U.S. patent application number 12/215297 was filed with the patent office on 2009-06-25 for diagnosis, prevention, and/or treatment of atherosclerosis and underlying and/or related diseases.
This patent application is currently assigned to Crossbeta Biosciences B.V.. Invention is credited to Manuel Castro Cabezas, Hans van Dijk, Petronella Maria van.
Application Number | 20090162357 12/215297 |
Document ID | / |
Family ID | 8172012 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090162357 |
Kind Code |
A1 |
Cabezas; Manuel Castro ; et
al. |
June 25, 2009 |
Diagnosis, prevention, and/or treatment of atherosclerosis and
underlying and/or related diseases
Abstract
Complement is recognized as an important, humoral defense system
involved in the innate (nonspecific) recognition and elimination of
microbial invaders, other foreign particles or molecules, and
antigen-antibody complexes from the body. The present invention
makes use of the surprising notion that the handling of lipids by
the body, rather than its antimicrobial activity, is the primary
and most ancient function of the complement system. Consequently,
atherosclerosis as observed in disorders associated with disturbed
lipid metabolism (familial combined hyperlipidemia (FCHL),
postprandial hyperlipidemia, hypertriglyceridemia with low levels
of HDL cholesterol, and insulin resistance associated with type-II
diabetes and obesity), is ascribed to either genetic or acquired
defects in ancient (activatory and/or regulatory) complement
components. Based on this new insight, novel preventive measures
and treatment modalities of disturbed lipid metabolism are
introduced.
Inventors: |
Cabezas; Manuel Castro;
(Zeist, NL) ; Dijk; Hans van; (De Bilt, NL)
; Maria van; Petronella; (De Bilt, NL) |
Correspondence
Address: |
TRASKBRITT, P.C.
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Assignee: |
Crossbeta Biosciences B.V.
|
Family ID: |
8172012 |
Appl. No.: |
12/215297 |
Filed: |
June 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10327604 |
Dec 20, 2002 |
7498147 |
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12215297 |
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PCT/NL01/00673 |
Sep 12, 2001 |
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10327604 |
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Current U.S.
Class: |
424/136.1 ;
424/133.1; 424/152.1; 424/172.1; 435/29; 530/389.1 |
Current CPC
Class: |
A61K 39/39 20130101;
A61P 37/02 20180101; G01N 33/564 20130101; A61K 2039/505 20130101;
G01N 2800/323 20130101; A61K 38/39 20130101; G01N 2800/044
20130101; A61P 37/06 20180101; A61P 29/00 20180101; A61P 43/00
20180101; A61P 3/00 20180101; A61P 35/00 20180101; A61K 38/1709
20130101; A61K 48/00 20130101; G01N 33/92 20130101; G01N 2333/4716
20130101; A61P 3/06 20180101; A61P 7/00 20180101; A61P 9/10
20180101; A61P 37/00 20180101; A23L 33/30 20160801; A61P 31/00
20180101 |
Class at
Publication: |
424/136.1 ;
424/172.1; 424/133.1; 424/152.1; 435/29; 530/389.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/02 20060101 C12Q001/02; C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 12, 2000 |
EP |
00203156.5 |
Claims
1. A method for the treatment and/or prophylaxis of diseases
associated with disturbances in the complement/lipid pathway, said
method comprising: modulating the activity of one or more elements
in the complement/lipid pathway of a subject.
2. The method according to claim 1, wherein the activity of one or
more elements of a lectin pathway, a classical pathway and/or an
alternative pathway for complement activation are modulated.
3. The method according to claim 1, wherein the disease is
atherogenic.
4. The method according to claim 1, wherein the disease is
atherosclerosis and/or an underlying and/or related disease.
5. The method according to claim 1, wherein said modulating the
activity of one or more elements is achieved through administering
one or more modulators to the subject.
6. The method according to claim 5, wherein the modulator is
selected from the group consisting of MBL and MBL-replacement
factors, C4A, C4B, C2, C3, IgG- and IgM-antibodies raised against
triglyceride-rich particles and LDL or parts thereof, C3adesArg,
factor B, factor D, factor P, serum carboxypeptidases, sCP-N,
erythrocyte-bound CR1, free CR1, CR1 mimetics, C3b antibodies,
vitronectin, clusterin, and apo B (48 and 100) and apo B
replacement factors, esterases, an MASP-protein, and a functional
equivalent or mixture of any thereof.
7. The method according to claim 5, wherein the modulator is
selected from the group consisting of MBL-replacement factors and
apo B replacement factors.
8. The method according to claim 1, wherein said modulator is an
antibody.
9. The method according to claim 8, where said antibody is selected
from the group consisting of IgG antibodies, IgM antibodies, and
mixtures thereof.
10. The method according to claim 6, wherein the modulator
comprises apo B replacement factors and a heavily mannosylated IgA
or IgD antibody directed against an apo B lipoprotein.
11. The method according to claim 6, wherein the modulator
comprises apo B replacement factors and a heavily
N-acetylglucosaminylated IgA or IgD antibody directed against an
apo B lipoprotein.
12. The method according to claim 6, wherein the modulator
comprises apo B replacement factors and a heavily fucosylated IgA
or IgD antibody directed against an apo B lipoprotein.
13. The method according to claim 8, wherein said antibody is
selected from the group consisting of polyclonal antibodies,
humanized monoclonal antibodies, combinatorial antibody, and
mixtures thereof.
14. The method according to claim 8, wherein said antibody
comprises bi-specific antibodies reactive towards both an apo B and
CR1.
