown by this invention that not only does IDAAT (immune defenseactivated antithrombin, patent application No. DE 100 45 047.4) bind to HIV GP 120 and toCD4, but it also binds to other oxidized proteins. HTV GP 120 comprises a CD26 homologuesequence (Crombie et al., 1998). 5

It has also been shown by this invention that not only IDAAT but also other oxidized proteinsmédiate the binding of thrombospondin to cells like thrombocytes, monocytes, endothélialcells, and T cells. Therefore, there is a compelling reason to believe that oxidized proteins ingeneral induce thrombospondin mediated cell reactions, like the inhibition of angiogenesis, 10 the defense of HIV infections, the régulation of inflammatory processes by e.g. downrégulation of IL-12 in monocytes, and upregulation of IL-10. Hence, oxidized proteinsthemselves can hâve a therapeutically useful activity in certain disease processes.

Further, it has been shown in the context of the présent invention that pathological cell 15 functions mediated by oxidized proteins can be inhibited by substances that inhibit theinteraction between CD36-oxidized proteins, or which can interfère with TSP bound to CD36.

Examples of such substances include soluble thrombospondin-1 and monoclonal anti-CD36antibody clones 37, 13, and 7, which were manufactured by us and are disclosed herein. 20

Examples: 1. Isolation ofhuman thrombospondin-1

Isolation of human thrombospondin-1 from thrombocytes was carried out as described in 25 Kehrel et al., 1991. The description of Kehrel et al., 1991 is incorporated herewith byreference. However, in contrast thereto, the platelets were activated with thrombin, andEDTA in the wash buffer was substituted by Na-citrate (0.08 M). Additionally, theaggregation buffer and ail buffers for the following purification steps were substituted withCa2+ at a concentration of 2 mM. 30 2. Hybridoma culture for the préparation of monoclonal antibodies

The préparation of monoclonal antibodies against CD36 was broadly carried out according to the instructions of Peters and Baumgartner (1990). 10 01243 1 ( 8-12 weeks old Balb/c female mice were immunized with purifïed CD36 (50 pg/boost). Forimmunization, the long immunization protocol (4 months) according to Baumgartner et al.,1990, was used. Approximately 14 days before the planned fusion time point, blood wastaken from the animais, and the IgM and IgG titers against CD36 in the sérum of the mice 5 were determined. If the IgG titer was still signifîcantly different from the control at dilutionsof 1:100,000, spleen cells were fused with Ag 8,653 cells. Ag 8,653 cells were selected inculture medium containing 0.13 M 8-azaguanine. Lymphocytes from the spleen wereprepared and fused with Ag 8,653. Directly after the fusion, fused cells (1 x 106 spleencells/ml) wae incubated for 24 hours in sélection medium (culture medium with the addition 10 of hypoxanthine, 27.2 pg/ml, and azaserine (50 pg/ml)) in a cell culture flask (75 ml).Thereby, macrophages dérivé from the spleen attach to the plastic surface and no longerinterfère with the actual culture of hybridoma in a 96 well plate.

The cloning steps were carried out by limited-dilution-cloning with a seed probability of 0.5 15 cells per well of the 96 well plate according to Würzner 1990. Supematants of hybridomawere tested in an ELISA for the production of IgG and IgM, respectively. IgG positive cellculture supematants were tested for their specificity against CD36 with different methods: 19 CD36 spécifie clones were obtained, which did not react with CD36 déficient platelets 20 (description: Kehrel et al., 1993, Saelman et al., 1994, Kehrel et al., 1995), but which showedsignificant binding to control platelets. This was proven by flow-through cytometry and bythe dot blot method and immunoprécipitation. AH antibodies tested recognized purifïedCD36. Three of these clones (clone 37, 13, and 7) inhibited the reaction of oxidized proteinswith human cells (see below). 25

Isolation of CD36

Isolation of CD36 was carried out as described by our group (Kronenberg et al., 1998), byphase séparation of the membrane proteins with Triton X-114, and subséquent affinitychromatography using cholesterol-hemisuccinate agarose. 30

Examples for the préparation of oxidized proteins 348 pg protein (commercially available fibrinogen, human albumin, bovine sérum albumin (BSA), or antithrombin) was incubated with 832 pg NaOCl (sodium hypochlorite) in 1 ml phosphate buffered saline with the addition of EDTA (0.1 mM) for 10 minutes on ice. Protein 11 012431 and oxidants were immediately separated after the end of the reaction by a gel filtration at 4°Cwith Sepharose G25-Coarse (PD-10 column, Amersham Pharmacia).

Examples for the activity of the invention: 5 1. Oxidized proteins (ox fïbrinogen, ox antithrombin III, ox BSA, ox human albumin) bind specifïcally to the CD36 homologue domain of HIV GP 120 protein (see figure 1).The interaction between oxidized proteins and HTV GP 120 was determined byplasmon résonance technique in BIACORE System 2000. 10 2 15 20 25

Oxidized proteins activate thrombocytes. The activation of thrombocytes is inhibitedby substances that inhibit the interaction of CD36 with oxidized proteins, or thatinterfère with thrombospondin bound to CD36. a) Oxidized proteins (ox fibrihogen, ox antithrombin III, ox BSA, and ox humanalbumin) increase, in a dose dépendent manner, the adhesion of thrombocytesto adhesion proteins, like thrombospondin, vitronectin, fïbrinogen, fibrin,fibronectin, and collagen (see figure 2a).

This increase in adhesion is inhibited by soluble thrombospondin-1 (see figure2b). This increase in adhesion is also inhibited by monoclonal anti-CD36antibody clones 37, 13, and 7 (see figure 2c/d), while VCTG peptide, whichinhibits binding of thrombospondin to CD36, has no effect (see figure 2e). Thecommercially available anti-CD36 antibody FA6/152 shows no inhibition (seefigure 2f). b) Oxidized proteins (ox fïbrinogen,. ox antithrombin III, ox BSA, ox humanalbumin) induce, in the presence of thrombospondin-1 (10 pg/ml) and in a dosedépendent manner, the binding of thrombospondin to thrombocytes (see figure3 a). This activation is inhibited by soluble thrombospondin-1 at a concentrationof > 20 pg/ml (see figure 3b).

This activation is also inhibited by monoclonal antibodies against CD36, clone37, 13, and 7 (see figure 3c-e). c) Oxidized proteins (ox fïbrinogen, ox antithrombin III, ox BSA, ox humanalbumin) cause, in the presence of thrombospondin-1 (10 pg/ml), theaggregation of platelets (see figure 4a). This aggregation is inhibited bymonoclonal antibodies against CD36, clones 37, 13, and 7, in a dose dépendentmanner (see figure 4b). 30 12 012431 3 10 15 20 25 4. d) Oxidized proteins (ox fibrinogen, ox antithrombin III, ox BSA, ox humanalbumin) cause, in the presence of thrombospondin-1 (10 pg/ml), the pro-coagulant condition of the platelets (see figure 5a-c), and lead to micro particleformation (see figure 5d). This activation is inhibited by solublethrombospondin-1 at concentrations of > 20 pg/ml (see figure 5e).

This activation is also inhibited by monoclonal antibodies against CD36,clones 37, 13, and 7, in a dose dépendent manner (see figures 5f and g).

Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox humanalbumin) activate monocytes. This activation is inhibited in a dose dépendent mannerby certain substances, e.g., soluble thrombospondin or monoclonal antibodies againstCD36, which inhibit the interaction between CD36 and oxidized proteins or whichinterfère with thrombospondin bound to CD36. a) Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox humanalbumin) induce a Ca2+ signal in monocytes (see figure 6). b) Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox humanalbumin) induce the oxidative burst in PMNL (see figure 7). c) Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox humanalbumin) induce an increased transmigration of monocytes, PMNL andlymphocytes through endothélial monolayers (see figure 8a). This reaction isinhibited by substances that inhibit the binding of CD36 to oxidized proteinssuch as soluble thrombospondin or monoclonal antibodies against CD36,clones 37, 13, and 7 (see figure 8b).

Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox human albumin) play a causal rôle in the development of arteriosclerosis. a) Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox human albumin) induce a homing of macrophages in arteriosclerotic plaques. Homingwas carried out according to Patel et al., 1998. In C57BL/6 mice, the migrationof monocytes/macrophages into the peritoneum was induced by intraperitonealinjection of thioglycolate. After 4 days, the peritoneum was washed andactivated peritoneal macrophages were obtained. In the probes, érythrocyteswere lysed. Macrophages were resuspended in RP MI 1640 medium andtransferred to cell culture dishes to allow adhesion. Fluorescence-marked 13 012431 microspheres (2 μπι yellow-green fluorescence latex microspheres, molecular probes) were opsonized for 30 minutes with 50 % mouse sérum for better uptake by the macrophages, and were then added to the plated macrophages.

