Note: Descriptions are shown in the official language in which they were submitted.
translation vw t12/3244M2
Applicant: KEHREL, B and BRODDE, M.
CA 02453140 2004-01-09
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
Description
The present invention relates to substances that inhibit the binding of
oxidized proteins to
CD36 or that inhibit functions of CD36 induced by the interaction of CD36 with
oxidized
proteins, and their use as a medicament for humans and animals.
Oxidative modification of proteins is regarded as a critical step for the
pathogenesis of various
diseases, which range from atherosclerosis (arteriosclerosis) and
neurodegenerative diseases
to the process of aging itself (Holvoet and Cohen, 1997 Curr Opin Lipidol,
Witztum and
Steinberg 1991, J Clin Invest 88, 1785-92, Smith MA et al., 1996, Nature,
Oxidative damage
in 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 the
development of arteriosclerotic vessel diseases (Brown MS, Goldstein IL, 1986,
Science 232:
34-37).
However, today there is overwhelming evidence to believe that not LDL itself
but its oxidized
form is the decisive 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 that
measurement of cholesterin, triglycerides, and lipoproteins, as risk markers
for developing
arteriosclerosis is not sufficient, because approximately half of all heart
diseases based on
arteriosclerosis is present in patients who show normal plasma triglyceride
and normal
cholesterin values, and because arteriosclerosis can also be demonstrated
angiographically in
patients with normal lipid values. Therefore, processes that have not yet been
published must
play a causal role in the development of arteriosclerosis.
Oxidation of lipids in LDL, either in vitro, e.g. by copper induced oxidation,
or in vivo, leads
to the formation of reactive aldehydes (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).
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CA 02453140 2004-01-09
Oxidation of the lipid portion of LDL is regarded as responsible for this
process. Therefore,
oxLDL is induced by almost all scientists in order to investigate the
forrnation of
arteriosclerosis, by the addition of CuSO4 or malonedialdehyde, an end product
in lipid
peroxidation. The concentration of oxLDL in plasma of patients with coronary
primary heart
disease or transplantation associated coronary heart disease corresponds
closely to the
progression of the disease, while in healthy control persons no elevated oxLDL
levels can be
measured (Holvoet et al., Circulation 98: 1487-94, 1998). Also chronic kidney
diseases and
transplant rejections are associated with high oxLDL levels (Holvoet, Cohen
Thromb
Haemost 76(5): 663-9, 1996 + ATVB 18(1) 100-7, 1998).
Also oxidations of the protein portion of LDL can lead to
physiological/pathophysiological
modifications. Thus, delipidated HOC1-oxLDL induces oxidative burst in
macrophages
(Nguyen-Khoa et al., BBRC 263: 804-9, 1999) and HOC1-oxLDL leads to
thrombocyte
aggregation (Volf et al., ATVB 20(8): 2011-18, 2000).
Reactive oxidants can be released from the body by phagocytes and play a
decisive role in the
defense against pathogenic agents, tumor monitoring and all inflammatory
processes (Babior,
NEJMed 298: 659-663, 1978, Weiss J, NEJMed 320: 365-376, 1989). Besides
"professional"
phagocytes, like granulocytes and monocytes, other cells like e.g. endothelial
cells or smooth
muscle cells also produce and release reactive oxidants.
Among these oxidative substances are 02, superoxide, hydrogen peroxide,
peroxynitrite, OH-
radicals, hypochloric acid HOC1, C12-gas and chloramine. It remains unclear
which processes
in detail contribute to the development of these oxidative substances.
Ceruloplasmine, 15-
lipooxygenase, myeloperoxidase (MPO) and nitric oxide synthase (NO-S) were
found in
arteriosclerotical lesions in animals and humans and may contribute to
oxidation of LDL
(Carr et al., ATVB 20:1716-23, 2000). A possible oxidation pathway involves
MPO. MPO, a
heme-protein enzyme can halogenate and peroxidate (Carr et al.,). The best-
described product
of myeloperoxidase is hypochloric acid HOC1-, Cl- + H202 + H4 -4 HOC1 + H2O.
Hypochlorite-modified proteins, in particular HOC1-oxLDL, are found in
arteriosclerotical
lesions (Haze11 et al., 1996). HOC1 modification of proteins also plays a role
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
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CA 02453140 2004-01-09
90(6): 2565-73, 1992), tissue destruction mediated by granulocytes in
inflammatory reactions
in general (Schraufstatter et al., J Clin Invest 85(2): 554-62, 1990),
ischemia, reperfusion
damage (Samanta et al., Fee Radic Res Commun 7(2): 73-82, 1989),
glomerulonephritis
(Shah et al., J Clin Invest 79(1): 25-31, 1987), and immune regulation (NK-
cell-apoptosis)
(Hansson et al., J Immunol 156(1): 42-7, 1996).
CD36, also named glycoprotein II% (GPIIlb) or glycoprotein IV (GB IV), is a
major
glycoprotein of platelets, endothelial cells, monocytes, erythroblasts,
epithelial cells, and
some tumor cell lines such as melanoma cells and osteosarcoma cells (Asch et
al J. Clin.
Invest. 79:1054-1061 (1987), Knowles et al., J. Immunol. 132, 2170-2173
(1984), Kieffer et
al., Biochem. J. 262:835-842 (1989)). CD36 belongs to the family of class B
scavenger
receptors. Members of the family also include the integral lysosomal membrane
protein
LIMP-II (lysosomal integral membrane protein IL Vega et at., 1991), the CLA-1
(CD36 and
LIMP-II analogous, Calvo and Vega 1993), the FAT protein of adipocyte membrane
(Abumrad et al., 1993), PAS IV from breast epithelial 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 integral membrane protein of lactating breast epithelial
cells, and is
concentrated in apical plasmalemma. With secretion of triglycerides, it
reaches the milk and is
found in the milk fat globule membrane (MFGM) fraction. The sequence of PAS IV
is almost
identical to CD36, but there are differences in glycosylation.
SR-B1 is a scavenger receptor for LDL (Acton et al., 1994). CD36 consists of a
single heavy
glycosylated polypeptide chain with an apparent molecular weight of 88,000 in
reduced and
non-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 determine a clearly
defined
isoelectric point is the variable content of sialylic acid (McGregor et al.,
1981). The
carbohydrate portion of 26 % and a strong hydrophobicity provides CD36 with a
high
resistance towards degradation 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 regions in which inflammatory processes take place. CD36 has N- and
0-linked
glycosidic modifications. The amino acid sequence for CD36 derived from
placenta cDNA
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(Oquendo et al., 1989) shows multiple hydrophobic regions and two presumably
transmembrane regions.
Certain functions have been postulated for CD36. It has been described as a
receptor for
collagen (Tandon et al., 1989). Purified CD36 binds to fibrils of collagen
type I, and Fab
fragments of a polyclonal antibody directed against CD36 inhibit collagen-
induced
aggregation.
However, analysis of platelets, which lack CD36, show that CD36 is not
strictly required for
activation of platelets by collagen. Our joint experiments with colleagues of
the group of J.J.
Sixma (Utrecht) showed no difference in adhesion of CD36-deficient platelets
and control
platelets to bovine or human collagen type I or III in a static system or
under the influence of
low, intermediate, and high shear rates in a perfusion chamber when using
heparin blood and
physiological Ca2+ concentrations (Saelman et al., 1994).
CD36 deficient platelets aggregate normal with horn collagen, a mixture of
equine type I and
type III collagen, and with purified bovine and human collagen type I and III
(Kehrel et al.,
1991 and 1993). The secretion of a-granulae and dense granulae induced by type
I or III
collagen is not different in CD36 deficient platelets and control platelets
(Kehrel et al., 1993).