15. The method according to claim 5, wherein the modulator is
administered via parenteral feeding in an intralipid carrier.
16. The method according to claim 15, wherein the intralipid
carrier is olive oil.
17. The method according to claim 5, wherein the modulator is
generated in vivo in the subject.
18. The method according to claim 1 for the treatment and/or
prophylaxis of diseases selected from the group consisting of
diseases associated with impaired complement-dependent lipid
metabolism, atherosclerosis and/or underlying and/or related
diseases, and atherogenic processes of concomitant (infectious,
autoimmune, or neoplastic) diseases that at least partially occupy
the lipid eliminating complement activation pathway.
19. A method for diagnosing disturbances in the complement/lipid
pathway or an underlying or related defect of atherosclerosis, said
method comprising: determining the presence and/or abundance of at
least one element of the complement/lipid pathway in a sample.
20. (canceled)
21. The method according to claim 19, wherein said at least one
element of the complement/lipid pathway is selected from the group
consisting of MBL, C4A, C4B, C2, factor B, factor D, C3adesArg,
serum carboxypeptidase N, vitronectin, clusterin, chylomicron-bound
sialic acid, and erythrocyte-bound complement receptor 1 (CR1).
22. The method according to claim 21, wherein additionally at least
one concomitant (infectious, autoimmune, or neoplastic) disease
that may at least partially occupy the lipid eliminating complement
activation pathway is diagnosed.
23. The method according to claim 21, wherein additionally a
subject's lipid profile is determined by using whole blood.
24. The method according to claim 21, for discovering
pharmaceutical and/or nutritional compounds for the treatment
and/or prophylaxis of atherogenic disturbances of lipid metabolism
related to disturbances in the complement/lipid pathway.
25. (canceled)
26. A composition for the treatment and/or prophylaxis of diseases
selected from the group consisting of diseases associated with
disturbances in the complement/lipid pathway, atherosclerosis
and/or an underlying and/or a related disease associated with
disturbances in the complement/lipid pathway, and disturbances of
lipid metabolism, said composition comprising: at least one
modulator of the complement/lipid pathway.
27. The composition of claim 26, wherein said composition modulates
the activity of one or more elements of the lectin pathway and/or
the alternative pathway for complement activation.
28. The composition of claim 26, wherein said modulator is selected
from the group consisting of MBL and MBL-replacement factors, C4A,
C4B, C2, C3, IgG- and IgM-antibodies raised against
triglyceride-rich particles and LDL or parts thereof, C3adesArg,
factor B, factor D, factor P, serum carboxypeptidases, sCP-N,
erythrocyte-bound CR1, free CR1, CR1 mimetics, C3b antibodies,
vitronectin, clusterin, apo B (48 and 100) and apo B replacement
factors, esterases, an MASP-protein, and functional equivalents and
mixtures of any thereof.
29. The composition of claim 26, wherein said modulator is selected
from the group consisting of MBL-replacement factors and apo B
replacement factors.
30. The composition of claim 26, wherein said modulators are
metabolic precursors of modulators.
31. The composition of claim 26, further comprising a
pharmaceutically acceptable carrier selected from the group
consisting of natural lipid carriers, artificial lipid carriers,
synthetic lipid carriers, mineral oil, natural oil, processed
mineral oil, natural oil, and mixtures thereof.
32. A kit for diagnosing atherogenic disturbances of lipid
metabolism or diagnosing atherosclerosis by a method according to
any of the claims 18, said kit comprising: means for receiving a
sample, and means for carrying out an assay for the detection of at
least one modulator and/or element of the complement/lipid pathway
in the sample.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/327,604 filed Dec. 20, 2002, pending, which application
is a continuation-in-part of PCT International Patent Application
PCT/NL01/00673, filed on Sep. 12, 2001, designating the United
States of America, and published, in English, as WO 02/22161 A2 on
Mar. 21, 2002, the contents of the entirety of each of which is
incorporated herein by this reference. This application also claims
benefit under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent
Application Ser. No. 60/253,465 filed on Nov. 28, 2000.
TECHNICAL FIELD
[0002] The invention relates to the diagnosis, prevention, and/or
treatment of atherosclerosis and/or underlying and/or associated
diseases.
BACKGROUND
[0003] According to the classical view, atherosclerosis is a
condition ultimately leading to the narrowing of blood vessels,
impaired circulation, and restricted oxygenation of
tissues..sup.(1) If this process occurs in heart vessels (coronary
arteries), consequences are the clinical conditions of angina
pectoris and myocardial infarction; in the brain, atherosclerosis
leads to cerebrovascular accidents; in the legs, the clinical
presentation is claudicatio intennittens. Classical risk factors
Associated with atherosclerosis are: obesity, hypertension,
smoking, diabetes, male gender, fasting hyperlipidemia, and
especially increased cholesterol concentrations. Novel risk factors
have emerged during the last decennia, these including
hyperhomocysteinemia, hypertriglyceridemia with low HDL cholesterol
concentrations, postprandial hyperlipidemia, the insulin resistance
syndrome, and a positive family history for cardiovascular disease,
among others.