The adhered macrophages phagocytosed the microspheres. Non-adhered cells 5 and microspheres were removed from the dish by washing. Macrophages were removed from the dish on ice and were resuspended in Hanks balanced saitsolution (HBSS). 50 week old ApoE déficient mice were each injectedintraperitoneally with 3 times 50 μg oxidized protein, in this case oxidizedantithrombin III, and placebo (only HBSS), respectively, 6 hours before 10 intravenous injection of marked macrophages, 2 hours after injection of macrophages and 10 hours after injection of macrophages. 10 x 106macrophages were suspended in 0.2 to 0.3 ml HBSS and injected into the tailvein. 24 hours after macrophage injection, the animais were sacrifîced. Theheart basis and the aorta ascendens were embedded in OCT, stored al -80°C, 15 and 7 pm cryo-sections were prepared. Fluorescence-marked macrophages of

140 serial sections per mice from a 1 mm range of the aorta ascendens on thelevel of the sinus valsalva were counted. In comparison to the placebo treatedApoE7' control mice, the migration of marked macrophages intoarteriosclerotic plaques in mice treated with oxidized antithrombin III 20 increased from 100 + 15 % (n = 14) to 156 + 9.2 % (n = 5) signifîcantly (p = 0.008) ("altemate welch test”). As this migration is causal to the development,the progression, and the danger of rupture of arteriosclerotic plaques, thisexample élucidâtes the importance of oxidize proteins with respect toarteriosclerosis (figure 9). 25 b) Substances that inhibit the interaction of CD36 and oxidized proteins, or interfère with thrombospondin bound to CD36, prevent/reduce the pro-arteriosclerotic activity of oxidized proteins. Soluble thrombospondin inhibitsadhesion of macrophages to arteriosclerotically altered endothélial cells. This isa prerequisite for migration of microphages into arteriosclerotic plaque. 30 Murine-immortalized endothélial cells were stimulated with β-VLDL (50 pg protein/ml) from plasma of ApoE déficient mice for 6 hours, and thensuspended with 2 x 105 peritoneal monocytes/macrophages per ml with a shearrate of 400s'1 in a flow through chamber. β-VLDL induced a signifîcantrelative increase of the rolling leucocytes by 224 + 44 % in comparison to the 14 012431 f control. Addition of 10 gg/ml soluble thrombospondin inhibited this increaseof adhesion completely and additionally inhibited partly the basic adhesion ofmacrophages to the endothélium (75+37 % adhesion in comparison to thecontrol, n = 4-7; p < 0.01) (see figure 10a). Significantly induced permanent 5 adhesion of macrophages by β-VLDL was also reduced by thrombospondin-1 after a 5 minute wash period (100 + 21 % control versus 300 + 119% β-VLDLversus 157 ± 70 % β-VLDL + TSP-1; n = 4-7; p < 0.01) (figure 10b). 5. Soluble thrombospondin-1 inhibits inflammatory reaction in vivo. In the ear of Balb/c- 10 mice, an Arthus reaction was induced by local injection of anti-BSA at time point 0 and simultaneous injection of FITC coupled BSA into the peritoneum. Control animais(négative Controls) were only injected with FITC (without BSA) into the peritoneum.After 6 hours, animais treated with anti-BSA and BSA-FITC showed a clearlydeveloped inflammatory reaction with ear swelling (edema), FITC incorporation, 15 migration of PMNL and pétéchial bleeding into the tissue. 18 mice were additionally intraperitoneally injected at time point 0, and after 0 + 3 hours, with 50 pgthrombospondin-1 in buffer (PBS). 16 control mice received only PBS at time point 0+ 3 hours. The Arthus reaction was almost completely prevented by thrombospondin.Thrombospondin-treated mice showed significantly less FITC incorporation, 20 significantly less ear thickness (less oedems), and almost no petechia in comparison to PBS treated control animais (see figures 1 la-c). 6. Examples for quantification of oxidized pro'teins

Préparation of monoclonal antibodies that recognize epitopes on proteins or peptides, 25 and that are directly or indirectly modified by oxidation processes:

Human albumin, antithrombin III and fibrinogen were, as described in this dsiclosure,oxidized with HOC1, and 8-12 weeks old Balb/c female mice were immunizedtherewith. Préparation of hybridoma and hybridoma culture was carried according toclassic procedures. Supematants of hybridoma were tested for the production of IgG 30 and IgM, respectively. IgG positive cell culture supematants were tested for positive reaction with oxidized protein and simultaneously négative reaction with the unalteredinitial protein. Clones that produced antibodies against oxidized protein (ox humanalbumin, ox antithrombin III, ox fibrinogen) and simultaneously did not react with 15 5 10 15 20 25 30 7. 01243 1 non-oxidized mother protein, were tested for cross-reaction with other oxidized proteins and peptides.

Quantification of oxidized proteins with the help of monoclonal antibodies, asdisclosed above:

Such quantification is easily possible with processes like ELISA, RIA, quantitativeflow-through cytometry on cell surfaces, and similar routine procedures,e.g.: quantification of oxidized human albumin with ELISA. A polyclonal antibodyfrom rabbit against human albumin (préparation - routine procedure) was bound to thebottom of an ELISA dish (Nunc-Maxisorb) as catcher antibody. The dish wasthoroughly washed with PBS pH 7.4, 0.5 % Tween 20, and spaces on the plasticsurface were blocked with 3 % BSA for 1 hour at room température (RT). The dishwas washed again and then incubated with differently diluted plasma, sera,supematants of blood products (e.g. thrombocyte concentrâtes, érythrocytesconcentrâtes, FFP) or buffer solutions to which defined amounts of ox human albuminhâve been added, for 1 hour at RT. Probe material and standard solutions, respectively,were removed, the dish was washed thoroughly and incubated with the above-described monoclonal antibody, which recognizes oxidized human albumin and whichwas marked with biotin, in a dilution of 1:15000 in PBS, 1 % NGS (normal goatsérum). The dish was again washed thoroughly for several times and incubated withStreptavidin peroxidase (1 hour, RT). After again washing the dish, it was treated withsubstrate solution (100 pg/well) (20 mg ortho-phenyldiamine, 5 % H2O2 in a buffer of 12.15 ml 0.1 M citric acid and 12.85 ml 0.2 M Na2HPÛ4 plus 25 ml H2O dest.). Theextinction at 405 nm, measured in an ELISA photometer, indicated the amount ofoxidized human albumin. The reaction was stopped with 50 μΐ/well 4 N H2SO4, andthe extinction was measured at 490 nm HOCl-oxidized human albumin in the probe was quantified with a calibration sériés.HOCl-modified fibrinogen and HOC1 modified antithrombin III were treated similarly.

Example for the documentation of the activated condition of the receptor for oxidizedproteins, CD36.

Préparation of polyclonal antibodies against threonine (92) phosphorylated CD36.Peptides (15 AS) (1) Lys, Gin, Arg, Gly, Pro, Tyr, Thr, Tyr, Arg, Val, Arg, Phe, Leu,Ala, Lys and (2) Lys, Gin, Arg, Gly, Pro, Tyr, PhosphoThr, Tyr, Arg, Val, Arg, Phe, 16 01243 1 (

Leu, Ala, Lys (as peptide (1), but phosphoiylated at threonine), were synthesized andcoupled to KLH (Keyhole Limpet Hemocyanin). Polyclonal antibodies from rabbitwere prepared according to standard procedures with these coupled peptides. For this,rabbits (New Zealand, white) were essentially immunized with 250 pg peptide 1-KLH 5 and peptide 2-KLH plus the addition of complété Freund adjuvans s.c., respectively, and boostered with 250 pg peptide 1-KLH and peptide 2-KLH with the addition ofAlu-Gel-S (1.3 % aluminium hydroxide in water, SIGMA) 3 times, respectively.Blood was drained from the ear veins of the animais, and sérum was prepared andtested for antibodies against the peptides that were used for the immunization. The 10 antibody against phosphorylated CD36 peptide was cross-absorbed on an affinity column with non-phosphorylated CD36 peptide, so that the resulting antibody mixtureonly contained antibodies that specifîcally recognized phosphorylated non-activatedCD36 (AK CD36P). (The préparation of spécifie monoclonal antibodies againstthreonine phosphorylated/dephosphorylated CD36 is possible with these peptides 15 according to the described préparation of anti-CD36 protein antibodies.)

Both monoclonal antisera reacted with CD36 on the surface of platelets in flow-through cytometry. While antibody "CD36P" directed against phosphorylated CD36preferably recognizes CD36 on non-activated thrombocytes, antibody "CD36 total"recognizes non-phosphorylated CD36 and phosphorylated CD36. With activation of 20 the thrombocytes, CD36 is dephosphorylated and the binding capacity for antibody "CD36P" decreases (see figure 12). Quantitative flow-through cytometry with bothantibodies allowed the calculation of the portion of activated CE36. 8. The organism of patients with type I diabètes is particularly susceptible to oxidized 25 proteins : e.g.: thrombocytes of patients with diabètes type I react more sensitively to oxidizedprotein as agonist in comparison to thrombocytes of healthy control persons.Activation-dependent fibrinogen binding can be induced on thrombocytes of patientswith diabètes with reduced concentrations of ox protein in comparison to 30 thrombocytes of control persons (see figure 13). 9. Oxidized proteins inhibit HIV infection.

Oxidized protein (ox antithrombin III and ox human albumin were tested) bind withhigh affinity to both HIV-GP120 and its receptor CD4. 17 01243 1 e.g.: Binding of oxidized antithrombin III and oxidized human albumin to HIV-GP120 and to CD4 was shown using a BIACORE 2000 System. Running buffer: 25 mM Tris; 100 mM NaCl pH 7.4; 1 mM CaCl2; 1 mM MgCl2; 0.005 % Surfactant P-20.

Protein HIV-GP120: c = 100 gg/ml, 200 μΐ 5 Storage buffer: Tris/HCl, NaCl pH 7.6

Protein CD4: c = 63 pg/ml, 318 μΐStorage buffer: 10 mM Tris; 300 mM NaCl pH 8Protein ox antithrombin III: c = 1 mg/ml, 120 μΐProtein ox human albumin: c = 1 mg/ml, 120 μΐ 10 Cl-chip (BIACORE AB)

Amine coupling kit (BIACORE AB) HIV-GP120 and CD4 were immobilized on the Cl sensor surface. For this 10 mMNaAc pH 4 was used as coupling buffer.