Daniel and coworkers showed that the signal transduction after activation with
collagen type I
in CD36 deficient 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.
Dephosphorylation allows
the binding of thrombospondin (Asch, Science 1993). Purified CD36 binds
specifically to
thrombospondin. This binding is Ca2+-dependent and cannot be inhibited by RGE
peptides.
The monoclonal antibody OKM5, which is directed against CD36, inhibits binding
of
thrombospondin to platelets activated by thrombin (Asch et al., 1989). Leung
and coworkers
reported that two peptide regions on CD36 influence binding of thrombospondin.
Peptide
139-155 enhances platelet aggregation in platelet-rich plasma, which has been
induced by
ADP or collagen. However, peptide 93-110 partly inhibits collagen-induced
aggregation, and
also blocks binding of CD36 to immobilized thrombospondin. This peptide is not
able to bind
thrombospondin 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
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affinity (Li et al., 1993). Silverstein et al., (1992) demonstrated the
relevance of CD36 for
thrombospondin binding by experiments with "sense" and "antisense" transfected
melanoma
cells. The binding site for thrombospondin on CD36 is between amino acids 93-
120 (Frieda et
al., 1995).
CD36 is also described as a binding mediator between platelets, endothelial
cells, monocytes,
or C32 melanoma cells on the one hand and erythrocytes infected with malaria
parasite
Plasmodium falciparum on the other hand (Barnwell et al., 1989). Binding of
infected
erythrocytes to caterpillar endothelial cells, called sequestration, is of
decisive significance for
the often deadly end of malaria tropica, if sequestration takes place in the
brain (celebral
malaria). Infected erythrocytes bind to immobilized purified CD36 (Ockenhouse
et al., 1989).
COS cells in which cDNA for CD36 is expressed are capable of binding to
malaria-infected
erythrocytes (Oquendo et al., 1989). With the help of anti-idiotype antibodies
against the
monoclonal anti-CD36 antibody OKM5, a binding partner on infected
erythrocytes, the
sequestrine was found (Ockenhouse et al., 1991). Contact with infected
erythrocytes activates
platelets and monocytes (Ockenhouse et al., 1989). CD36-deficient platelets do
not show any
binding capability for infected erythrocytes in the hands of Tandon et al.,
(1991). In contrast,
we have observed that binding is only disrupted in the presence of EDTA. In
the presence of
Ca and and Mg2+ we could clearly observe rosetting of Plasmodium faktparum-
infected
erythrocytes and CD36-deficient platelets (Kronenberg et al., 1992). Besides
CD36, further
binding mediators for malaria infected erythrocytes have been described and
among these are
thrombospondin, which needs bivalent cations for this function (Berendt et
al., 1989, Roberts
et al., 1985). Thrombospondin might be able to mediate the binding of CD36-
deficient
platelets to infected erythrocytes, which was observed by us. Binding of
Plasmodium
falciparum-infected erythrocytes to CD36 is inhibited by CD36 peptides 145-171
and 156-
184 (Baruch et al., 1999). Also, a role of CD36 in signal transduction is
often discussed
(Shattil and Brugge 1991). In immunoprecipitation of CD36 from resting
platelets, tyrosine
kinases of the src gene family pp6OfY", pp62Yes and pp54/581" are
coprecipitated, which
indicates a close association with CD36 (Huang et al., 1991). The meaning of
this discovery is
still unclear. Some antibodies against CD36 activate platelets and monocytes
(Aiken et al.,
1990, Schiiepp et al., 1991). IgM antibody NLO7 activates platelets with the
help of the
complement system (Alessio et al., 1993). As a further function of CD36, its
role in
thrombotic thrombocytopenic purpura (TTP) is described (Lian et al., 1991). A
protein (p37),
which is present in plasma of TTP patients, agglutinates platelets, mediated
by CD36. The
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CA 02453140 2004-01-09
meaning of this finding is still unknown. Peptide VTCG from thrombospondin
inhibits the
phosphatidylinositol (3,4) bisphosphate synthesis in platelets activated with
thrombin. CD36
is one of the thrombospondin receptors, which mediate the later activation of
PI-3-kinase and
phospholipase C (Trumel, Payrastre, Mazarguil, Chap, Plantavid, personal
communication).
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 of
fatty acids, increased fatty acid oxidation by contractile muscles, and
reduced the
concentration of triglycerides and free fatty acids in the plasma. The mice
had reduced body
weight, 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 are
the main energy source for the heart muscle, in heart muscle cells, and
consequently to
elevated appearance of innate hypertrophic cardiomyopathies (Fuse et al.,
1998). Also, CD36-
deficient mice show a defect in the transport of fatty acids in cells and a
disturbed lipoprotein
metabolism (Febrraio et al., 1999).
CD36 mediates the arachidonic acid-mediated platelet aggregation (Dutta-Roy et
al., 1996)
and binds to negatively charged phospholipids in cell membranes (Ryeom et al.,
1996). In
particular, phosphatidylserine (PS) and phosphatidylinositol (PI) are
specifically bound by
CD36 with high affinity (Rigotti et al., 1995). Because apoptotic cells
express
phosphatidylserines on their surface, contact with phagocytotic cells can be
mediated by
CD36 (Fadok et al., 1998, Alberts et al., 1998). Phosphatidylserine presumably
binds to the
CD36 sequence 162-183 (Yamaguchi et al., 2000). CD36 binds to cholesteryl
hemisuccinate
and can be easily purified by this reaction (Kronenberg et al., 1998).
The main function of CD36 might be its role as receptor for oxidized LDL. This
role was first
described by Endemann et al., 1993. Transfection experiments with a cDNA
clone, which
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 OKM5 inhibits binding of Cu2+-oxLDL to platelets by 54 %. The binding
site for
Cu2+-oxLDL lies in the region of the 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
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al., 1995). Macrophages from blood donors, which are deficient for CD36 on
monocytes,
have significantly reduced (-40%) uptake of oxLDL in comparison to controls
(Nozaki et al.,
1995).
Vitamin E (alpha-tocopherol) inhibits the uptake of Cu2+-oxLDL in smooth
muscle cells of
the aorta by inhibiting CD36 (Ricciarelli et al., 2000). The binding of oxLDL
to murine CD36
is partly prevented by oxidized phospholipids, which are associated with the
lipid and protein
portion (Boullier et al., 2000). It has recently been found that CD36 is not
only the receptor
for Cu2+-oxLDL, but also for NO2-LDL, and that CD36 is responsible for NO2-LDL-
mediated
foam cell formation (Podrez et al., 2000).
This binding is competitively inhibited by oxidation products of the lipid 1-
palmitoy1-2-
arachidonyl-sn-glycero-3-phosphocholine. Therefore, the authors speculate that
the
myeloperoxidase-catalyzed peroxidation of lipids is required to mark
phospholipid containing
targets for phagocytosis by CD36 containing cells. In accord with its role as
receptor for
oxLDL, CD36 is found on macrophages loaded with lipids in arteriosclerotic
plaques (Nakata
et al., 1999), and smooth muscle cells may develop to foam cells by expression
of CD36 in
vivo (Ohya et al., 2000).
Lack of CD36 seems to be a protection against arteriosclerosis. Therefore,
mice that lack
ApoE protein develop arteriosclerotic plaques under a corresponding diet. If
the CD36 gene in
these mice is also knocked out then, under the same conditions as their CD36
containing
control relatives, the mice develop 76 % less arteriosclerotic lesions in the
aortic tree under a
fat rich diet, and 45 % less plaques in the aortic sinus under normal diet.