[0004] According to epidemiological surveys, coronary heart disease
(CHD) is the leading cause of death in western societies. In the
United Kingdom in 1987, 31% of all deaths in males (280,177 total
deaths in men) and 24% in females (total number: 286,817) were due
to CHD. More than one quarter of CHD deaths in men (total CHD
mortality in men: 86,978) occurred before the age of 65 years. In
women (68,257 CHD deaths), the vast majority (almost 75%) occurred
at ages beyond 75 years. The Dutch situation is similar and
representative for other countries in Western society. In The
Netherlands in 1997, there were 135,783 deaths in total (67,242
males and 68,541 females). In men, 37% of total mortality (24,664
deaths) was due to CHD and in women 38% (25,881 deaths). From 1972
to 1997, mortality due to CHD in The Netherlands decreased by 44%
(age-corrected); however, hospital admissions related to CHD
increased by 53%. This decrease in CHD-associated mortality is
probably ascribed to improved care in coronary-care and
intensive-care units. In addition, the early recognition of the
above-mentioned risk factors for CHD and improved treatment of
these risk factors may have led to increased survival in patients
at risk.
[0005] The classical drugs for the treatment of these risk factors
are cholesterol-lowering drugs (mainly statins),.sup.(3) drugs
aiming at the reduction of blood pressure like
angiotensin-converting-enzyme inhibitors.sup.(4) and drugs like
aspirin which act on clot formation. The effects of lifestyle
change to reduce body weight and stopping smoking have been
disappointing so far, although their impact has not been
established adequately on a population basis. Improvement of
regulation of diabetes has resulted in decreased morbidity (less
amputations, less diabetics with end-stage renal failure
necessitating dialysis, and less diabetics becoming blind),.sup.(5,
6) but the incidence of cardiovascular disease in diabetics did not
decrease by these measures..sup.(6, 7)
[0006] Many investigators point at the need for the recognition of
concealed risk factors for CHD in diabetes (and obesity) and a more
aggressive treatment of these factors should result in improved
outcome. Moreover, landmark trials with lipid-lowering drugs in
secondary and primary prevention settings have resulted in
significantly decreased mortality in treated patients (30% risk
reduction),.sup.(2) but there were still significant numbers of
patients that could not be saved by these drugs. Therefore, the
identification of additional risk factors and the development of
novel therapeutic interventions are expected to result in a
significant reduction of total mortality due to CHD.
[0007] It has been postulated that atherosclerosis is associated
with an impaired clearance of chylomicron remnants, i.e., partially
hydrolyzed chylomicrons (intestinally derived triglyceride-rich
lipoproteins). Also, it was suggested (PCT International Patent
Publication WO 00/34469) that clusterin could be used as a
migration inhibitor of vascular smooth muscle cells in arteries
whose migration and proliferation may lead to vessel injury and
arterial lesion and whose migration and proliferation can be
induced by atherosclerosis. Rosenberg and Silkensen,.sup.(10) in
reviewing the multifunctional protein role of clusterin state the
determination of a common mechanism underlying its various
functions would lead to a key in comprehending an important area of
biology. Other researchers.sup.(11) have demonstrated that
clusterin (apo J) may have a protective role against
atherosclerosis as it participates in cholesterol transport.
[0008] One of the recently recognized mechanisms in the development
of atherosclerosis is inflammation..sup.(8) Several studies have
demonstrated that slightly elevated concentrations of C-reactive
protein (CRP; a well-known acute-phase reactant named after its
reactivity with the so-called C-polysaccharide of pneumococci) are
predictive of coronary events in middle-aged and elderly men and
women. However, the precise mechanism by which complement is
involved in atherosclerosis is not known. In discussing a possible
relationship between infections with pathogenic micro-organisms,
MBL (an innate immune-defense plasma protein) deficiency and
atherosclerosis, Madsen et al..sup.(12) suggested the presence of
unexpected non-infective mechanisms relevant to the development of
atherosclerosis but could not conclusively exclude a relationship
with other pathogens. The role of MBL in the immune system and the
use of recombinant MBL in treating deficiencies in the immune
system is well known (PCT International Patent Publication WO
00/70043). The present invention teaches how lipid metabolism,
complement activation, atherogenic processes and immune responses
are physiologically related.
SUMMARY OF THE INVENTION
[0009] The present inventors have elucidated a mechanism providing
an explanation for the insufficient protective effects of
lipid-lowering drugs, the persistently high incidence of coronary
heart disease in western societies, and the relationship with
markers of inflammation like CRP. As a result of this new insight,
a novel approach for the diagnosis, prevention, and/or therapy of
atherosclerosis and underlying or related disease(s) is presented
which comprises a method for the treatment and/or prophylaxis of
diseases associated with disturbances in the complement/lipid
pathway by modulating the activity of one or more elements in the
pathway. Such a new method may for instance be implemented at a
large scale in combination with current strategies to lower
mortality and morbidity by CHD.
[0010] The present invention makes use of the surprising notion
that the primary and most ancient function of the complement system
is the transport and targeting of lipoproteins (i.e., chylomicrons,
VLDLs, LDLs, and their remnants) to the liver, rather than its
antimicrobial activity. Consequently, atherosclerosis as observed
in disorders associated with disturbed lipid metabolism (familial
combined hyperlipemia (FCHL), postprandial hyperlipidemia,
hypertriglyceridemia with low levels of HDL cholesterol, and
insulin resistance associated with type-II diabetes and obesity),
must be ascribed to either genetic or acquired defects in ancient
(activatory and/or regulatory) complement components. Based on this
new insight, novel preventive measures and treatment modalities of
disturbed lipid metabolism are introduced.