Coupling conditions: HIV-GP120: 20 μΐ; 10 pg/ml GP120 in 10 mM NaAc pH 4;15 immobilized amount: 1158 RU, 300 pg CD4: 30 μΐ; 6.3 pg/ml CD4 in 10 mM NaAc pH 4; immobilized amount: 568 RU, 148Pg

The chip surface was saturated with BSA, and the binding of oxidized proteins wasinvestigated. For this, e.g. 50 μΐ oxidized antithrombin III were injected in different 20 concentrations at a flow rate of 20 μΐ/minute. The protein solutions were diluted with sample buffer. With increasing concentrations of oxidized antithrombin III theresulting signais increase. Figure 14 shows an overlay plot of 12 sensorgrams, whichshow binding of oxidized antithrombin III to immobilized CD4 and subséquent dissociation. 25 The quantitative analysis resulted in the following values for the binding of a) oxidized antithrombin III to HIV-GP120:

Ko„ (1/Ms): 6.38 x 105

Kofr(l/s): 4.44 x 10'4and a Kd[M] of 7.01 x 10'10, 30 b) oxidized antithrombin III to CD4:

KoB(1/Ms): 7.13 x 105

Koff(l/s): 1.12 x 10'3 andaKD[M] of 1.63 x1ο’9, 18 0Î243) c) non-modified antithrombin III bound neither to HIV-GP120 nor to CD4. Oxidizedhuman albumin bound with a higher affmity to HIV-GP120 and CD4 than to oxantithrombin III. Non-oxidized human albumin bound neither to HTV-GP120 nor toCD4. Oxidized protein inhibited HIV-1 infection of monocyteous cells from 5 peripheral blood (PBMC). PHA-activated PBMC were incubated together with négative human sérum 1:100(négative control), with neutralizing V3-loop spécifie antibodies (positive control),with oxidized protein (150 pg/ml) and a CCR5 tropic HIV-1 primary isolate (903) 10 from a patient, and after 5 days the virus production was tested by P24 ELIS A. For this, freshly PHA-activated PBMC were suspended in RPMI 1640 medium plus 20 %FCS plus 100 U/ml IL-2 in a cell concentration of 2 x 106 cells/ml, and 200,000cells/well/100 μΐ were distributed on a 96 well plate. Tested substances for inhibition:Positive control: neutralizing human anti-V3-loop antibody (1:100) 15 Négative control: négative human sérum 1:100

Experiment: oxidized protein (150 pg/ml) was added to the cells in RPMI medium and incubated for 30 minutes at 37°C/5 %CO2. Subsequently, HIV-1 virus was added to the samples: each contains 10 μΐ/well ofHTV-1 primary isolate 903 supematant (CCR5 trop) with 20,000 TCID50 (50 % tissue 20 culture infective dose)/ml = 1000 TCIDso/ml per well. These samples were incubated ovemight at 37°C/5 % CO2. The following day, the cells were washed 3 times withRPMI 1640, and new culture medium was added. On day 5 after infection, P24-ELISAtests were carried out. P24-ELISA: 25 Anti-P24 antibody (11-G7 [Niedrig, Berlin] and D7320 [Biochrom]) recognize the P24 protein of the primary isolate variant 903. Maxi-Sorb-ELISA plates (Nunc) wereoverlayed with these antibodies ovemight. The virus supematant from the inhibitionassay was inactivated by 1 % Triton X-100. After washing the treated cells with PBS,the inactivated virus supematant and alkaline phosphatase conjugated détection 30 antibody (BC1071-AP[Aalto]) were transferred together in wells and incubated for 5 hours at 37°C. Wells were again washed with PBS, dissolved substrate for alkaline phosphatase p-nitrophenyl-phosphate (Sigma) was added to the wells, and the color development was measured after 20 minutes at 405 nm in an ELISA photometer. The parallel values in the P24-ELISA varied up to 0.02 optical density (OD) units about a 19 012431 common mean value. While OD 405 nm for the négative control - no inhibition was at0.8, the neutralizing antibody (positive control) reduced the OD to 0.12. 150 gg/mloxidized protein reduced the OD to 0.10. The addition of oxidized proteins effectivelyinhibitedHIV-1 infection ofPBMCs. 5 10. Oxidized proteins induced TSP binding to cells

Oxidized proteins (for example, ox human albumin, ox antithrombin III, and oxfîbrinogen, were used herein) induced spécifie and dose dépendent binding of TSP-1to CD36 containing cells (see figure 15ar-d). 10

Therefore, on the one hand, objects of the présent invention are médicaments comprisingsubstances that inhibit the binding of oxidized proteins to CD36 or inhibit the fonctions ofCD36 that are induced by the interaction of CD36 with oxidized proteins. 15 In a preferred embodiment of the invention, the médicaments comprise antibodies that inhibitbinding of oxidized proteins to CD36, comprising particularly preferred monoclonalantibodies and antibody fragments like F(ab)2, F(ab), or of the antibody récognition région.

In a forther preferred embodiment, the médicament comprises peptides of CD36, peptide 20 mimetics, or peptide analogues that inhibit binding of CD36 to oxidized proteins or thatinhibit cell fonctions of CD36 induced by interaction of CD36 with oxidized proteins.Preferably, these substances are identified and selected by monoclonal anti-CD36 antibodiesdisclosed in this invention, and in particular, they react with clones 37, 13, or 7, or inhibitbinding of oxidized proteins/peptides to CD36, or inhibit a characteristic fonction of CD36, 25 induced by oxidized protein/peptide as, but not limited to, the fonctions described in theexamples.

In forther preferred embodiment, the médicament comprises proteins or protein componentsthat inhibit binding of CD36 to oxidized proteins or that interfère with thrombospondin bound 30 to CD36. In a particularly preferred embodiment, such protein is soluble thrombospondin.

In a forther preferred embodiment, the médicament comprises peptides or peptide mimetics that bind to CD36 and thereby inhibit the interaction of CD36 with oxidized proteins. Such 20 01243 1 peptides or peptide mimetics can be easily identified, e.g. using the so-called “phage display”procedure.

On the other hand, the object of the présent invention is the use of médicaments according to5 the invention for prophylaxis of thrombosis, in particular in inflammatory diseases, forsupport of an anti-thrombotic therapy, for preventing a transplant rejection, for preventingtransplantation associated arteriosclerosis, for preventing high blood pressure in kidneydiseases, and in particular renin associated high blood pressure, for preventing thedevelopment and the progression of arteriosclerotic (atherosclerotic) diseases, for the 10 treatment of chronic inflammatory reactions, for preventing early vessel reocclusion afterbypass surgery, stent, PTCA, or the like, for preventing of vessel restenosis after bypasssurgery, stent, PTCA, or the like, for preventing reperfusion damages, such as, but not limitedto myocardial ischemia, organ transplantation, stroke, peripheral occlusive disease aftersurgery, and/or multi organ failure after successful réanimation, for preventing vessel damage, 15 in particular in patients with diabètes mellitus, for preventing the inhibition of endothélialprolifération and angiogenesis induced by oxidized proteins through CD36/TSP-1, and forsupporting wound healing.

Still another object of the présent invention is a method for quantifying oxidized proteins (in 20 particular ox antithrombin III, ox human albumin or ox fibrinogen), individually or together,for the évaluation of individual indications for thérapies with médicaments according to theprésent invention, for the diagnosis of diseases, in which inflammatory reactions play a rôle,such as, but not limited to, arteriosclerosis, diabetic vasculopathy, rheumatic arthritis,Goodpasture Syndrome, sepsis, Colitis ulcerosa, graft-versus-host diseases, pemphigus, 25 cancer, neurodermatitis, HIV infections, ARDS, glomerulonephritis, reperfusion damages,and for quality control of blood products.

Another object is the characterization of the activated condition of CD36, which is thereceptor for oxidized proteins, as a diagnostics for diseases in which inflammatory reactions 30 play a rôle such as, but not limited to, arteriosclerosis, diabetic vasculopathy, rheumaticarthritis, Goodpasture syndrome, sepsis, Colitis ulcerosa, graft-versus-host diseases,pemphigus, cancer, neurodermatitis, HIV infections, ARDS, glomerulonephritis, reperfusiondamages or for monitoring a CD36/ox protein inhibition therapy with médicaments accordingto the présent invention, by measuring the phosphorylation condition of CD36. 21 01243 1

Still another object of the présent invention is a médicament that comprises oxidizedprotein/oxidized proteins, oxidized peptide, oxidized structural analogues or structuralmimetics thereof. Médicaments according to the présent invention are also characterized in 5 that they may contain further pharmaceutically acceptable fillers and/or excipients. Themédicaments according to the présent invention are preferably suitable for local, intradermaltopical, intraperitoneal, intravenous, oral or intramuscular administration, or they can beapplied as vesicles. Further, it is preferred that the médicaments according to the présentinvention further comprise substances as e.g. antibiotics, immunosuppressants, or interaction 10 partners of oxidized proteins in the body. By the addition of such substances, the activities ofthe médicaments according to the présent invention can be further supported and assisted.

In the context of the présent invention, oxidized proteins or peptides are generated accordingto the présent invention preferably by reaction with HOC1 or peroxynitrites. 15 A further use of the médicaments according to the présent invention, and in particular of amédicament compris ing oxidized proteins/peptides or analogues or mimetics thereof, lies inthe prophylaxie or therapy of acute infections, the inhibition of angiogenesis, and for theimprovement of hemostasis. Thereby, the médicament is preferably used for the prophylaxis 20 or therapy of an HIV infection. In another preferred embodiment, the use of a médicamentaccording to the présent invention comprising oxidized protein inhibits tumor angiogenesis bymeans of induction of TSP binding to CD36.

In still another preferred embodiment, the médicament is used for hemostasis, in particular in 25 patients with innate or acquired blood coagulation disorders, or innate or acquiredthrombocytopathia, under anticoagulation therapy or thrombosis prophylaxis, or is used insurgery under heart-lung-machine.