Macrophages of CD36- and ApoE-double knock-out mice internalize less than 40%
of copper
oxidized LDL and NO2-LDL (Febbraio et al., 2000).
Blocking thrombotic and arteriosclerotic functions of CD36, while
simultaneously not
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 present invention. In the context of the present
invention it has
been found that cell functions that oxLDL triggers through CD36 can also be
triggered by
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other substances. Surprisingly, these other substances are oxidized proteins,
which do not
need to have 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 not
only triggered by oxLDL, but are also triggered by other oxidized proteins and
CD36:
(1) Activation of thrombocytes
¨> on the one hand, thrombosis, heart attack, and stroke, but on
the other hand,
also hemostasis
(2) Damage of endothelial cells
¨> ischemia, inflammatory reactions, edema formation, and
disturbance of
prostacyclin release
(3) Activation of leucocytes, e.g.:
a) elevated adhesion of leucocytes to endothelial cells;
b) support of transmigration of leucocytes through endothelium 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
reaction
triggered by lipopolysaccharide (LPS).
¨> damages by inflammatory reaction, thrombosis etc.
(4) Activation of smooth muscle cells (SMCs):
a) proliferation of SMCs, and
b) intimal swelling in arteriosclerotic plaques.
reocclusion after bypass, stent, PTCA; development of arteriosclerosis
(5) Stimulation of renin release from juxta-glomerulic cells
¨> renin dependent high blood pressure in kidney diseases.
(6) Formation of foam cells by stimulation of the uptake of coincubated
LDL by
macropinocytosis.
¨> development/increase of arteriosclerosis
(7) Apoptosis of vessel cells in the center of arteriosclerotic lesions.
¨> necrosis, plaque rupture
(8) Stimulation of the expression of matrix metalloproteinases in
endothelial cells.
¨> stimulation of rupture of arteriosclerotic plaques.
CA 02453140 2010-04-28
9
Additionally, it is shown by this invention that not only does IDAAT (immune
defense
activated antithrombin, patent application No. DE 100 45 047.4) bind to HIV
GP120 and to
CD4, but it also binds to other oxidized proteins. HIV GP120 comprises a CD26
homologue
sequence (Crombie et al., 1998).
It has also been shown by this invention that not only IDAAT but also other
oxidized proteins
mediate the binding of thrombospondin to cells like thrombocytes, monocytes,
endothelial
cells, and T cells. Therefore, there is a compelling reason to believe that
oxidized proteins in
general induce thrombospondin mediated cell reactions, like the inhibition of
angiogenesis,
the defense of HIV infections, the regulation of inflammatory processes by
e.g. down
regulation of IL-12 in monocytes, and upregulation of IL-10. Hence, oxidized
proteins
themselves can have a therapeutically useful activity in certain disease
processes.
Further, it has been shown in the context of the present invention that
pathological cell
functions mediated by oxidized proteins can be inhibited by substances that
inhibit the
interaction between CD36-oxidized proteins, or which can interfere with TSP
bound to CD36.
Examples of such substances include soluble thrombospondin-1 and monoclonal
anti-CD36
antibody clones 37, 13, and 7, which were manufactured by us and are disclosed
herein.
Examples:
1. Isolation of human thrombospondin-1
Isolation of human thrombospondin-1 from thrombocytes was carried out as
described in
Kehrel et al., 1991. However, in contrast thereto, the platelets were
activated with thrombin,
and EDTA in the wash buffer was substituted by Na-citrate (0.08 M).
Additionally, the
aggregation buffer and all buffers for the following purification steps were
substituted with
Ca2+ at a concentration of 2 mM.
2. Hybridoma culture for the preparation of monoclonal antibodies
The preparation of monoclonal antibodies against CD36 was broadly carried out
according to
the instructions of Peters and Baumgarter (1990).
CA 02453140 2010-04-28
8 - 12 weeks old Balb/c female mice were immunized with purified CD36 (50
pg/boost). For
immunization, the long immunization protocol (4 months) according to
Baumgartner et al.,
1990, was used. Approximately 14 days before the planned fusion time point,
blood was
taken from the animals, and the IgM and IgG titers against CD36 in the serum
of the mice
5 were determined. If the IgG titer was still significantly different from
the control at dilutions
of 1:100,000, spleen cells were fused with Ag 8,653 cells. Ag 8,653 cells were
selected in
culture medium containing 0.13 M 8-azaguanine. Lymphocytes from the spleen
were
prepared and fused with Ag 8,653. Directly after the fusion, fused cells (1 x
106 spleen
cells/nil) were incubated for 24 hours in selection medium (culture medium
with the addition
10 of hypoxanthine, 27.2 pg/ml, and amserine (50 1.tg/m1)) in a cell
culture flask (75 ml).
Thereby, macrophages derive from the spleen attach to the plastic surface and
no longer
interfere 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
cells per well of the 96 well plate according to Wiirzner 1990. Supernatants
of hybridoma
were tested in an ELISA for the production of IgG and IgM, respectively. IgG
positive cell
culture supernatants were tested for their specificity against CD36 with
different methods:
19 CD36 specific clones were obtained, which did not react with CD36 deficient
platelets
(description: Kehrel et al., 1993, Saelman et al., 1994, Kehrel et al., 1995),
but which showed
significant binding to control platelets. This was proven by flow-through
cytometry and by
the dot blot method and immunoprecipitation. All antibodies tested recognized
purified
CD36. Three of these clones (clone 37, 13, and 7) inhibited the reaction of
oxidized proteins
with human cells (see below).
Isolation of CD36
Isolation of CD36 was carried out as described by our group (Konenberg et al.,
1998), by
phase separation of the membrane proteins with Triton X-114, and subsequent
affinity
chromatography using cholesterol-hemisuccinate agarose.
Examples for the preparation of oxidized proteins
348 pg protein (commercially available fibrinogen, human albumin, bovine serum
albumin
(BSA), or antithrombin) was incubated with 832 pg Na0C1 (sodium hypochlorite)
in 1 ml
phosphate buffered saline with the addition of EDTA (0.1 mM) for 10 minutes on
ice. Protein
CA 02453140 2010-04-28
11
and oxidants were immediately separated after the end of the reaction by a gel
filtration at 4 C
with Sepharose G25-Coarse (PD-10 column, Atnersham Pharmacia).
Examples for the activity of the invention:
1.
Oxidized proteins (ox fibrinogen, ox antithrombin III, ox BSA, ox human
albumin)
bind specifically to the CD36 homologue domain of HIV GP120 protein (see
figure 1).
The interaction between oxidized proteins and HIV GP120 was determined by
plasmon resonance technique in BIACORETM System 2000.
2. Oxidized
proteins activate thrombocytes. The activation of thrombocytes is inhibited
by substances that inhibit the interaction of CD36 with oxidized proteins, or
that
interfere with thrombospondin bound to CD36.
a) Oxidized proteins (ox fibrinogen, ox antithrombin III, ox BSA, and ox
human
albumin) increase, in a dose dependent manner, the adhesion of thrombocytes
to adhesion proteins, like thrombospondin, vitronectin, fibrinogen, fibrin,
fibronectin, and collagen (see figure 2a).
This increase in adhesion is inhibited by soluble thrombospondin-1 (see figure
2b). This increase in adhesion is also inhibited by monoclonal anti-CD36
antibody clones 37, 13, and 7 (see figure 2c/d), while VCTG peptide, which
inhibits binding of thrombospondin to CD36, has no effect (see figure 2e). The
commercially available anti-CD36 antibody FA6/152 shows no inhibition (see
figure 2f).
b) Oxidized proteins (ox fibrinogen, ox antithrombin III, ox BSA, ox human
albumin) induce, in the presence of thrombospondin-1 (10 gimp and in a dose
dependent manner, the binding of thrombospondin to thrombocytes (see figure
3a). This activation is inhibited by soluble thrombospondin-1 at a
concentration
of > 20 p.g/ml (see figure 3b).