[0011] In accordance with the invention, it has surprisingly been
found that clearance of chylomicron remnants and in general
clearance of all triglyceride-rich particles (chylomicrons, VLDL,
IDL and their remnants) and LDL particles is positively regulated
by the complement system; that is to say by the most ancient
complement activation pathways, the "lectin" and "alternative"
pathways. Delayed clearance of triglyceride-rich particles, in
particular those containing apolipoprotein B as a structural
protein, is related to deficiencies in the ancient complement
activation pathways. Moreover, in one embodiment the invention
predicts that low serum levels of the intercellular matrix proteins
vitronectin and/or clusterin, which function as regulators of the
"terminal" or "lytic" pathways of complement, lead to decreased
intravascular integrity of chylomicron remnants. Such a decreased
integrity is typically atherogenic.
[0012] Accordingly, the invention relates to the use of purified or
enriched physiologic complement components, physiologic complement
regulators and/or extrinsic complement modulators of natural (e.g.,
plant-derived), synthetic, or semi-synthetic origin in the
prevention and/or treatment of atherosclerosis and underlying
and/or related diseases by substituting for and/or at least
diminishing deficiencies in the complement activation pathways.
[0013] Because a thorough and mechanistic insight has now been
achieved, the invention provides novel diagnostic tools and
formulations of specific and highly effective primary and secondary
prevention strategies for disturbances leading to atherosclerosis.
Dependent on what is (are) the weakest link(s) in the specific
pathways of the complement system in an individual patient, a
physician can, based on the considerations of the invention,
modulate the activity of the complement system of the patient in
order to prevent and/or treat manifestations of disease.
[0014] The present invention has as an objective to provide new and
improved manners of prevention and/or treatment of atherosclerosis
and underlying/related diseases. The invention further provides new
and improved manners of determining the occurrence (diagnosis) of
atherosclerosis and related diseases, in particular those which are
associated with disturbed lipid metabolism and to classify these
diseases accordingly.
[0015] A further object of the present invention is to provide for
coordinated design and discovery of new drugs for the treatment of
atherosclerosis and related diseases as well as providing
compositions comprising modulators of the complement activation
pathways which can serve as a basis or an ingredient of a
pharmaceutical composition or a food product. The present invention
therefore also relates to pharmaceutical products or food products
that comprise such modulating compositions. As a further object,
the present invention provides the use of at least one complement
factor or modulator for the manufacture of a medicament for the
treatment and/or prevention of atherosclerosis or an underlying
and/or related disease.
[0016] In order to appreciate the importance of the invention, the
inventors deem it necessary to explain the newly developed concept
in much more detail. The surprisingly intricate relationship
between the complement system and the clearance of chylomicron
remnants unraveled by the inventors signifies a pathway not
hitherto known. This unexpected finding gives rise to measures for
treatment and prophylaxis of atherosclerosis that are themselves
surprising, and that lead to the identification of additional risk
factors and the development of novel therapeutic interventions
which results in a significant reduction of total mortality due to
CHD.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows the complement system in schematic
representation.
[0018] FIG. 2 shows the two most ancient pathways of the complement
system in a schematic representation.
[0019] FIG. 3 shows the relationship between triglyceride-rich
particles (TRP), their remnants (TRP-R), the position of
lipoprotein lipase (LPL), free fatty-acids (FFA),
acylation-stimulating protein (ASP), several complement components
(C3, factor B and factor D) in relation to triglyceride (TG) uptake
by adipocytes and liver-derived very low density lipoproteins
(VLDL).
[0020] FIGS. 4A to E show the binding to chylomicrons of C3, MBL,
clusterin and vitronectin as determined in Example 1.
[0021] FIGS. 5A and B show the effect of immune adherence of
triglyceride-rich particles to erythrocytes in blood after staining
with Sudan Black as determined in Example 2.
[0022] FIG. 6 shows the flow cytogram obtained after staining apo B
on human erythrocytes as determined in Example 3.
[0023] FIG. 7 shows the internalization of triglyceride-rich
particles in a blood leukocyte as determined in Example 4.
[0024] FIG. 8 shows the complement/lipid pathway in schematic
representation.
[0025] FIG. 9 shows the potency of different substances to activate
complement in vitro.
[0026] FIG. 10 shows the effects of glycosylated plant stanols on
fasting triglycerides, plasma apoB and plasma cholesterol levels
over a four month period in a patient with heterozygous Familial
Hypercholesterolemia.
[0027] FIG. 11a shows the effect of vitamin A on postprandial C3
plasma concentration after two hours in healthy lean
volunteers.
[0028] FIG. 11b shows the effect of vitamin A on postprandial
plasma triglyceride concentration after two hours in healthy lean
volunteers.
DETAILED DESCRIPTION OF THE INVENTION
The Complement System
General Description
[0029] The complement system.sup.(9) is a complex signaling system
comprising enzymes present in blood. The complement system is
involved in the early recognition and clearance of foreign bodies
and antigen-antibody complexes (also called immune complexes) from
the circulation and tissues. Complement is recognized as an
important, humoral defense system involved in the innate
(nonspecific) recognition and elimination of microbial invaders,
other foreign particles or molecules, and antigen-antibody
complexes from the body.
[0030] Upon the recognition of foreign material in tissue or blood,
the most crucial and abundant complement component, C3, is
activated by C3 convertases. This activation triggers a cascade of
events that ultimately leads to the clearance of the foreign
material. C3, consisting of an a chain and a .beta. chain, is
activated through a split-conversion into C3b and C3a (FIG. 1). C3a
represents the N-terminus (77 amino acids) of the .alpha. chain and
C3b represents the C-termini of the .alpha. and .beta. chains. C3
convertases, of which various forms exist, can be generated through
three different complement activation pathways (FIG. 1) and its
synthesis is well regulated. The C3-convertase-generating pathways
include, in order of descending evolutionary age, the so-called
"lectin" pathway (LP), the "alternative" pathway (AP) which is also
known as the "amplification loop," and the relatively young
"classical" pathway (CP). During evolution, an additional system
known as "terminal" or "lytic" pathway has developed on top of the
complement activation system, which can destabilize membranes of,
e.g., Gram-negative bacteria, virus-infected body cells, or even
tumor cells by pore formation, resulting in their killing.