Because oxidized proteins induced TSP binding, médicaments according to claim 21 30 inevitably induce indirect effects of TSP bound to cell surfaces, as, e.g., inhibition ofangiogenesis (see figure 16a) or inhibition of HIV infection. On the other hand, inhibition ofthe interaction between oxidized proteins and CD36 consequently induces the repression offunctions that are induced by the reaction chain ox protein-CD36 cell bound TSP. Inhibitors 22

°’243J according to daims 1-5 thereby also inhibit TSP-mediated processes, as the inhibition ofangiogenesis and therefore are proangiogenetic (see figure 16b).

Legends of the figures: 5

Figure 1 : Oxidized proteins bind to a CD36 homologue domain in HIV-1 GP 120 protein 1) Overlay-plot of 12 sensorgrams, which show the binding of oxidized antithrombin III toimmobilized HTV-GP120 and the dissociation of oxidized antithrombin III. HIV-GP120 is 10 immobilized (300 pg); the concentration of oxidized antithrombin III varied (from the bottomto the top: 0 nM; 1 nM; 5.1 nM; 10.2 nM; 17 nM; 20.4 nM; 23 nM; 34 nM; 40.8 nM; 51 nM;85 nM; 119 nM). With an increasing concentration of oxidized AT III the resulting signalincreases. 15 Figure 2: Oxidized proteins are hemostatic/prothrombotic - increase of thrombocyte adhesion/inhibition of this reaction 2a) Oxidized proteins increase platelet adhesion to collagen type I. Adhesion of thrombocyteswas carried out according to Santoro et al., 1994. A 96 well cell culture plate was coated with 20 collagen type I (25 pg/ml; 100 μΐ/well) ovemight at 4°C, and the plates were blocked withBSA. Human thrombocytes were purified from plasma proteins by gel filtration in HEPES-Tyrode buffer pH 7.4 with the addition of 2 mM Mg2+, 1 mM Mn2+, 0.9 % glucose and 0.35% BSA. 100 μΐ gel-filtered platelets (300000/μ1) were incubated with and without oxidizedproteins for 1 hour at RT in a humid chamber in the wells. Non-adhered thrombocytes were

25 thoroughly washed away. The number of adhered platelets with determined after lysis of theplatelets with Triton X-100 and détermination of the lyzosomal enzyme hexosaminidase. Forcalibration of the adhesion assay, a calibration sériés with known increasing platelets numberis given on a microtiter plate, and the extinction of the substrate P-nitrophenyl-N-acetyl-p-D-glucosamide is determined in relation to the number of platelets. Oxidized antithrombin III 30 increased the thrombocyte adhesion in a dose dépendent manner. 2b) Thrombocytes were used in the above-described adhesion assay and were activated with 50 pg/ml oxidized ATIII. Addition of soluble purified thrombospondin inhibited increased thrombocyte adhesion mediated by oxidized ATIII in a dose dépendent manner. 23 0 7243 ί 2c) Thrombocytes were used in the above described adhesion assay, and were activated withoxidized ATIII. Antibodies that inhibit binding of oxidized proteins to CD36, like clones 37,13, and 7, inhibit the activity of oxidized ATIII. Ail experiments for the measurement of theinfluence of antibodies on the thrombocyte functions were carried out in the presence ofsaturating, completely blocking, concentrations of Fab fragments of an antibody against theFcRIIA receptor (clone IV.3), in order to avoid Fc receptor effects. 2d) This effect is dose dépendent. 2e) Inhibition of thrombocyte adhesion by soluble thrombospondin-1 is not mediated directlythrough its binding site on CD36 (peptide VTCG). VTCG shows no influence on the increaseof thrombocyte adhesion by oxidized proteins (herein oxidized ATIII). 2f) Antibodies against CD36 that do not inhibit binding of CD36 to oxidized proteins, likeclone FA6/152, however, do not induce any signifïcant inhibition. Ail experiments todétermine the influence of antibodies on thrombocyte functions were carried out in thepresence of saturated, completely blocking, concentrations of Fab fragments of an antibodyagainst the FcRIIA receptor (clone IV. 3) in order to avoid Fc receptor effects.

Figure 3: Oxidized proteins are hemostatic/prothrombotic - increase of fibrinogen binding to thrombocytes/inhibition of this reaction FITC-conjugated fibrinogen and 10 gg/ml thrombospondin was added to gel-filtered platelets(50000/pg) in HEPES-Tyrode-BSA buffer. A portion of the sample was added with oxidizedproteins in increasing concentrations. After incubation for 30 minutes at RT, the fibrinogenbinding was determined in flow-through cytometry. 3a) Oxidized proteins (herein as examples, oxidized fibrinogen, oxidized human albumin, andoxidized antithrombin III) increase the fibrinogen binding to thrombocytes. 3b) Soluble thrombospondin-1 inhibits fibrinogen binding induced by ox. protein in a dose- dependent manner. 24 01243 1 ί 3c) Antibodies that inhibit binding of oxidized proteins to CD36, inhibit in a dose dépendentmanner the fibrinogen binding to thrombocytes induced by oxidized proteins.

Oxidized protein: oxidized ATIII; antibody: anti-CD36 antibody, clone 37. 5 3d) Oxidized protein: oxidized fibrinogen;antibody: anti-CD36 antibody, clone 37. 3e) Oxidized protein: oxidized human albumin10 antibody: anti CD36 antibody, clone 37.

Ail experiments for the détermination of the influence of antibodies on thrombocyte fonctionswere carried out in the presence of saturated, completely blocking, concentrations of Fabfragments of an antibody against the FcRIIA-receptor (clone IV.3), in order to avoid Fcreceptor effects. 15

Figure 4: Oxidized proteins act hemostatic/prothrombotic/induction of thrombocyte aggregation/inhibition of this reaction

4a) Oxidized proteins induce thrombocyte aggregation. Thrombocyte aggregation was carried20 out according to Bom 1962. To gel-filtered platelets (20000/μ1) in HEPES-Tyrode buffer pH 7.4 with 100 pg/ml fibrinogen, TSP-1 (25 pg/ml) was pipetted in an aggregation cuvette.Soluble TSP-1 alone did not induce aggregation. Simultaneous addition of oxidized proteins(herein oxidized fibrinogen or oxidized antithrombin III) led to a strong aggregate formation.Soluble thrombospondin inhibits in high concentrations > 50 pg/ml the aggregation induced 25 by oxidized proteins. 4b) Antibodies that inhibit binding of oxidized proteins to CD36, inhibit in a dose dépendentmanner the platelet aggregration induced by oxidized proteins. Ail experiments concerningthe influence of antibodies on thrombocyte fonctions were carried out in the presence of 30 saturating, completely blocking, concentrations of Fab fragments of an antibody against theFcRIIA receptor (clone IV.3) in order to avoid Fc receptor effects. 25 012431 ί'

Figure 5: Oxidized proteins act hemostatic/prothrombotic - induction of the pro-coagulated condition of thrombocytes and microparticle formation/inhibition of this reaction 5a) Oxidized proteins (herein as an example oxidized fibrinogen) induce binding of factor5 V/Va to thrombocytes. Factor V/Va binding was carried out as described in Dôrmann et al., 1998. 5b) Oxidized proteins (herein as an example oxidized fibrinogen) induced binding of factorX/Xa to thrombocytes. Factor X/Xa binding was carried out as described by us according to 10 Dôrmann et al., 1998. 5c) Oxidized proteins (herein as an example oxidized fibrinogen) induce phospholipid flip-flop in the membrane and binding of annexin V to thrombocytes. Annexin V binding wascarried out described by us according to Dôrmann et al., 1998. 15 5d) Oxidized proteins (herein as an example oxidized fibrinogen) induce microparticleformation of thrombocytes. Gel-filtered platelets (50000/μ1) were incubated with oxidizedprotein for 30 minutes at RT under slight agitation. Then, the platelets and microparticlesresulting from platelets were incubated for 30 minutes with an anti-GPIX-PE antibody, and 20 the number of resulting microparticles was measured in the ratio to 5000 counted particles ina flow through cytometer. 5e) Soluble thrombospondin inhibits microparticle formation induced by oxidized proteins. Inthis experimental example microparticle formation from thrombocytes was induced by 25 oxidized human albumin. Gel-filtered platelets in HEPES-Tyrode buffer pH 7.4 wereactivated with each 50 g/ml oxidized human albumin for 1 h at RT. Before activation, theplatelet suspension was added with soluble TSP in concentrations as depicted. Microparticleformation was analyzed as described in 5d). Soluble thrombospondin inhibited the formationof microparticle in a dose dépendent manner. 30 5f) Anti-CD36 clone 37 inhibits the ox protein-induced formation of the pro-coagulated condition of the platelets. Annexin V binding as an indicator for the formation of a pro- ► coagulated membrane surface of the thrombocytes, was measured as described under 5c).

Preincubation of the platelets with anti-CD36 antibodies (30 minutes, RT), which inhibits 26 01243 1 r binding of oxidized proteine to CD36, inhibited the subséquent activation of these platelets byoxidized proteins (herein as an example oxidized fibrinogen). Ail experiments regarding theinfluence of antibodies on thrombocyte fonctions were carried out in the presence ofsaturating completely blocking concentrations of Fab fragments of an antibody against the 5 FcRIIA receptor (clone IV.3) in order to avoid Fc receptor effects. 5g) Anti-CD36 clone 37 inhibits the oxidized protein induced microparticle formation ofthrombocytes. Microparticle formation was determined as described under 5d). Preincubationof platelets with anti-CD36 antibodies (30 minutes, RT), which inhibit binding of oxidized 10 proteins to CD36, before activation with oxidized proteins (herein as an example oxidizedfibrinogen) inhibits microparticle formation.