This activation is also inhibited by monoclonal antibodies against CD36, clone
37, 13, and 7 (see figure 3c-e).
c) Oxidized
proteins (ox fibrinogen, ox antithrombin III, ox BSA, ox human
albumin) cause, in the presence of thrombospondin-1 (10 1.1g/m1), the
aggregation of platelets (see figure 4a). This aggregation is inhibited by
monoclonal antibodies against CD36, clones 37, 13, and 7, in a dose dependent
manner (see figure 4b).
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CA 02453140 2004-01-09
d) Oxidized proteins (ox fibrinogen, ox antithrombin III, ox BSA, ox human
albumin) cause, in the presence of thrombospondin-1 (10 gimp, the pro-
coagulant condition of the platelets (see figure 5a¨c), and lead to micro
particle
formation (see figure 5d). This activation is inhibited by soluble
thrombospondin-1 at concentrations of > 20 ig/m1 (see figure 5e).
This activation is also inhibited by monoclonal antibodies against CD36,
clones 37, 13, and 7, in a dose dependent manner (see figures 5f and g).
3.
Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox human
albumin) activate monocytes. This activation is inhibited in a dose dependent
manner
by certain substances, e.g., soluble thrombospondin or monoclonal antibodies
against
CD36, which inhibit the interaction between CD36 and oxidized proteins or
which
interfere with thrombospondin bound to CD36.
a) Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox
human
albumin) induce a Ca2+ signal in monocytes (see figure 6).
b) Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox
human
albumin) induce the oxidative burst in PMNL (see figure 7).
c) Oxidized proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox
human
albumin) induce an increased transmigration of monocytes, PMNL and
lymphocytes through endothelial monolayers (see figure 8a). This reaction is
inhibited by substances that inhibit the binding of CD36 to oxidized proteins
such as soluble thrombospondin or monoclonal antibodies against CD36,
clones 37, 13, and 7 (see figure 8b).
4. Oxidized
proteins (e.g. ox fibrinogen, ox antithrombin III, ox BSA, ox human
albumin) play a causal role 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. Homing
was carried out according to Patel et al., 1998. In C57BL/6 mice, the
migration
of monocytes/macrophages into the peritoneum was induced by intraperitoneal
injection of thioglycolate. After 4 days, the peritoneum was washed and
activated peritoneal macrophages were obtained. In the probes, erythrocytes
were lysed. Macrophages were resuspended in RPMI 1640 medium and
transferred to cell culture dishes to allow adhesion. Fluorescence-marked
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microspheres (2 gm yellow-green fluorescence latex microspheres, molecular
probes) were opsonized for 30 minutes with 50 % mouse serum for better
uptake by the macrophages, and were then added to the plated macrophages.
The adhered macrophages phagocytosed the microspheres. Non-adhered cells
and microspheres were removed from the dish by washing. Macrophages were
removed from the dish on ice and were resuspended in Hanks balanced salt
solution (HBSS). 50 week old ApoE deficient mice were each injected
intraperitoneally with 3 times 50 gg oxidized protein, in this case oxidized
antithrombin III, and placebo (only HBSS), respectively, 6 hours before
intravenous injection of marked macrophages, 2 hours after injection of
macrophages and 10 hours after injection of macrophages. 10 x 106
macrophages were suspended in 0.2 to 0.3 ml HBSS and injected into the tail
vein. 24 hours after macrophage injection, the animals were sacrificed. The
heart basis and the aorta ascendens were embedded in OCT, stored at ¨80 C,
and 7 gm cryo-sections were prepared. Fluorescence-marked macrophages of
140 serial sections per mice from a 1 mm range of the aorta ascendens on the
level of the sinus valsalva were counted. In comparison to the placebo treated
ApoE control control mice, the migration of marked macrophages into
arteriosclerotic plaques in mice treated with oxidized antithrombin III
increased from 100 + 15 % (n = 14) to 156 + 9.2 % (n = 5) significantly (p =
0.008) ("alternate welch test"). As this migration is causal to the
development,
the progression, and the danger of rupture of arteriosclerotic plaques, this
example elucidates the importance of oxidize proteins with respect to
arteriosclerosis (figure 9).
b) Substances that inhibit the interaction of CD36 and oxidized proteins,
or
interfere with thrombospondin bound to CD36, prevent/reduce the pro-
arteriosclerotic activity of oxidized proteins. Soluble thrombospondin
inhibits
adhesion of macrophages to arteriosclerotically altered endothelial cells.
This is
a prerequisite for migration of microphages into arteriosclerotic plaque.
Murine-immortalized endothelial cells were stimulated with 13-VLDL (50 gg
protein/ml) from plasma of ApoE deficient mice for 6 hours, and then
suspended with 2 x 105 peritoneal monocytes/macrophages per ml with a shear
rate of 400s-1 in a flow through chamber. P-VLDL induced a significant
relative increase of the rolling leucocytes by 224 + 44 % in comparison to the
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CA 02453140 2004-01-09
control. Addition of 10 g/ml soluble thrombospondin inhibited this increase
of adhesion completely and additionally inhibited partly the basic adhesion of
macrophages to the endothelium (75+37 % adhesion in comparison to the
control, n = 4-7; p < 0.01) (see figure 10a). Significantly induced permanent
adhesion of macrophages by p-VLDL was also reduced by thrombospondin-1
after a 5 minute wash period (100 + 21 % control versus 300 + 119 % P-VLDL
versus 157 + 70 % P-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-
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
animals
(negative controls) were only injected with FITC (without BSA) into the
peritoneum.
After 6 hours, animals treated with anti-BSA and BSA-FITC showed a clearly
developed inflammatory reaction with ear swelling (edema), FITC incorporation,
migration of PMNL and petechial bleeding into the tissue. 18 mice were
additionally
intraperitoneally injected at time point 0, and after 0 + 3 hours, with 50 pig
thrombospondin-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,
significantly less ear thickness (less oedems), and almost no petechia in
comparison to
PBS treated control animals (see figures lla¨c).
6. Examples for quantification of oxidized proteins
Preparation of monoclonal antibodies that recognize epitopes on proteins or
peptides,
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 immunized
therewith. Preparation of hybridoma and hybridoma culture was carried
according to
classic procedures. Supernatants of hybridoma were tested for the production
of IgG
and IgM, respectively. IgG positive cell culture supernatants were tested for
positive
reaction with oxidized protein and simultaneously negative reaction with the
unaltered
initial protein. Clones that produced antibodies against oxidized protein (ox
human
albumin, ox antithrombin III, ox fibrinogen) and simultaneously did not react
with
15
CA 02453140 2004-01-09
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, as
disclosed above:
Such quantification is easily possible with processes like ELISA, RIA,
quantitative
flow-through cytometry on cell surfaces, and similar routine procedures.
e.g.: quantification of oxidized human albumin with ELISA. A polyclonal
antibody
from rabbit against human albumin (preparation ¨ routine procedure) was bound
to the
bottom of an ELISA dish (Nunc-Maxisorb) as catcher antibody. The dish was
thoroughly washed with PBS pH 7.4, 0.5
Tween 20, and spaces on the plastic
surface were blocked with 3 % BSA for 1 hour at room temperature (RT). The
dish
was washed again and then incubated with differently diluted plasma, sera,
supernatants of blood products (e.g. thrombocyte concentrates, erythrocytes
concentrates, FFP) or buffer solutions to which defined amounts of ox human
albumin
have 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
which
was marked with biotin, in a dilution of 1:15000 in PBS, 1 % NGS (normal goat
serum). The dish was again washed thoroughly for several times and incubated
with
Streptavidin peroxidase (1 hour, RT). After again washing the dish, it was
treated with
substrate solution (100 1.1g/well) (20 mg ortho-phenyldiamine, 5 % H202 in a
buffer of
12.15 ml 0.1 M citric acid and 12.85 ml 0.2 M Na2HPO4 plus 25 ml H20 dest.).