Phylogenetic studies have pointed out that the "lectin" and
"alternative" pathways are by far the most ancient complement
activation pathways (about 700 million years; FIG. 2), whereas the
"classical" and "lytic" pathways are relatively young (400 to 350
million years).
[0031] The complex nature of the complement system can be
appreciated when following the fate of the split-products of C3.
One of the split products, C3a, is a spasminogen and anaphylatoxin,
which induces the release of histamine from basophilic cells,
including tissue mast cells and basophilic granulocytes. Histamine,
in turn, helps phagocytes to leave the blood vessels in order to
arrive at the site of complement activation, i.e., the accumulation
site of foreign material or immune complex. In blood, C3a is
rapidly (in about 15 minutes) inactivated by serum
carboxypeptidases. The most prominent serum carboxypeptidase (sCP)
in blood is the constitutively expressed sCP-N. All other sCP types
are inducible and are less abundant than the N type. Upon the
inactivation of C3a by carboxypeptidases, the C-terminal arginine
is removed, resulting in the generation of C3adesArg. This compound
is (probably identical to) an acylation-stimulating protein (ASP),
a hormone that can stimulate fat accumulation in the body.
[0032] C3b and its inactivated form C3bi are opsonins, which means
that they can bind covalently to sugar OH groups (via ester bonds)
or protein NH.sub.2 groups (via amide bonds) on material identified
as "foreign." In case of such binding events, foreign material is
also termed "substrate." Other complement components can also
function as opsonins, among these are other C3-derivatives and the
complement component C4 (see below) and derivatives thereof.
Opsonins promote the clearance of foreign material by the
blood-based monocytes and tissue-based macrophages, both known as
mononuclear phagocytes. The mononuclear phagocytic system is
present in the liver, spleen, lymph nodes, or the affected tissue
itself. These specialized cells carry specific complement receptors
on their surface that can bind the opsonins. Known complement
receptors on phagocytes are CR1, CR3, and possibly also CR4. CR1 is
an exclusive receptor of C3b whereas CR3 and CR4 are also able to
bind C3bi. In contrast to mononuclear phagocytes, polymorphonuclear
phagocytes (PMNs) are relatively inefficient in eliminating foreign
material, at least in the absence of antibodies.
[0033] In primates, immune complexes are eliminated by the
mononuclear phagocytic system in liver, spleen and bone after
erythrocyte-mediated transport via the blood stream. The
erythrocytes carry a restricted number of CR1 molecules on their
surface to which C3b(i)-coated immune complexes can adhere. This
phenomenon is called "immune adherence." Erythrocytes of
non-primate species are CR1 negative and consequently do not
mediate the transport of immune complexes to liver, spleen and
bone. In primates suffering from systemic autoimmune diseases and
neoplastic diseases (cancer), the clearance of immune complexes
involves antibody-mediated activation of the complement system.
[0034] Microbial pathogens in the circulation are also cleared by
the mononuclear phagocytic system, but only after MBL ("lectin"
pathway)-mediated or antibody/C1 ("classical" pathway)-mediated
activation of complement components C4, C2, and C3. This process is
known to involve erythrocyte-mediated clearance as well.
[0035] The phenomenon of "immune adherence," as it has turned out,
is one of importance to the present invention, as the present
inventors have found that these CR1 complement receptors do not
only bind immune complexes or microbial pathogens, but also
chylomicrons and other triglyceride-rich particles and their
remnants. Based on this finding, new methods for the treatment and
prophylaxis of atherosclerosis and related disease have emerged
that are based on intervention or modulation of the complement
pathways involved.
[0036] As mentioned, the complement system comprises several
pathways each with a multitude of protein compounds, signaling
molecules, receptors, regulators and activators. To appreciate the
scope of the present invention, the various pathways of complement
activation will be described in some more detail.
The Complement System: The "Lectin" Pathway
[0037] Activation of the "lectin" pathway (LP) starts with the
recognition and binding of foreign bodies by a serum lectin, called
mannose-binding lectin (MBL). MBL is a high-molecular-weight,
sugar-binding protein, present in minute amounts (about 2 .mu.g per
ml) in blood plasma. MBL and the lung surfactant proteins A (LspA)
and D (LspD), belongs to the family of the collagenous lectins
(collectins). C1, the first component in the "classical" pathway is
a collectin-like activator of C4 and C2. Upon binding of MBL to
foreign bodies, a number of MBL-associated proteins (MASPs--which
are themselves esterases) become coordinately activated, ultimately
leading to the generation of the active forms of the associated
proteins, the LP-dependent C4, C2 and/or C3 convertases. These
convertases, which have C3, C4 and C2 as their natural substrates,
generate essentially five products: C3b and C3a, C4bC2a and split
products C4a and C2b. Like C3b, the C4b portion of C4bC2a binds
covalently to its substrate (e.g., polysaccharides or
(glyco)proteins on bacteria) via ester or amide bonds, and is
therefore known as an opsonin. The two split products, C4a and C2b,
are released in the fluid phase. Substrate-bound C4bC2a is the
LP-dependent C3 convertase, causing the conversion of C3 into C3b
and C3a. Like C3a, C4a is a spasminogen and anaphylatoxin
(histamine liberator), whereas C2b has kinin-like activity.