Figure 6: Oxidized proteins activate leucocytes - Ca2+ signal 15 Oxidized proteins (shown herein oxidized antithrombin III) induce a Ca2+ signal inmonocytes. The Ca2+ measurement was carried out according to Sozzani et al., 1993. Elutedmonocytes (5 x 106Zml) were washed at RT with HEPES-Tyrode buffer pH 7.4 andsubsequently marked for 15 minutes with 1 μΜ Fura2/AM at 37°C, washed twice withHEPES-Tyrode buffer without Ca2+ and then suspended in HEPES-Tyrode with 1 mM Ca2+. 20 Ca2+ signal, induced by oxidized proteins and as positive or négative control effectivesubstances were fluorimetrically determined in Hitachi F-2000. Oxidized antithrombin III(100 pg/ml) activâtes monocytes and induces a clear Ca2+ signal.

Figure 7: Oxidized proteins activate leucocytes - oxidative burst 25

Oxidized proteins increase, in a dose-dependent manner, the activating effect of fMLF on theoxidative burst of PMNL, and they even induce oxidative burst as autonomous, independentagonists. Induction of oxidative burst was essentially carried out according to themanufacturera instructions with phagotest/burst test of the company Orpegen (Heidelberg) 30 using a flow-through cytometer. However, PMNL were fïrst incubated with substrateDHR123 and then PMNL were activated. Oxidized antithrombin III increased the activatingeffect of fLMF and itself induced an ox. burst reaction. 27 01243 1

Figure 8: Oxidized proteins activate leucocytes - transmigration through the endothelium/inhibition of this reaction 8a) In a transwell cell culture chamber (Costar, Bodenheim) a transwell insert spanned with a5 microporous polycarbonate membrane was placed on eacb of the 24 wells. The polycarbonatemembrane with a pore size of 5 pm was coated with fïbronectin and human microvascularendothélial cells (HMEC-1) were cultured until confluence. Human monocytes isolated bydensity-gradient-centrifugation (200 μΐ with 2 x 107 cells/ml in DMEM from peripheralblood) were incubated as 37°C, 7 % CO2 with the HMEC-1 monolayer. As a degree for the 10 transmigration rate, the number of monocytes in the lower transwell compartment below thetranswell insert was determined. In order to investigate the influence of different oxidizedproteins, of thrombospondin, or of anti-CD36 antibodies, the test substances were added tothe medium in the upper transwell chamber, or the endothélial cells were preincubated for 10minutes with the test substances and washed. After 4-7 hours of transmigration time the 15 inserts were carefully removed, the cell culture plate were placed on ice for 30 minutes toremove adhered monocytes, and the number of transmigrated monocytes was counted.Oxidized protein (herein oxidized ATIII) promûtes the transmigration of monocytes throughthe HMEC-1 monolayer while the non-oxidized parent protein did not show this reaction(transmigration period 4 h). 20 8b) Preincubation of the endothélial layers for 10 minutes with TSP-1 and addition of TSP-1to the cell culture medium during the transmigration experiment, respectively couldsignificantly inhibit the transmigration of monocytes (transmigration period 7 h). 25 Figure 9: Oxidized proteins induce processes that promote arteriosclerosis

Oxidized antithrombin III increases homing of macrophages in arteriosclerotic plaques. Therealization of the experiment was described in detail in the description of the example. 30 Figure 10: Thrombospondin inhibits proarteriosclerotic processes

Thrombospondin inhibits the adhesion of macrophages, to arteriosclerotically altered endothélial cells. The realization of the experiment is described in detail in the description of the example. 28 012431 ( 10a) Thrombospondin-1 inhibits the transient adhesion of macrophages, which ischaracterized by rolling of macrophages to arteriosclerotically altered endothélium. 5 10b) Thrombospondin inhibits the permanent stable adhesion of macrophages to arteriosclerotically altered endothélium.

Figure 11: Thrombospondin inhibits inflammatorv processes in vivo 10 Soluble thrombospondin-1 inhibits the Arthus reaction in the ear of Balb/c mice. 1 la) Mouse treated twice at time point 0 and 0 + 3 hours with each 50 pg TSP-1 i.p. - inhibitsArthus reaction in left ear. 15 11b) Mice treated twice at time point 0 and 0 + 3 hours with control buffer i.p. - Arthus reaction in left ear. 11c) Incorporated BSA-FITC in the ear as a measure for the Arthus reaction in mice treatedwith TSP-1 and control buffer - Arthus reaction in left ear. 20

Figure 12:

Gel filtered human thrombocytes were diluted with Hepes-Tyrode buffer pH 7.4 to 50000/μ1and activated at room température. In order to avoid Fc receptor effects on the platelets byantibodies, the experiments were carried out in the presence of completely saturating blocking 25 concentrations of Fab fragments of an antibody against the FcRIIA receptor (clone IV.3).Affcer activation, thrombocytes were fixed with 0.1 % paraformaldéhyde in Hepes-Tyrodebuffer pH 7.4 for 30 minutes and washed. Fixed, resting, and activated thrombocytes wereincubated ovemight anti-CD36 "AK36P" and anti-CD36 "AK36-total" in saturatingconcentrations ovemight, respectively, and the thrombocytes were washed and incubated with 30 a secondary, FITC-marked antibody (goat anti-rabbit IgG-FITC, "minimal X reaction withhuman IgG") for 1 h at RT. The thrombocytes were again washed and the binding of anti-CD36 "AK36P" and anti-CD36 "AK36-total" antibodies, respectively, were quantified byFITC fluorescence in a flow-through cytometer (FACScan-Becton Dickinson) (according toDôrmann et al., 1998). 29 072437 (

Decrease of binding of anti-CD36 "AK36P" to thrombocytes by activation.

Figure 13: 5

Blood was drained from patients with diabètes mellitus type I and control persons, and theblood was coagulated with citrate. Platelet-rich plasma (PRP) was prepared by centrifugation.PRP of patients and healthy control persons was mixed with fibrinogen (150 pg/ml) that wascoupled to FITC, and the thrombocytes were activated with oxidized protein (herein oxidized 10 human albumin) in increasing concentrations for 30 minutes. Fibrinogen binding wasmeasured in flow-through cytometer as described above in detail. The figure shows acharacteristic example of the simultaneous détermination of activation with PRP of patientswith diabètes mellitus type I in comparison to Controls. Thrombocytes of patients withdiabètes mellitus type I are particularly sensitive for the activation with oxidized proteins. 15

Figure 14:

The interaction of oxidized proteins and HIV receptor CD4 was determined as described indetail in the description of the example by plasmon résonance technique in the BIACORESystem 2000. 20

Overlay plot of 12 sensorgrams that show the binding of oxidized antithrombin III toimmobiîized CD4 and the dissociation of oxidized antithrombin III. CD4 is immobilized (148pg); the concentration of oxidized antithrombin III was varied (from the bottom to the top: 0nM; 1 nM; 5.1 nM; 10.2 nM; 17 nM; 20.4 nM; 23 nM; 34 nM; 40.8 nM; 51 nM; 85 nM; 119 25 nM). With increasing concentrations of oxidized AT ΠΙ, the resulting signal increases.

Figure 15: 15a) Oxidized protein médiates TSP-1 binding to thrombocytes 30 Gel-filtered human thrombocytes were diluted with Hepes-Tyrode buffer pH 7.4 to 50000/μ1and FITC-conjugated purified thrombospondin-1 (50 pg/ml) was added. The thrombocyteswere incubated for 1 h at RT with oxidized protein (herein oxidized fibrinogen), and TSP-1binding to thrombocytes was measured in flow through cytometer. Oxidized proteins induceTSP-1 binding to thrombocytes. 30 01243 1 ( 15b) Oxidized protein (herein oxidized antithrombin III) induces binding of thrombospondinto endothélial cells.

Human microvascular endothélial cells (HMEC-1) were dissolved from the cell culture plate5 according to standard procedure, dissolved, and the suspension was incubated for 1 h at RTwith oxidized protein and oxidized protein plus TSP-1 addition, respectively. The cells werewashed and bound TSP-1 was marked with mAK anti-TSP-1 (clone P10) coupled tophycoerythrin (PE) and quantified in a flow-through cytometer. Oxidized protein induced TSP-1 binding to endothélial cells. 10 15c) While without the addition of purifïed TSP-1 and without addition of oxidizedantithrombin III only approximately 1 % of the eluted monocytes were detected by anantibody (clone P10), which recognizes TSP-1 on the cell surface, in flow-through cytometer,the amount increased by the addition of purifïed TSP-1 (10 pg/ml) to approximately 5 %. The 15 addition of oxidized AT III (without the addition of exogenous TSP-1) médiates binding ofendogenous TSP-1 to monocytes. About 18 % of the monocytes were TSP-1 positive. By thesimultaneous addition of TSP-1 and oxidized ATIII almost ail of the peripheral bloodmonocytes were strongly positive for TSP-1. 20 15d) Oxidized protein induces binding of TSP-1 to T cells. Cultured human T cells (Jurkat cells) were incubated for 1 h at RT with oxidized protein (herein oxidized antithrombin III) oroxidized protein plus TSP-1 addition (25 gg/ml). TSP-1 bound to T cells was marked by themonoclonal PE conjugated anti-TSP antibody (clone P10) and was measured in the flow-through cytometer. Oxidized proteins induced binding of endogenously présent and 25 exogenously added TSP to T cells.