The
extinction at 405 nm, measured in an ELISA photometer, indicated the amount of
oxidized human albumin. The reaction was stopped with 50 1/well 4 N H2SO4,
and
the extinction was measured at 490 nm.
HOC1-oxidized human albumin in the probe was quantified with a calibration
series.
HOC1-modified fibrinogen and HOC1 modified antithrombin III were treated
similarly.
7. Example for the documentation of the activated condition of the receptor
for oxidized
proteins, CD36.
Preparation of polyclonal antibodies against threonine (92) phosphorylated
CD36.
Peptides (15 AS) (1) Lys, Gln, 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,
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CA 02453140 2004-01-09
Leu, Ala, Lys (as peptide (1), but phosphorylated at threonine), were
synthesized and
coupled to KLH (Keyhole Limpet Hemocyanin). Polyclonal antibodies from rabbit
were prepared according to standard procedures with these coupled peptides.
For this,
rabbits (New Zealand, white) were essentially immunized with 250 lig peptide 1-
KLH
and peptide 2-KLH plus the addition of complete Freund adjuvans s.c.,
respectively,
and boostered with 250 [tg peptide 1-KLH and peptide 2-KLH with the addition
of
Alu-Gel-S (1.3 % aluminium hydroxide in water, SIGMA) 3 times, respectively.
Blood was drained from the ear veins of the animals, and serum was prepared
and
tested for antibodies against the peptides that were used for the
immunization. The
antibody against phosphorylated CD36 peptide was cross-absorbed on an affinity
column with non-phosphorylated CD36 peptide, so that the resulting antibody
mixture
only contained antibodies that specifically recognized phosphorylated non-
activated
CD36 (AK CD36P). (The preparation of specific monoclonal antibodies against
threonine phosphorylated/dephosphorylated CD36 is possible with these peptides
according to the described preparation 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 CD36
preferably recognizes CD36 on non-activated thrombocytes, antibody "CD36
total"
recognizes non-phosphorylated CD36 and phosphorylated CD36. With activation of
the thrombocytes, CD36 is dephosphorylated and the binding capacity for
antibody
"CD36P" decreases (see figure 12). Quantitative flow-through cytometry with
both
antibodies allowed the calculation of the portion of activated CE36.
8. The organism of patients with type I diabetes is particularly
susceptible to oxidized
proteins:
e.g.: thrombocytes of patients with diabetes type I react more sensitively to
oxidized
protein as agonist in comparison to thrombocytes of healthy control persons.
Activation-dependent fibrinogen binding can be induced on thrombocytes of
patients
with diabetes with reduced concentrations of ox protein in comparison to
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
with
high affinity to both HIV-GP120 and its receptor CD4.
17
CA 02453140 2004-01-09
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 CaC12; 1 mM MgC12; 0.005 % Surfactant P-20.
Protein HIV-GP120: c = 100 g/ml, 200 I
Storage buffer: Tris/HC1, NaC1 pH 7.6
Protein CD4: c = 63 g/ml, 318 pl
Storage buffer: 10 mM Tris; 300 mM NaC1 pH 8
Protein ox antithrombin III: c = 1 mg/ml, 120 p.1
Protein ox human albumin: c = 1 mg/ml, 120 pl
Cl-chip (BIACORE AB)
Amine coupling kit (BIACORE AB)
HIV-GP120 and CD4 were immobilized on the Cl sensor surface. For this 10 mM
NaAc pH 4 was used as coupling buffer.
Coupling conditions: HIV-GP120: 20 1; 10 g/ml GP120 in 10 mM NaAc pH 4;
immobilized amount: 1158 RU, 300 pg
CD4: 30 I; 6.3 g/m1 CD4 in 10 mM NaAc pH 4; immobilized amount: 568 RU, 148
Pg
The chip surface was saturated with BSA, and the binding of oxidized proteins
was
investigated. For this, e.g. 50 pl oxidized antithrombin III were injected in
different
concentrations at a flow rate of 20 p.1/minute. The protein solutions were
diluted with
sample buffer. With increasing concentrations of oxidized antithrombin III the
resulting signals increase. Figure 14 shows an overlay plot of 12 sensorgrams,
which
show binding of oxidized antithrombin III to immobilized CD4 and subsequent
dissociation.
The quantitative analysis resulted in the following values for the binding of
a) oxidized antithrombin III to HIV-GP120:
Kon (1/Ms): 6.38 x 105
Koff (Vs): 4.44 x 10.4
and a KD[M] of 7.01 x
b) oxidized antithrombin III to CD4:
Kon (1/Ms): 7.13 x 105
Koff (l/s): 1.12 x 10-3
and a KD[M] of 1.63 x 10-9,
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c) non-modified antithrombin III bound neither to HIV-GP120 nor to CD4.
Oxidized
human albumin bound with a higher affinity to HIV-GP120 and CD4 than to ox
antithrombin III. Non-oxidized human albumin bound neither to HIV-GP120 nor to
CD4. Oxidized protein inhibited HIV-1 infection of monocyteous cells from
peripheral blood (PBMC).
PHA-activated PBMC were incubated together with negative human serum 1:100
(negative control), with neutralizing V3-loop specific antibodies (positive
control),
with oxidized protein (150 g/ml) and a CCR5 tropic HIV-1 primary isolate
(903)
from a patient, and after 5 days the virus production was tested by P24 ELISA.
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
200,000
cells/well/100 I were distributed on a 96 well plate. Tested substances for
inhibition:
Positive control: neutralizing human anti-V3-loop antibody (1:100)
Negative control: negative human serum 1:100
Experiment: oxidized protein (150 g/m1)
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
l/well of
HIV-1 primary isolate 903 supernatant (CCR5 trop) with 20,000 TCED50 (50 %
tissue
culture infective dose)/m1 1000 TCID50/m1 per well. These samples were
incubated
overnight at 37 C/5 % CO2. The following day, the cells were washed 3 times
with
RPMI 1640, and new culture medium was added. On day 5 after infection, P24-
ELISA
tests were carried out.
P24-ELISA:
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)
were
overlayed with these antibodies overnight. The virus supernatant from the
inhibition
assay was inactivated by 1 % Triton X-100. After washing the treated cells
with PBS,
the inactivated virus supernatant and alkaline phosphatase conjugated
detection
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
CA 02453140 2004-01-09
common mean value. While OD 405 nm for the negative control no inhibition was
at
0.8, the neutralizing antibody (positive control) reduced the OD to 0.12. 150
ps/ml
oxidized protein reduced the OD to 0.10. The addition of oxidized proteins
effectively
inhibited HIV-1 infection of PBMCs.
10. Oxidized proteins induced TSP binding to cells
Oxidized proteins (for example, ox human albumin, ox antithrombin III, and ox
fibrinogen, were used herein) induced specific and dose dependent binding of
TSP-1
to CD36 containing cells (see figure 15a¨d).
Therefore, on the one hand, objects of the present invention are medicaments
comprising
substances that inhibit the binding of oxidized proteins to CD36 or inhibit
the functions of
CD36 that are induced by the interaction of CD36 with oxidized proteins.