Furthermore, one of the MBL-associated proteins is capable of
direct activation of C3.
[0038] MBL recognizes foreign bodies by its six identical
sugar-binding moieties with specificity for mannose,
N-acetyl-glucosamine, and fucose. This makes sense, because
microbial pathogens like fungi, yeasts, and, e.g., Mycobacteria
carry relatively high amounts of mannose, while peptidoglycan of
gram-positive bacteria contains N-acetyl-glucosamine as one of its
major building blocks.
The Complement System: The "Alternative" Pathway
[0039] Until the discovery of the "lectin" pathway in 1989, the
"alternative" pathway (AP, also known as alternate pathway or
alternative complement pathway), first described in 1956, was
considered the most ancient complement activation route. The main
function of this "alternative" pathway is to increase (amplify) the
number of C3-converting sites on the substrate of complement
activation: the foreign body or the immune complex. This means
that, once "non-self" material has been identified by MBL and
activation of the "lectin" pathway has consequently taken place,
the LP-dependent C3 convertase C4bC2a present on the substrate will
be amplified by AP-dependent C3 convertases in the following manner
(FIG. 1): Substrate-bound C3b, generated by the LP-dependent C3
convertase C4bC2a, will bind AP component factor B which, in turn,
will be activated to Bb by AP component factor D (also known as
adipsin) to form the AP-dependent C3 convertase (C3bBb). Along with
the formation of this new C3 convertase, the factor-B part loses a
split product called Ba. The enzymic function of the AP-dependent
C3 convertase is considerably stabilized upon the binding of AP
component "properdin" (factor P), resulting in the AP-dependent C3
convertase complex C3bBbP. Split product Ba is a leukotaxin, which
helps to direct the movement of phagocytes to the site of
complement activation (primary inflammation site).
[0040] The net result of AP activation is an increase in the number
of C3b and inactivated C3b (C3bi) moieties on the substrate, which
promote the recognition and clearance of foreign bodies and immune
complexes by, predominantly, mononuclear phagocytes
(monocytes/macrophages).
The Complement System: The "Classical" Pathway
[0041] The "classical" pathway (CP) is generally considered the
youngest complement activation route, since it is dependent on
antibodies (IgM and IgG), which appeared relatively late in
phylogeny (from about 350 million years ago). The CP is very
similar to, and therefore probably derived from the ancient
"lectin" pathway, since the first CP component (C1; consisting of a
complex of the collectin-like C1q and two MASP-like proteins called
C1r and C1s) is both phenotypically and functionally very much
related to the MBL/MASPs complex. In addition, the "classical"
pathway involves "lectin" pathway complement components C4 and C2.
Like the sugar-bound MBL/MASPs complex, C1 (composed of C1q, C1r,
and C1s) bound to IgM- or IgG-type immune complexes becomes
coordinately activated to form a C1-esterase which has C4 and C2 as
its natural substrates and which gives rise to the generation of
CP-dependent C3 convertases, which are identical to LP-dependent C3
convertases (substrate-bound C4bC2a complexes).
[0042] C4 exists in two isoforms known as C4A and C4B. C4A is
involved in the clearance phenomenon, whereas C4B is mainly
involved in the killing of bacteria and cell destruction (e.g.,
hemolysis). In the present description, C4 is understood to relate
to the C4A isoform unless otherwise stated.
The Complement System: The Terminal or "Lytic" Pathway
[0043] When a newly formed C3b molecule does not bind to the
substrate directly, but to another, substrate-bound C3 convertase
(C4bC2a or C3bBbP), triple or quadruple complexes consisting of
C4bC2aC3b or C3bBbC3bP are formed. These complexes have
C5-converting activity indicating that they are able to split
complement component C5 into C5b and C5a. This is the starting
point of the so-called "terminal" or "lytic" complement pathway.
Like Ba, C5a is a leukotaxin, but more potent than Ba. C5b forms a
complex with C6 and C7, the resultant of which is a soluble C5b-7
complex, which has affinity for membranous bilayers. Upon insertion
into a membrane of, e.g., a gram-negative bacterium, complement
component C8 will bind to the complex, which results in a new
enzyme, the membrane-bound C9 polymerase (C5b-8). Under the
influence of one C5b-8 complex, some 13 C9 molecules become
polymerized, resulting in a cylindrical pore in the membrane that
is under attack. Depending on the total number of membrane-bound
poly-C9 pores, and on whether the bacterium is encapsulated or not,
the Gram-negative bacterium will either be killed or be able to
resist and survive membrane attack.
The Complement System: Complement Regulation and Complement
Regulators
[0044] In order to prevent unwanted activation of the complement
cascade, e.g., by cells of the body itself (homologous cells, in
contrast to foreign or heterologous cells), complement activation
on homologous cells is heavily regulated by both cell-bound
complement inhibitors and regulators in the fluid phase (e.g.,
serum or plasma).