Figure 16:

This figure shows the mechanism by which médicaments according to daims 1-5 andaccording to claim 21 inhibit or médiate functions that are induced by the reaction of 30 thrombospondin with CD36 (example angiogenesis). The working group of N. Bouckidentified thrombospondin-1 and dérivatives thereof as a potent endogenous inhibitor oftumor angiogenesis, and they showed that this reaction is mediated by CD36 (Dawson et al.,1997; Jimenez et al., 2000). 31 012431 ( 16a) It has been shown by this invention that oxidized proteins médiate binding ofthrombospondin to CD36. Médicaments according to claim 21 therefore induce reactions thatare mediated by this binding, as e.g. inhibition of angiogenesis, a process which can betherapeutically used for the treatment of tumors. 5 16b) In this invention, substances are disclosed that inhibit the interaction of oxidized proteinswith CD36 and therefore processes that are induced in the body by oxidized proteins byCD36. Médicaments according to daims 1-5 inhibit these reactions and prevent angiogenesisinhibition that is induced by the reaction chain oxidized proteins (CD36-conformational 10 change-thrombospondin binding to CD36-CD36-»signal for angiogenesis inhibition. Thisreaction can be therapeutically used if angiogenesis is desired, e.g. in the heart muscle in theevent of an attack.

Reference List 15 1. Abumrad N, Cobum C, Ibrahimi A: Membrane proteins implicated in long-chain fatty aciduptake by mammalian cells: CD36, FATP and FABPm Biochim.Biophys.Acta 1441: 4-13,1999 2. Abumrad NA, el Maghrabi MR, Amri EZ, Lopez E, Grimaldi PA: Cloning of a rat 20 adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte différentiation. Homology with human CD36.J.Biol.Chem 268:17665-17668,1993 3. Acton SL, Scherer PE, Lodish HF, Krieger M: Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J.Biol.Chem 269: 21003-21009,1994 25 4. Aiken ML, Ginsberg MH, Byers-Ward V, Plow EF: Effects of 0KM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surfaceexpressioh [see comments]. Blood 76: 2501-2509,1990 5. Alberts GL, Pregenzer JF, Im WB: Contributions of cysteine 114 of the human D3 dopamine receptor to ligand binding and sensitivity to extemal oxidizing agents.

Br.J.Pharmacol. 125: 705-710,1998 30 32 $1243 ι ( . . 6. Alessio M, Roggero S, Bussolino F, Saitta M, Malavasi F: Characterization of the murinemonoclonal antibody NL07 spécifie for the human thrombospondin receptor (CD36molécule).Curr.Stud.Hematol.BloodTransfos. 182-186,1991 7. Alessio M, Greco NJ, Primo L, Ghigo D, Bosia A, Tandon NN, Ockenhouse CF, 5 Jamieson GA, Malavasi F: Platelet activation and Inhibition of malarial cytoadherence by the anti-CD36 IgM monoclonal antibody NL07. Blood 82: 3637-3647,1993 8. Asch AS, Bamwell J, Silverstein RL, Nachman RL: Isolation of the thrombospondinmembrane receptor. J.Clin.Invest 79:1054-1061,1987 9. Asch AS, Nachman RL: Thrombospondin: phenomenology to fonction. 10 Prog.Hemost.Thromb. 9:157-176,1989 10. Asch AS, Liu 1, Briccetti FM, Bamwell JW, Kwakye-Berko F, Dokun A, Goldberger J,Pemambuco M: Analysis of CD36 binding domains: ligand specificity controled bydéphosphorylation of an ectodomain. Science 262:1436-1440,1993 11. Babior BM: Oxygen-dependent microbial killing by phagocytes (second of two parts). 15 N.EngLJ.Med. 298: 721-725, 1978 12. Babior BM: Oxygen-dependent microbial killing by phagocytes (first of two parts).N.EngLJ.Med 298: 659-668,1978 13. Bamwell JW, Asch AS, Nachman RL, Yamaya M, Aikawa M, Ingravallo P: A human 88-kD membrane glycoprotein (CD36) fonctions in vitro as a receptor for a cytoadherence 20 ' ligand on Plasmodium falciparum-infected eiythrocytes. J.Clin.Invest 84: 765-772,1989 14. Baruch DI, Ma XC, Pasloske B, Howard RJ , Miller LH: CD36 peptides that blockcytoadherence define the CD36 binding région for Plasmodium falcipanim-mïectedérythrocytes. Blood 94: 2121-2127,1999 15. Berendt AR, Ferguson DJ, Gardner J, Turner G, Rowe A, McCormick C, Roberts D, Craig 25 A, Pinches R, Elford BC: Molecular mechanisms of Séquestration in malaria.

Parasitology 108 Suppl: S19-S28,1994 16. Bosmans JL, Holvoet P, Dauwe SE, Ysebaert DK, Chapelle T, Jürgens A, Kovacic V,Van Marck EA, De Broe ME, Verpooten G A: Oxidative modification of Low-density 33 01243 1 ( lipoprotéine and the outcome of rénal allografts at 1 1/2 years. Kidney Int. 59: 2346-2356,2001 17. Boullier A, Gillotte KL, Horkko S, Green SR, Friedman P, Dennis EA, Witztum JL,Steinberg D, Quehenberger O: The binding of oxidized low density lipoprotein to mouse 5 CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein. J.BioI.Chem. 275: 9163-9169,2000 18. Brown MS, Goldstein JL: A receptor-mediated pathway for cholestérol homeostasis.Science 232: 34-47,1986 19. Calvo D, Vega MA: Identification, primaiy structure, and distribution of CLA-1, a novel 10 member of the CD36/LIMPH gene family. J.BioLChem. 268:18929-18935,1993 20. Carr AC, McCall MR, Frei B: Oxidation of LDL by myeloperoxidase and reactivenitrogen species: reaction paîhways and antioxidant protection. Arterioscler.Thromb.Vasc.Biol. 20: 1716-1723,2000 21. Clemetson KJ: Biochemistry of platelet membrane glycoproteins. Prog. Clin. Biol. 15 Res. 283: 33-75,1988 22. Crombie R, Silverstein RL, MacLow C, Pearce SFA, Nachman RL, Laurence J:Identification of a CD36-related thrombospondin 1-binding domain in HIV-1 envelopeglycoprotein gpl20: relationship to HIV-1-spécifie inhibitory factors in human saliva.J.Exp.Med. 187: 25-35,1998 20 23. Daniel JL, Dangelmaier C, Strouse R, Smith JB: Collagen induces normal Signal transduction in platelets déficient in CD36 (platelet glycoprotein IV).Thromb.Haemost. 71: 353-356,1994 24. Davies JM, Horwitz DA, Davies KJ: Potential rôles of hypochlorous acid and N-chloroamines in collagen breakdown by phagocytic cells in synovitis. Free 25 Radic.Biol.Med. 15: 637-643,1993 25. Dawson DW, Pearce SF, Zhong R, Silverstein RL, Frazier WA, Bouck NP: CD36médiates the In vitro inhibitory effects of thrombospondin-1 on endothélial cells. J.CellBiol. 138: 707-717.1997 34 012431 ( 26. Dôrmann D, Kardoeus J, Zimmermann RE, Kehrel B: Flow cytométrie analysis ofagonist-induced annexin V, factor Va and factor Xa binding to human platelets. Platelets9: 171-177,1998 27. Dutta-Roy AK, Crosbie LC, Gordon MJ, Campbell FM: Platelet membrane 5 glycoprotein IV (CD36) is involved in arachidonic acid induced-platelet aggregation.

Biochem.Soc.Trans. 24:167S, 1996 28. Fadok VA, Warner ML, Bratton DL, Henson PM: CD36 is required for phagocytosis ofapoptotic cells by human macrophages that use either a phosphatidylserine receptor orthe vitronectin receptor (alpha v beta 3). J.lmmunol. 161:6250-6257,1998 10 29. Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM: Macrophages that hâve ingested apoptotic cells in vitro inhibit proinflammatory cytokine productionthrough autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.J.Clin.Invest 101: 890-898,1998