In a preferred embodiment of the invention, the medicaments comprise
antibodies that inhibit
binding of oxidized proteins to CD36, comprising particularly preferred
monoclonal
antibodies and antibody fragments like F(ab)2, F(ab), or of the antibody
recognition region.
In a further preferred embodiment, the medicament comprises peptides of CD36,
peptide
mimetics, or peptide analogues that inhibit binding of CD36 to oxidized
proteins or that
inhibit cell functions of CD36 induced by interaction of CD36 with oxidized
proteins.
Preferably, these substances are identified and selected by monoclonal anti-
CD36 antibodies
disclosed in this invention, and in particular, they react with clones 37, 13,
or 7, or inhibit
binding of oxidized proteins/peptides to CD36, or inhibit a characteristic
function of CD36,
induced by oxidized protein/peptide as, but not limited to, the functions
described in the
examples.
In further preferred embodiment, the medicament comprises proteins or protein
components
that inhibit binding of CD36 to oxidized proteins or that interfere with
thrombospondin bound
to CD36. In a particularly preferred embodiment, such protein is soluble
thrombospondin.
In a further preferred embodiment, the medicament comprises peptides or
peptide mimetics
that bind to CD36 and thereby inhibit the interaction of CD36 with oxidized
proteins. Such
20
CA 02453140 2004-01-09
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 present invention is the use of
medicaments according to
the invention for prophylaxis of thrombosis, in particular in inflammatory
diseases, for
support of an anti-thrombotic therapy, for preventing a transplant rejection,
for preventing
transplantation associated arteriosclerosis, for preventing high blood
pressure in kidney
diseases, and in particular renin associated high blood pressure, for
preventing the
development and the progression of arteriosclerotic (atherosclerotic)
diseases, for the
treatment of chronic inflammatory reactions, for preventing early vessel
reocclusion after
bypass surgery, stent, PTCA, or the like, for preventing of vessel restenosis
after bypass
surgery, stent, PTCA, or the like, for preventing reperfusion damages, such
as, but not limited
to myocardial ischemia, organ transplantation, stroke, peripheral occlusive
disease after
surgery, and/or multi organ failure after successful reanimation, for
preventing vessel damage,
in particular in patients with diabetes mellitus, for preventing the
inhibition of endothelial
proliferation and angiogenesis induced by oxidized proteins through CD36/TSP-
1, and for
supporting wound healing.
Still another object of the present invention is a method for quantifying
oxidized proteins (in
particular ox antithrombin III, ox human albumin or ox fibrinogen),
individually or together,
for the evaluation of individual indications for therapies with medicaments
according to the
present invention, for the diagnosis of diseases, in which inflammatory
reactions play a role,
such as, but not limited to, arteriosclerosis, diabetic vasculopathy,
rheumatic arthritis,
Goodpasture Syndrome, sepsis, Colitis ulcerosa, graft-versus-host diseases,
pemphigus,
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 the
receptor for oxidized proteins, as a diagnostics for diseases in which
inflammatory reactions
play a role such as, but not limited to, arteriosclerosis, diabetic
vasculopathy, rheumatic
arthritis, Goodpasture syndrome, sepsis, Colitis ulcerosa, graft-versus-host
diseases,
pemphigus, cancer, neurodermatitis, HIV infections, ARDS, glomerulonephritis,
reperfusion
damages or for monitoring a CD36/ox protein inhibition therapy with
medicaments according
to the present invention, by measuring the phosphorylation condition of CD36.
21
CA 02453140 2004-01-09
Still another object of the present invention is a medicament that comprises
oxidized
protein/oxidized proteins, oxidized peptide, oxidized structural analogues or
structural
mimetics thereof. Medicaments according to the present invention are also
characterized in
that they may contain further pharmaceutically acceptable fillers and/or
excipients. The
medicaments according to the present invention are preferably suitable for
local, intradermal
topical, intraperitoneal, intravenous, oral or intramuscular administration,
or they can be
applied as vesicles. Further, it is preferred that the medicaments according
to the present
invention further comprise substances as e.g. antibiotics, immunosuppressants,
or interaction
partners of oxidized proteins in the body. By the addition of such substances,
the activities of
the medicaments according to the present invention can be further supported
and assisted.
In the context of the present invention, oxidized proteins or peptides are
generated according
to the present invention preferably by reaction with HOC1 or peroxynitrites.
A further use of the medicaments according to the present invention, and in
particular of a
medicament comprising oxidized proteins/peptides or analogues or mimetics
thereof, lies in
the prophylaxis or therapy of acute infections, the inhibition of
angiogenesis, and for the
improvement of hemostasis. Thereby, the medicament is preferably used for the
prophylaxis
or therapy of an HIV infection. In another preferred embodiment, the use of a
medicament
according to the present invention comprising oxidized protein inhibits tumor
angiogenesis by
means of induction of TSP binding to CD36.
In still another preferred embodiment, the medicament is used for hemostasis,
in particular in
patients with innate or acquired blood coagulation disorders, or innate or
acquired
thrombocytopathia, under anticoagulation therapy or thrombosis prophylaxis, or
is used in
surgery under heart-lung-machine.
Because oxidized proteins induced TSP binding, medicaments according to claim
21
inevitably induce indirect effects of TSP bound to cell surfaces, as, e.g.,
inhibition of
angiogenesis (see figure 16a) or inhibition of HIV infection. On the other
hand, inhibition of
the interaction between oxidized proteins and CD36 consequently induces the
repression of
functions that are induced by the reaction chain ox protein-CD36 cell bound
TSP. Inhibitors
22
CA 02453140 2004-01-09
according to claims 1-5 thereby also inhibit TSP-mediated processes, as the
inhibition of
angiogenesis and therefore are proangiogenetic (see figure 16b).
Legends of the figures:
Figure 1: Oxidized proteins bind to a CD36 homologue domain in HIV-1 GP120
protein
1) Overlay-plot of 12 sensorgrams, which show the binding of oxidized
antithrombin III to
immobilized HIV-GP120 and the dissociation of oxidized antithrombin III. HIV-
GP120 is
immobilized (300 pg); the concentration of oxidized antithrombin III varied
(from the bottom
to 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 signal
increases.
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 thrombocytes
was carried out according to Santoro et al., 1994. A 96 well cell culture
plate was coated with
collagen type I (25 pg/m1; 100 l/well) overnight at 4 C, and the plates were
blocked with
BSA. 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 1 gel-filtered platelets (300000411) were incubated with and
without oxidized
proteins for 1 hour at RT in a humid chamber in the wells. Non-adhered
thrombocytes were
thoroughly washed away. The number of adhered platelets with determined after
lysis of the
platelets with Triton X-100 and determination of the lyzosomal enzyme
hexosaminidase. For
calibration of the adhesion assay, a calibration series with known increasing
platelets number
is given on a microtiter plate, and the extinction of the substrate P-
nitrophenyl-N-acety1-13-D-
glucosamide is determined in relation to the number of platelets. Oxidized
antithrombin III
increased the thrombocyte adhesion in a dose dependent manner.
2b) Thrombocytes were used in the above-described adhesion assay and were
activated with
50 g/m1 oxidized ATIII. Addition of soluble purified thrombospondin inhibited
increased
thrombocyte adhesion mediated by oxidized ATIII in a dose dependent manner.
23
CA 02453140 2004-01-09
2c) Thrombocytes were used in the above described adhesion assay, and were
activated with
oxidized ATIII. Antibodies that inhibit binding of oxidized proteins to CD36,
like clones 37,
13, and 7, inhibit the activity of oxidized ATM. All experiments for the
measurement of the
influence of antibodies on the thrombocyte functions were carried out in the
presence of
saturating, completely blocking, concentrations of Fab fragments of an
antibody against the
FcRIIA receptor (clone IV.3), in order to avoid Fc receptor effects.