[0045] The most important soluble regulators are: [0046] For the
"lectin" pathway: .alpha..sub.2-Macroglobulin (.alpha.2M), serpines
and C4-binding protein (C4BP), which interfere with the formation
of the LP-dependent C4/C2-convertase (activated MBL/MASPs complex)
and the subsequent activation of C4 and C2; [0047] For the
"alternative" pathway: Factor H (also known as .beta.1H) and factor
H-like molecules, acting at the level of factor B binding to
target-bound C3b (preventing the formation of AP-dependent C3
convertases), C3b inactivator (factor I), acting in conjunction
with factor H, to convert C3b in its enzymatically inactive, but as
opsonin still active form C3bi; [0048] For the "classical" pathway:
C1INH, an inhibitor of complement component C1, acting at the level
of activated C1, the C1-esterase (C1INH is also an inhibitor of
other serine esterases such as kallikrein, the clotting factors XIa
and XIIa, and the fibrinolysis product plasmin); and [0049] For the
"lytic" pathway: Vitronectin (S protein) and clusterin (also known
as apolipoprotein J or apo J). These proteins act at the level of
C5b-7 complexes, preventing their insertion into bilayer membranes
and inhibiting C9 polymerization and consequently, the lysis of
bacteria, viruses and body cells.
[0050] Cell-bound complement regulators include: [0051] Complement
receptor 1 (CR1) which has factor-H-like co-enzyme function versus
factor I; CR1 is present on phagocytes, platelets, but also as a
carrier protein on erythrocytes; [0052] Decay-accelerating factor
(DAF, which is also known as cluster of differentiation protein
CD55) and membrane cofactor protein (MCP=CD46), both acting at the
level of AP activation; [0053] Homologous restriction factor with
20-k molecular mass (HRF20=CD59) and HRF60, both inhibitory at the
level of C9 polymerase (C5b-8) formation; and [0054] Sialic acid,
which acts similar to CD55 and CD46 at the level of the
AP-dependent C3-convertase formation, but also on C9
polymerization.
The Complement System: Complement Activation and the Innate and
Specific Immune System
[0055] Apart from the physiological activatory and regulatory
complement components mentioned above, different substances of
bacterial, plant, animal, or (semi)synthetic origin are known to
either activate or inhibit the complement cascade(s). These
components include, i.e., bacterial lipopolysaccharides,
.beta.-glycyrrhetinic acid, phytosterols, bovine conglutinin, and
polymeric substances like dextran sulphate and glucans.
[0056] Bacterial lipopolysaccharides have recently been recognized
as potent activators of the "lectin" pathway. Likewise,
.beta.-glycyrrhetinic acid, as a possible activator of C4, was
suggested to be able to activate the "lectin" pathway, while the
phytosterols with as most important repesentatives
.beta.-sitosterol, stigmasterol, and campesterol, have been shown
to activate the "alternative" pathway. Dextran sulphate functions
as an acceptor site for "alternative" pathway regulatory protein
factor H, and thus facilitates the "alternative" pathway-mediated
activation of C3 and subsequent deposition of C3b on a
substrate.
[0057] Based on their complement-activating capacity, a number of
these substances, including bacterial lipopolysaccharides, dextran
sulphates, and glucans, as well as lipidated muramyl-dipeptides and
lipophilic quaternary ammonium compounds like dimethyldioctadecyl
ammonium bromide, show potent immunological adjuvant activity,
which means that they are able to stimulate antigen-specific T- and
B-cell responses.
Lipid Metabolism the Physiology of Lipid Metabolism
[0058] Under physiologic conditions, about 90% of the ingested fat
(triglycerides) is taken up by the epithelial cells of the small
intestine, resulting in the generation of intestinally derived
triglyceride-rich lipoproteins, called chylomicrons. These
chylomicrons are transcytosed through the epithelial cells and
delivered at their basolateral side to the sub-epithelial
interstitium. The structure of chylomicrons is stabilized by a
large, highly glycosylated protein, called apolipoprotein B48 (apo
B48), of which the most dominant glucoses residues are: mannose
(17.8%), N-acetyl-glucosamine (16.8%), galactose (13.4%), and
fucose (3.4%) which, in fact, fully matches with the binding
specificities of MBL. Apo B48 is the 5' splice product of a larger
apob gene, which, in human intestinal epithelial cells, is
post-transcriptionally modified by a unique editing enzyme. This
modification results in a premature stop codon leading to the
translation of only 48% of the apob mRNA. Since the human liver
lacks the unique editing enzyme, apob transcription in the liver
results in the synthesis of full-length apo B100. This protein is
the structural protein of the liver-derived triglyceride-rich
particles known as VLDL (very low density lipoproteins) and their
remnants (IDLs and LDLs).
[0059] From the sub-epithelial interstitium, chylomicrons are
collected in tissue fluid (lymph). Via lymph vessels, they are
transported to subsequent draining lymph nodes and, through the
thoracic duct and the left subclavian vein, they finally arrive in
the blood stream. Once in the circulation, chylomicrons are rapidly
converted into chylomicron remnants by the action of
vascular-endothelium-associated lipoprotein lipase (LPL).
Chylomicron remnants are present in blood in different sizes.
[0060] Chylomicrons and chylomicron remnants are subsequently
cleared efficiently by the liver from where they can undergo
bile-mediated excretion via the stools. However, the efficiency of
the process of chylomicron and chylomicron-remnant targeting to the
liver is far from understood, while the subsequent hepatic
clearance of these triglyceride-rich particles has not completely
been elucidated either. In the liver, it involves at least the
activity of the hepatic triglyceride lipase (HTGL), interaction
with specific apo E receptors, and non-receptor binding to the
cellular surface in the hepatic space of Disse. Several local
receptors may be involved including low-density-lipoprotein
receptor-related protein/.alpha..sub.2-Macroglobulin receptor
(LRP-.alpha..sub.2M), a parenchymal liver cell "chylomicron remnant
receptor," the asialoglycoprotein receptor, the
lipolysis-stimulated receptor, and the IDL (low density
lipoprotein) receptor. Recently, the VLDL receptor, a new member of
the LDL receptor supergene family, which is not present in the
liver, has been recognized as a physiological receptor for
chylomicron remnants.