30. Febbraio M, Abumrad NA, Hajjar DP, Sharma K, Cheng W, Pearce SF, Silverstein RL: A 15 null mutation in murine CD36 reveals an important rôle in fatty acid and lipoprotein metabolism. J.Biol.Chem 274: 19055-19062,1999 31. Febbraio M, Podrez EA, Smith JD, Hajjar DP, Hazen SL, Hoff HF, Sharma K,Silverstein RL: Targeted disruption of the class B scavenger receptor CD36protects against atherosclerotic lésion development in mice [see commente]. 20 J.Clin.Invest 105: 1049-1056,2000 32. Frieda S, Pearce A, Wu J, Silverstein RL : Recombinant GST/CD36 fusion proteinsdefine a thrombospondin binding domain. Evidence for a single calcium-dependentbinding site on CD36. J.Biol.Chem. 270:2981-2986,1995 33. Glaser CB, Morser J, Clarke JH, Blasko E , McLean K, Kühn 1, Chang RJ, Lin JH, 25 Vilander L, Andrews WH,.: Oxidation of a spécifie méthionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism formodulation of coagulation. J.Clinlnvest 90:2565-2573,1992 34. Greenwalt DE, Watt KW, So O Y, Jiwani N: PAS IV, an intégral membrane protein ofmammary épithélial cells, is related to platelet and endotnelial cell CD36 (GP IV). 30 Biochemistry 29: 7054-7059,1990 35 012431 35. Greenwalt DE, Lipsky RH, Ockenhouse CF, Ikeda H, Tandon NN, Jamieson GA:Membrane glycoprotein CD36: a review of its rôles in adhérence, signal transduction, andtransfusion medicine. Blood80: 1105-1115,1992 36. Hansson M, Asea A, Ersson U, Hermodsson S, Hellstrand K Induction of apoptosis in 5 NK cells by monocyte-derived reactive oxygen métabolites. J.lmmunol. 156: 42-47,1996 37. Hazell LJ, Arnold L, Flowers D, Waeg G, Malle E, Stocker R: Presence of hypochlorite-modified proteins in human atherosclerotic lésions. J.Clin.Invest 97: 1535-1544,1996 38. Holvoet P, Collen D: Oxidized lipoproteins in atherosclerosis and thrombosis.FASEB J. 8: 1279-1284,1994 10 39. Holvoet P, Collen D: Oxidation of Iow density lipoproteins in the pathogenesis of atherosclerosis. Atherosclerosis 137 Suppi: S33-S38,1998 40. Holvoet P, Stassen UM, Van Cleemput J, Collen D, Vanhaecke J: Oxidized low densitylipoproteins in patients with transplant-associated coronaiy artery disease.Arterioscler.Thromb.Vasc.Biol. 18: 100-107,1998 15 41. Holvoet P, Van Cleemput J, Collen D, Vanhaecke J: Oxidized low density lipoprotein is a prognostic marker of transplant- associated eoronary artery disease.Arterioscler.Thromb.Vasc.Biol. 20: 698-702,2000 42. Holvoet P, Mertens A, Verhamme P, Bogaerts K, Beyens G, Verhaeghe R, Collen D,Muls E, Van de WF: Circulating oxidized LDL is a useful marker for identifying patients 20 . with eoronary artery disease. Arterioscler.Thromb.Vasc.Bi.ol. 21: 844-848,2001 43. Huang MM, Bolen JB, Bamwell. JW, Shattil SJ, Brügge JS: Membrane glycoproteinIV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosinekinases in human platelets. Proc.Natl.Acad.Sci.U.S.A 88: 7844-7848,1991 44. Jimenez B, Volpert OV, Crawford SE, Febbraio M, Silverstein RL, Bouck N: 25 Signais leading to apoptosis-dependent Inhibition of neovascularization by thrombospondin-1. Nat.Med. 6: 41-48, 2000 45. Kehrel B, Kronenberg A, Schwippert B, Niesing-Bresch D, Niehues U, Tschope D,van de LJ, Clemetson KJ: Thrombospondin binds normally to glycoprotein Illbdéficient platelets. BiochemBiophys.Res.Commun. 179:985-991,1991 36 01243 1. 46. Kehrel B, Kronenberg A, Rauterberg J, Niesing-Bresch D, Niehues U, Kardoeus J,

Schwippert B, Tschope D, van de LJ, Clemetson KJ: Platelets déficient in glycoprotein Illb aggregate normally to collagens type 1 and III but not to Collagen typeV. Blood 82: 3364-3370,1993 5 47. Kehrel B: Platelet-collagen interactions. Semin.Thromb.Hemost. 21:123-129,1995 48. Kieffer N, Bettaieb A, Legrand C, Coulombel L, Vainchenker W, Edelman L,Breton-Gorius J: Developmentally regulated expression of a 78 kDa erythroblastmembrane glycoprotein immunologically related to the platelet thrombospondinreceptor. BiochemJ. 262: 835-842,1989 10 49. Kielbassa K, Schmitz C, Gerke V: Disruption of endothélial microfilaments selectively reduces the transendothelial migration of monocytes. Exp.Cell Res. 243: 129-141,1998 50. Knowles DM, Tolidjian B, Marboe C, D'Agati V, Grimes M, Chess L: Monoclonal anti-human monocyte antibodies 0KM1 and 0KM5 possess distinctive tissue distributionsincluding differential reactivity with vascular endothélium J. Immunol. 132: 2170-2173, 15 1984 51. Kronenberg A, Grahl H, Kehrel B: Human platelet CD36 (GPIllb, GPIV) binds tocholesteryl-hemisuccinate and can be purified by a simple two-step method making useofthis property. Thromb.Haemost. 79:1021-1024,1998 52. Leung LL, Li WX, McGregor JL, Albrecht G, Howard RJ: CD36 peptides enhance or 20 inhibit CD36-thrombospondin binding. A two-step process of ligand-receptor interaction. J.Biol.Chem 267:18244-18250,1992 53. Li WX, Howard RJ, Leung LL: Identification of SVTCG in thrombospondin as theconformation- dépendent, high affinity binding site for its receptor, CD36. J.Biol.Chem268: 16179-16184,1993 25 54. Lian EC, Siddiqui FA, Jamieson GA, Tandon NN: Platelet agglutinating protein p37 causes platelet agglutination through its binding to membrane glycoprotein IV.Thromb.Haemost. 65: 102-106,1991 55. McGregor JL, Clemetson KJ, James E, Luscher EF, Dechavannne M: Characterization of human blood platelet membrane proteins and glycorproteins by their isoelectric point „ 072431 ( . (pl) and apparent molecular weight using two-dimensional electrophoresis and surface- labelling techniques. BiochimBiophys. Acta 599:473-483,1980 56. McGregor JL, Clemetson KJ, James E, Capitanio A, Greenland T, Luscher EF,Dechavanne M: Glycoproteins of platelet membranes from Glanzmann's thrombasthenia. 5 A comparison with normal using carbohydrate-specific or protein-specifïc labelling techniques and high-resolution two-dimensional gel electrophoresis. Eur.J.Biochem 116:379-388,1981 57. Nakaia A, Nakagawa Y, Nishida M, Nozaki S, Miyagawa J, Nakagawa T, Tamura R,Matsumoto K, Kameda-Takemura K, Yamashita S, Matsuzawa Y: CD36, a novel 10 receptor for oxidized Iow-density lipoproteins, is highly expressed on lipid-laden macrophages in human atherosclerotic aorta ArterioscIer.Thromb.Vasc.Biol. 19:1333-1339,1999 58. Nguyen-Khoa T, Massy ZA, Witko-Sarsat V, Canteloup S, Kebede M, Lacour B , DruekeT, Descamps-Latscha B: Oxidized Iow-density lipoprotein induces macrophage 15 respiratory burst via its protein moiety: A novel pathway in atherogenesis"

Biochem.Biophys.Res.Commun. 263: 804-809,1999 59. Nicholson AC, Frieda S, Pearce A, Silverstein RL: Oxidized LDL binds to CD36 onhuman monocyte-derived macrophages and transfected cell lines. Evidence implicatingthe lipid moiety of the lipoprotein as the binding site. ArterioscIer.Thromb.Vasc.Biol. 15: 20 269-275,1995 60. . Nozaki S, KashiwagiH, Yamashita S, Nakagawa T, KostnerB, Tomiyama Y, Nakata A,

Ishigami M, Miyagawa J, Kameda-Takemura K: Reduced uptake of oxidized Iow densitylipoproteins in monocyte-derived macrophages from CD36-defïcient subjects.J.Clin.Invest 96: 1859-1865,1995 25 61. Nozaki S, Tanaka T, Yamashita S, Sohmiya K, Yoshizumi T, Okamoto F, Kitaura Y,

Kotake C, Nishida H, Nakata A, Nakagawa T, Matsumoto K, Kameda-Takemura K,

Tadokoro S, Kurata Y, Tomiyama Y, Kawamura K, Matsuzawa Y: CD36 médiates long- chain fattv acid transport in human myocardium: complété myocardial accumulation defect of radiolabeled. long-chain fatty acid analog in subjects with CD36 deficiency. 30 Mol.Cell Biochem. 192: 129-135,1999 38 fil 24 3 1 62. Ockenhouse CF, Tandon NN, Magowan C, Jamieson GA, Chulay JD: Identification of aplatelet membrane glycoprotein as a falciparum malaria séquestration receptor [seecomments]. Science 243:1469-1471,1989 63. Oquendo P, Hundt E, Lawler J, Seed B: CD36 directly médiates cytoadherence of 5 Plasmodium falciparum parasitized érythrocytes. Cell 58:95-101,1989 64. Patel SS, Thiagarajan R, Willerson JT, Yeh ET: Inhibition of alpha4 integrin and ICAM-1 markedly attenuate macrophage homing to atherosclerotic plaques in ApoE-deficientmice. Circulation 97: 75-81,1998 65. Pearce SF, Roy P, Nicholson AC, Hajjar DP, Febbraio M, Silverstein RL: Recombinant 10 glutathione S-transferase/CD36 fusion proteins define an oxidized low density lipoprotein-binding domain. J.BioLChem 273:34875-34881,1998 66. Podrez EA, Febbraio M, Sheibani N, Schmitt D, Silverstein RL, Hajjar DP , Cohen PA,Frazier WA, Hoff HF, Hazen SL: Macrophage scavenger receptor CD36 is the majorreceptor for LDL modifïed by monocyte-generated reactive nitrogen species [see 15 comments] [published erratum appears in J Clin Invest 2000 May;105(10):1483], J.Clin.Invest 105:1095-1108,2000 67. Puente N, Daviet L, Ninio E, McGregor JL : Identification on human CD36 of a domain(155-183) implicated in bihding oxidized low-density lipoproteins (Ox-LDL).Arterioscler.Thromb.Vasc.Biol. 16:1033-1039,1996 20 68. Ricciarelli R, Zingg JM, Azzi A: Vitamin E reduces the uptake of oxidized LDL by inhibiting CD36 scavenger receptor expression in cultured aortic smooth muscle cells.Circulation 102: 82-87,2000 69. Rigotti A, Acton SL, Krieger M: The class B scavenger receptors SR-B1 and CD36 arereceptors for anionic phospholipids. J.Biol.Chem 270: 16221-16224,1995 25 70. Roberts DD, Sherwood JA, Spitalnik SL, Panton LJ, Howard RJ, Dixit VM, Frazier WA,