2d) This effect is dose dependent.
2e) Inhibition of thrombocyte adhesion by soluble thrombospondin-1 is not
mediated directly
through its binding site on CD36 (peptide VTCG). VTCG shows no influence on
the increase
of thrombocyte adhesion by oxidized proteins (herein oxidized ATIII).
20 Antibodies against CD36 that do not inhibit binding of CD36 to oxidized
proteins, like
clone FA6/152, however, do not induce any significant inhibition. All
experiments to
determine the influence of antibodies on thrombocyte functions were carried
out in the
presence of saturated, completely blocking, concentrations of Fab fragments of
an antibody
against 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 ps/m1 thrombospondin was added to gel-
filtered platelets
(50000/jig) in HEPES-Tyrode-BSA buffer. A portion of the sample was added with
oxidized
proteins in increasing concentrations. After incubation for 30 minutes at RT,
the fibrinogen
binding was determined in flow-through cytometry.
3a) Oxidized proteins (herein as examples, oxidized fibrinogen, oxidized human
albumin, and
oxidized antithrombin III) increase the fibrinogen binding to thrombocytes.
3b) Soluble thrombospondin-1 inhibits fibrinogen binding induced by ox.
protein in a dose-
dependent manner.
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CA 02453140 2004-01-09
3c) Antibodies that inhibit binding of oxidized proteins to CD36, inhibit in a
dose dependent
manner the fibrinogen binding to thrombocytes induced by oxidized proteins.
Oxidized protein: oxidized ATIII;
antibody: anti-CD36 antibody, clone 37.
3d) Oxidized protein: oxidized fibrinogen;
antibody: anti-CD36 antibody, clone 37.
3e) Oxidized protein: oxidized human albumin
antibody: anti CD36 antibody, clone 37.
All experiments for the determination of the influence of antibodies on
thrombocyte functions
were carried out in the presence of saturated, completely blocking,
concentrations of Fab
fragments of an antibody against the FcRIIA-receptor (clone IV.3), in order to
avoid Fc
receptor effects.
Figure 4: Oxidized proteins act hemostatic/prothrombotic/induction of
thrombocyte
aggregation/inhibition of this reaction
4a) Oxidized proteins induce thrombocyte aggregation. Thrombocyte aggregation
was carried
out according to Born 1962. To gel-filtered platelets (20000/ 1) in HEPES-
Tyrode buffer pH
7.4 with 100 ps/m1 fibrinogen, TSP-1 (25 gimp 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 jig/m1 the
aggregation induced
by oxidized proteins.
4b) Antibodies that inhibit binding of oxidized proteins to CD36, inhibit in a
dose dependent
manner the platelet aggregration induced by oxidized proteins. All experiments
concerning
the influence of antibodies on thrombocyte functions were carried out in the
presence of
saturating, completely blocking, concentrations of Fab fragments of an
antibody against the
FcRIIA receptor (clone IV.3) in order to avoid Fc receptor effects.
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CA 02453140 2004-01-09
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 factor
VNa to thrombocytes. Factor VNa binding was carried out as described in
Dormann et al.,
1998.
5b) Oxidized proteins (herein as an example oxidized fibrinogen) induced
binding of factor
X/Xa to thrombocytes. Factor X/Xa binding was carried out as described by us
according to
Dormann 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 was
carried out described by us according to DOrmann et al., 1998.
5d) Oxidized proteins (herein as an example oxidized fibrinogen) induce
microparticle
formation of thrombocytes. Gel-filtered platelets (50000411) were incubated
with oxidized
protein for 30 minutes at RT under slight agitation. Then, the platelets and
microparticles
resulting from platelets were incubated for 30 minutes with an anti-GPIX-PE
antibody, and
the number of resulting microparticles was measured in the ratio to 5000
counted particles in
a flow through cytometer.
5e) Soluble thrombospondin inhibits microparticle formation induced by
oxidized proteins. In
this experimental example microparticle formation from thrombocytes was
induced by
oxidized human albumin. Gel-filtered platelets in HEPES-Tyrode buffer pH 7.4
were
activated with each 50 g/ml oxidized human albumin for 1 h at RT. Before
activation, the
platelet suspension was added with soluble TSP in concentrations as depicted.
Microparticle
formation was analyzed as described in 5d). Soluble thrombospondin inhibited
the formation
of microparticle in a dose dependent manner.
50 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
CA 02453140 2004-01-09
binding of oxidized proteins to CD36, inhibited the subsequent activation of
these platelets by
oxidized proteins (herein as an example oxidized fibrinogen). All experiments
regarding the
influence of antibodies on thrombocyte functions were carried out in the
presence of
saturating completely blocking concentrations of Fab fragments of an antibody
against the
FcRIIA receptor (clone IV.3) in order to avoid Fc receptor effects.
5g) Anti-CD36 clone 37 inhibits the oxidized protein induced microparticle
formation of
thrombocytes. Microparticle formation was determined as described under 5d).
Preincubation
of platelets with anti-CD36 antibodies (30 minutes, RT), which inhibit binding
of oxidized
proteins to CD36, before activation with oxidized proteins (herein as an
example oxidized
fibrinogen) inhibits microparticle formation.
Figure 6: Oxidized proteins activate leucocytes ¨ Ca2+ signal
Oxidized proteins (shown herein oxidized antithrombin III) induce a Ca2+
signal in
monocytes. The Ca2+ measurement was carried out according to Sozzani et al.,
1993. Eluted
monocytes (5 x 106/m1) were washed at RT with HEPES-Tyrode buffer pH 7.4 and
subsequently marked for 15 minutes with 1 11M Fura2/AM at 37 C, washed twice
with
HEPES-Tyrode buffer without Ca2+ and then suspended in HEPES-Tyrode with 1 mM
Ca2+.
Ca2+ signal, induced by oxidized proteins and as positive or negative control
effective
substances were fluorimetrically determined in Hitachi F-2000. Oxidized
antithrombin III
(100 g/ml) activates monocytes and induces a clear Ca2+ signal.
Figure 7: Oxidized proteins activate leucocytes ¨ oxidative burst
Oxidized proteins increase, in a dose-dependent manner, the activating effect
of fMLF on the
oxidative burst of PMNL, and they even induce oxidative burst as autonomous,
independent
agonists. Induction of oxidative burst was essentially carried out according
to the
manufacturers instructions with phagotest/burst test of the company Orpegen
(Heidelberg)
using a flow-through cytometer. However, PMNL were first incubated with
substrate
DHR123 and then PMNL were activated. Oxidized antithrombin III increased the
activating
effect of fLMF and itself induced an ox. burst reaction.
27
CA 02453140 2004-01-09
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 a
microporous polycarbonate membrane was placed on each of the 24 wells. The
polycarbonate
membrane with a pore size of 5 gm was coated with fibronectin and human
microvascular
endothelial cells (HMEC-1) were cultured until confluence. Human monocytes
isolated by
density-gradient-centrifugation (200 gl with 2 x 107 cells/ml in DMEM from
peripheral
blood) were incubated as 37 C, 7 % CO2 with the HMEC-1 monolayer. As a degree
for the
transmigration rate, the number of monocytes in the lower transwell
compartment below the
transwell insert was determined. In order to investigate the influence of
different oxidized
proteins, of thrombospondin, or of anti-CD36 antibodies, the test substances
were added to
the medium in the upper transwell chamber, or the endothelial cells were
preincubated for 10
minutes with the test substances and washed. After 4-7 hours of transmigration
time the
inserts were carefully removed, the cell culture plate were placed on ice for
30 minutes to
remove adhered monocytes, and the number of transmigrated monocytes was
counted.