Miller LH, Ginsburg V: Thrombospondin binds falciparum malaria parasitizedeiythrocytes and may médiate cytoadherence. Nature 318: 64-66,1985 71. Ryeom SW, Sparrow JR, Silverstein RL: CD36 participâtes in the phagocytosis of rod outer segments by retinal pigment epithelium. J.Cell Sei. 109 ( R 2): .387-395,1996 39 01243 1 72. Saelman EU, Kehre! B, Hese KM, de Groot PG, Sixma JJ, Nieuwenhuis HK: Plateletadhesion to collagen and endothélial cell matrix under flow conditions is not dépendenton platelet glycoprotein IV. Blood 83: 3240-3244,1994 73. Samanta A, Das DK, Jones R, George A, Prasad MR: Free radical scavenging by 5 myocardial fatty acid binding protein. Free Radic.Res. Commun. 7: 73-82,1989 74. Schraufstatter IU, Browne K, Harris A, Hyslop PA, Jackson JH, Quehenberger O,Cochrane CG: Mechanisms of hypochlorite injury of target cells. J.Clin.Invest 85: 554-562,1990 75. Schuepp BJ, Pfister H, Clemetson KJ, Silverstein RL, Jungi TW: CD36-mediated signal 10 transduction in human monocytes by anti-CD36 antibodies but not by anti- thrombospondin antibodies recognizing cell membrane-bound thrombospondin.Biochem.Biophys.Res.Commun. 175:263-270,1991 76. Shah MM, Aust SD: Oxidatiôn of halides by peroxidases and their subséquent réductions:Arch.BiochemBiophys. 300: 253-257,1993 15 77. Shattil SJ, Brügge JS: Protein tyrosine phosphorylation and the adhesive functions of platelets. Curr.Opin.Cell Biol. 3: 869-879,1991 78. Silverstein RL, Baird M, Lo SK, Yesner LM: Sense and antisense cDNA transfection ofCD36 (glycoprotein IV) in melanoma cells. Rôle of CD36 as a thrombospondin receptor.J.Biol.Chem. 267:16607-16612,1992 20 79. Smith MA, Rottkamp CA, Nunomura A, Raina AK, Perry G; Oxidative stress in

Alzheimer's disease. Biochim.Biophys.Acta 1502: 139-144,2000 80. Stadtman ER: Protein oxidation and aging. Science 257: 1220-1224, 1992 81. Steinberg D: Low density lipoprotein oxidation and its pathobiological significance.J.Biol.Chem. 272: 20963-20966, 1997 25 82. Tandon NN, Kralisz U, Jamieson GA: Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion. J.Biol.Chem. 264: 7576-7583, 1989 83. Tandon NN, Ockenhouse CF, Greco NJ, Jamieson GA: Adhesive functions of platelets lacking glycoprotein IV (CD36). Blood 78: 2809-2813,1991 40 01243) 84. Vega MA, Segui-Real B, Garcia JA, Cales C, Rodriguez F, Vanderkerckhove J,

Sandoval IV:.CIoning, sequencing, and expression of a cDNA encoding rat LIMP II, a novel 74-kDa lysosomal membrane protein related to the surface adhesion protein CD36. J.Biol.Chem 266:16818-16824,1991 5 85. Volf 1, Bielek E, Moeslinger T, Koller F, Koller E: Modification of protein moiety of human Iow density lipoprotein by hypochlorite generates strong plateletagonist. Arterioscler.Thromb.Vasc.Biol. 20: 2011-2018,2000 86. Weiss SJ: Tissue destruction by neutrophils. N.Engl.J.Med. 320: 365-376,1989 87. Witztum JL, Steinberg D: Rôle of oxidized Iow density lipoprotein in atherogenesis. 10 J.Clin.Invest 88: 1785-1792,1991 88. Yamaguchi A, Yamamoto N, Akamatsu N, Saldo TC, Kaneda M, Umeda M, TanoueK: PS-liposome and οχ-LDL bind to different sites of the immunodominant domain(#155-183) of CD36: a study with GS95, a new anti-CD36 monoclonal antibody.Thromb.Res. 97: 317-326,2000 15

Claims (18)

1. 41 012431 ί 1. 5 2. 10 3. 15 4. 20 5. 6. 25 -7. 8. 30 9. K 35 Claims Médicament for humans and animais, characterized in that it comprises substances that inhibit the binding of oxidized proteins to CD36 or that inhibit functions of CD36 induced by interaction of CD36 with oxidized proteins. The médicament of daim 1, in particular antibodies that inhibit the binding ofoxidized protein to CD36; most preferably monoclonal antibodies including fragmentsF(ab)2, F(ab), régions for antigen récognition. The médicament of claim 1, in particular peptides of CD36, peptide mimetics, analogs,characterized in that they inhibit binding of CD36 to oxidized proteins (these can e.g.be identified in that they react with monoclonal anti CD36 antibodies clone 37, 13 or7, which are mentioned in the invention). The médicament of claim 1, in particular proteins that inhibit binding of CD36 tooxidized proteins or which compete with thrombospondin bound to CD36; mostpreferably soluble thrombospondin. The médicament of claim 1, in particular peptides and peptide mimetics, characterizedin that they bind to CD36 or inhibit the functions that are mediated by interaction ofCD36 with oxidized proteins (these may be easily identified, e.g. by phage display). Use of a médicament of any of claims 1 - 5 for prophylaxis of thrombosis, inparticular in inflammatoiy diseases. Use of a médicament of any of claims 1 - 5 for support of an anti-thrombotic therapy. Use of a médicament of any of claims 1 - 5 for preventing rejection of a transplant. Use of a médicament of any of claims 1 - 5 for preventing a transplant mediatedarteriosclerosis. Use a of a médicament of any of claims 1 - 5 for preventing high blood pressures in kidney diseases, preferably of renin-mediated high blood pressure. 42 01243 1 (
2. 11. Use of a médicament of any of daims 1 - 5 for preventing the development and the progression of arteriosclerotic (atherosclerotic) diseases.
3. 12. Use of a médicament of any of daims 1 - 5 for treatment of chronic inflammatory reactions.
4. 13. Use of a médicament of any of daims 1 - 5 for preventing an early vessel reocclusionafter bypass surgery, stent, PTCA or the like. 10
5. 14. Use of a médicament of any of daims 1 - 5 for preventing a vessel restenosis afterbypass surgery, stent, PTCA or the like.
6. 15. Use of a médicament of any of daims 1 - 5 as a médicament for preventing 15 reperfusion damages, as with, but not limited to myocardial ischemia, organ transplantation, stroke, peripheral occlusive disease after surgery, multi organ failureafter successful réanimation.
7. 16. Use of a médicament of any of daims 1 - 5 as a médicament for preventing vessel 20 damages, preferably in patients with diabètes mellitus.
8. 17. Use of a médicament according to any of daims 1 - 5 as a médicament for preventingan inhibition of endothélial prolifération and angiogenesis mediated by oxidizedproteins by CD36/TSP-1. 25
9. 18. Use of a médicament of any of daims 1 - 5 as a médicament for support of woundhealing.
10. 19. Method for the quantification of ox. proteins (preferably ox human albumin, ox 30 fibrinogen), individually or together for the évaluation of the individual indication of therapy with médicaments of any of daims 1-5, for the diagnosis of diseases, in which inflammatory reactions play a rôle, as in, but not restricted to, arteriosclerosis, diabetic vasculopathy, rheumatic arthritis, Goodpasture Syndrome, sepsis, Colitis ulcerosa, graft-versus-host diseases, pemphigus, cancer, neurodermatitis, HIV 43 J01243 1 infection, ARDS, glomerulonephritis, reperfusion damages and for quality control of blood products.
11. 20. Characterization of the activated condition of CD36, the receptor for oxidized 5 proteins, as diagnostics for diseases, in which inflammatory reactions play a rôle, as with, but not limited to, arteriosclerosis, diabetic vasculopathy, rheumatic arthritis,Goodpasture Syndrome, sepsis, Colitis ulcerosa, graft-versus-host diseases,pemphigus, cancer, neurodermatitis, HIV infection, ARDS, glomerulonephritis,reperfusion damages, or for monitoring of a CD36/ox protein inhibition therapy with 10 médicaments of any of daims 1 - 5 by measuring the phosphorylation condition of CD36.
12. 21. Médicament comprising oxidized protein/oxidized proteins, oxidized peptide,structural analogs or "mimetics". 15
13. 22. The médicament of any of daims 1 - 5 and 21, characterized in that it furthercomprises pharmaceutically acceptable fïllers and/or excipients.
14. 23. The médicament of any of daims 1-5 and 21, characterized in that it is formulated 20 for local, intradermal, topical, intraperitoneal, intravenous, oral or intramuscular administration or it is administered by vesicles.
15. 24. The médicament of any of daims 1 - 5 and 21, characterized in that it comprisesfurther substances, as e.g. antibiotics, immunosuppressants or interaction partners of 25 oxidized proteins in the body.
16. 25. Method for manufacturing oxidized protein/peptide by reacting with HOC1 orperoxynitrites.
17. 26. The use of a médicament of daim 21 for prophylaxis or therapy of acute infections, for inhibition of angiogenesis and for improvement of hemostasis.
18. 27. The use of daim 26, characterized in that as an acute infection an HIV infection is prevented or treated. f 28. 5 29. 44 .01243 1 The use of claim 26, characterized in that by oxidized protein, a tumor angiogenesis isinhibited by induction of TSP binding to CD36. The use of claim 26, characterized in that it is used for hemostasis, preferably inpatients with innate or acquired blood coagulation disorders or innate or acquiredthrombocytopathia, under anticoagulation therapy or thrombosis prophylaxie or withsurgery under heart-lung-machine. 10