Oxidized protein (herein oxidized ATIII) promotes the transmigration of
monocytes through
the HMEC-1 monolayer while the non-oxidized parent protein did not show this
reaction
(transmigration period 4 h).
8b) Preincubation of the endothelial layers for 10 minutes with TSP-1 and
addition of TSP-1
to the cell culture medium during the transmigration experiment, respectively
could
significantly inhibit the transmigration of monocytes (transmigration period 7
h).
Figure 9: Oxidized proteins induce processes that promote arteriosclerosis
Oxidized antithrombin III increases homing of macrophages in arteriosclerotic
plaques. The
realization of the experiment was described in detail in the description of
the example.
Figure 10: Thrombospondin inhibits proarteriosclerotic processes
Thrombospondin inhibits the adhesion of macrophages, to arteriosclerotically
altered
endothelial cells. The realization of the experiment is described in detail in
the description of
the example.
28
CA 02453140 2004-01-09
10a) Thrombospondin-1 inhibits the transient adhesion of macrophages, which is
characterized by rolling of macrophages to arteriosclerotically altered
endothelium.
10b) Thrombospondin inhibits the permanent stable adhesion of macrophages to
arteriosclerotically altered endothelium.
Figure 11: Thrombospondin inhibits inflammatory processes in vivo
Soluble thrombospondin-1 inhibits the Arthus reaction in the ear of Balb/c
mice.
11a) Mouse treated twice at time point 0 and 0 + 3 hours with each 50 t.tg TSP-
1 i.p. ¨ inhibits
Arthus reaction in left ear.
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 treated
with TSP-1 and control buffer ¨ Arthus reaction in left ear.
Figure 12:
Gel filtered human thrombocytes were diluted with Hepes-Tyrode buffer pH 7.4
to 50000/ 1
and activated at room temperature. In order to avoid Fc receptor effects on
the platelets by
antibodies, the experiments were carried out in the presence of completely
saturating blocking
concentrations of Fab fragments of an antibody against the FcRIIA receptor
(clone IV.3).
After activation, thrombocytes were fixed with 0.1 % paraformaldehyde in Hepes-
Tyrode
buffer pH 7.4 for 30 minutes and washed. Fixed, resting, and activated
thrombocytes were
incubated overnight anti-CD36 "AK36P" and anti-CD36 "AK36-total" in saturating
concentrations overnight, respectively, and the thrombocytes were washed and
incubated with
a secondary, FITC-marked antibody (goat anti-rabbit IgG-FITC, "minimal X
reaction with
human 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 by
FITC fluorescence in a flow-through cytometer (FACScan-Becton Dickinson)
(according to
Dormann et al., 1998).
29
CA 02453140 2004-01-09
Decrease of binding of anti-CD36 "AK36P" to thrombocytes by activation.
Figure 13:
Blood was drained from patients with diabetes mellitus type I and control
persons, and the
blood was coagulated with citrate. Platelet-rich plasma (PRP) was prepared by
centrifugation.
PRP of patients and healthy control persons was mixed with fibrinogen (150
g/ml) that was
coupled to FITC, and the thrombocytes were activated with oxidized protein
(herein oxidized
human albumin) in increasing concentrations for 30 minutes. Fibrinogen binding
was
measured in flow-through cytometer as described above in detail. The figure
shows a
characteristic example of the simultaneous determination of activation with
PRP of patients
with diabetes mellitus type I in comparison to controls. Thrombocytes of
patients with
diabetes mellitus type I are particularly sensitive for the activation with
oxidized proteins.
Figure 14:
The interaction of oxidized proteins and HIV receptor CD4 was determined as
described in
detail in the description of the example by plasmon resonance technique in the
BIACORE
system 2000.
Overlay plot of 12 sensorgrams that show the binding of oxidized antithrombin
III to
immobilized CD4 and the dissociation of oxidized antithrombin III. CD4 is
immobilized (148
pg); the concentration of oxidized antithrombin III was varied (from the
bottom to 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 increasing concentrations of oxidized AT III, the resulting signal
increases.
Figure 15:
15a) Oxidized protein mediates TSP-1 binding to thrombocytes
Gel-filtered human thrombocytes were diluted with Hepes-Tyrode buffer pH 7.4
to 50000/ 1
and FITC-conjugated purified thrombospondin-1 (50 jig/m1) was added. The
thrombocytes
were incubated for 1 h at RT with oxidized protein (herein oxidized
fibrinogen), and TSP-1
binding to thrombocytes was measured in flow through cytometer. Oxidized
proteins induce
TSP-1 binding to thrombocytes.
30
CA 02453140 2004-01-09
15b) Oxidized protein (herein oxidized antithrombin III) induces binding of
thrombospondin
to endothelial cells.
Human microvascular endothelial cells (HMEC-1) were dissolved from the cell
culture plate
according to standard procedure, dissolved, and the suspension was incubated
for 1 h at RT
with oxidized protein and oxidized protein plus TSP-1 addition, respectively.
The cells were
washed and bound TSP-1 was marked with mAK anti-TSP-1 (clone P10) coupled to
phycoerythrin (PE) and quantified in a flow-through cytometer. Oxidized
protein induced
TSP-1 binding to endothelial cells.
15c) While without the addition of purified TSP-1 and without addition of
oxidized
antithrombin III only approximately 1 % of the eluted monocytes were detected
by an
antibody (clone P10), which recognizes TSP-1 on the cell surface, in flow-
through cytometer,
the amount increased by the addition of purified TSP-1 (10 gimp to
approximately 5 %. The
addition of oxidized AT III (without the addition of exogenous TSP-1) mediates
binding of
endogenous TSP-1 to monocytes. About 18 % of the monocytes were TSP-1
positive. By the
simultaneous addition of TSP-1 and oxidized ATIII almost all of the peripheral
blood
monocytes were strongly positive for TSP-1.
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) or
oxidized protein plus TSP-1 addition (25 pg/m1). TSP-1 bound to T cells was
marked by the
monoclonal PE conjugated anti-TSP antibody (clone P10) and was measured in the
flow-
through cytometer. Oxidized proteins induced binding of endogenously present
and
exogenously added TSP to T cells.
Figure 16:
This figure shows the mechanism by which medicaments according to claims 1-5
and
according to claim 21 inhibit or mediate functions that are induced by the
reaction of
thrombospondin with CD36 (example angiogenesis). The working group of N. Bouck
identified thrombospondin-1 and derivatives thereof as a potent endogenous
inhibitor of
tumor angiogenesis, and they showed that this reaction is mediated by CD36
(Dawson et al.,
1997; Jimenez et al., 2000).
31
CA 02453140 2004-01-09
16a) It has been shown by this invention that oxidized proteins mediate
binding of
thrombospondin to CD36. Medicaments according to claim 21 therefore induce
reactions that
are mediated by this binding, as e.g. inhibition of angiogenesis, a process
which can be
therapeutically used for the treatment of tumors.
16b) In this invention, substances are disclosed that inhibit the interaction
of oxidized proteins
with CD36 and therefore processes that are induced in the body by oxidized
proteins by
CD36. Medicaments according to claims 1-5 inhibit these reactions and prevent
angiogenesis
inhibition that is induced by the reaction chain oxidized proteins (CD36-
conformational
change-thrombospondin binding to CD36-CD36¨.signal for angiogenesis
inhibition. This
reaction can be therapeutically used if angiogenesis is desired, e.g. in the
heart muscle in the
event of an attack.
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