[0061] Cholesterol, delivered to the liver by chylomicrons and
chylomicron remnants, is largely re-secreted into the circulation
after incorporation into very-low density lipoproteins (VLDL). This
cholesterol is further employed by the adrenals and genitals as a
skeleton for their steroid-hormone synthesis.
[0062] Free fatty acids (FFA) arising from the breakdown of
chylomicrons by the endothelial LPL are transported over the mucosa
towards sub-endothelial fat cells (adipocytes) in which they become
re-esterified into intracellular triglycerides (FIG. 3). The uptake
and incorporation of FFA into adipocytes is under the positive
control of a hormone called acylation-stimulating protein
(ASP).
[0063] Similarly to the hydrolysis of triglycerides in
chylomicrons, VLDL may become VLDL remnants also called IDL
(intermediate-density lipoproteins) by the lipolytic action of LPL,
in this case under the positive and negative control of two other
apolipoproteins, apo CII and apo cIII,.sup.(11) respectively. IDL
are rich in apo E which functions as the ligand for the hepatic LDL
receptor and "remnant-receptor" (=LRP, LDL-receptor-related
protein, a member of the LDL-receptor family comprising complement
repeats; possibly older than the LDL-receptor itself). Apo E
(formerly "Arginine-Rich Apoprotein") is one of the protein
constituents of triglyceride-rich lipoproteins. Chylomicron
remnants depend on apo E for their binding to the receptors, since
the apo B48 structural protein does not contain the
(carboxy-terminal) binding site for the LDL-receptor and "remnant
receptor." Apo E is synthesized by almost all tissues but not by
the epithelium of the intestine. The major organ responsible for
apo E synthesis is the liver. As a result, chylomicrons receive apo
E from HDL in the circulation and, therefore, apo E is an
exchangeable apoprotein. In the liver-sinusoids, hepatocytes
secrete apo E resulting in an enrichment of remnant particles,
thereby facilitating their removal from the circulation. There are
three major apo E isoforms which are genetically determined: Apo E3
(the most common), apo E2 (which results in a minority of the cases
in dysbetalipoproteinemia in homozygotes), and apo E4. The latter
has the highest affinity for binding to the receptors, while apo E2
exhibits the lowest affinity. Apo E4-individuals are highly
responsive to dietary changes and cholesterol and fat enriched
diets lead to higher plasma cholesterol concentrations in these
individuals, due to down-regulation of LDL-receptors.
[0064] Under physiological conditions, IDL are taken up by
LDL-receptors in the liver, by which organ the lipoproteins are
degraded and cholesterol is removed from the body by excretion into
the bile.
[0065] Although much is known, the metabolic pathways of the
intestinally and liver-derived triglyceride-rich particles in
blood, chylomicrons and VLDL, respectively, and their remnants have
hitherto only partially been identified. It has been shown that
these pathways comprise common elements and show a certain overlap.
However, until the present invention, the very efficient targeting
to the liver of chylomicrons and chylomicron remnants under
physiological conditions and their clearance was far from
understood.
Lipid Metabolism Aberrant Lipid and/or Free-Fatty-Acid
Metabolism
[0066] Chylomicron remnants are potentially atherogenic
(atherosclerosis generating) particles due to their ability to
directly induce foam-cell formation, without any modification.
Low-density lipoprotein particles (LDL), in contrast, must be
oxidized before they induce transformation of mononuclear
phagocytes into foam cells. Mononuclear phagocytes have an LDL
receptor by which they are able to bind, take up, internalize and
subsequently degrade native LDL. As soon as the intracellular free
cholesterol levels reach a threshold value the IDL receptors are
down-regulated and the internalization process is stopped. Oxidized
LDL particles, on the other hand, are taken up by "scavenger"
receptors, which are not down-regulated by cholesterol.
[0067] Since chylomicrons, VLDL, and their remnants compete for the
same metabolic pathways, patients with delayed remnant clearance
may experience a temporary accumulation of chylomicrons and
chylomicron remnants in the circulation, which obviously
contributes to the process of atherogenesis. Such situations are
likely to occur in patients with familial combined hyperlipidemia
(FCHL), type-2 diabetes mellitus, insulin resistance, and obesity.
Enhanced plasma VLDL levels in these situations are associated with
delayed clearance of chylomicron remnants.
[0068] Similar mechanisms are involved in conditions in which the
clearance of remnant particles is impaired due to mutations in the
apo E ligand gene (type III hyperlipidemia=familial
dysbetalipoproteinemia), the LDL receptor (familial
hypercholesterolemia; FH), familial defective apo B100 (FDB) and
after menopause. In these conditions, which are all associated with
the development of (premature) atherosclerosis, a delayed clearance
of chylomicron remnants has been established due to an impaired
binding to receptors in the liver. Other disorders associated with
impaired remnant clearance are apo CII deficiency, (partial)
lipoprotein lipase (LPL) deficiency, and hepatic triglyceride
lipase (HTGL) deficiency. In these disorders, the conversion of
triglyceride-rich particles into their remnants is delayed, leading
to an accumulation in the circulation of triglyceride-rich
particles of different sizes and triglyceride content.
[0069] In many endocrinological disorders like hypothyroidism,
growth hormone deficiency, hypercortisolism by endogenous or
exogenous corticosteroids, and the postmenopausal state, a
decreased clearance of chylomicron remnants has been established
when compared with the control situation. Finally, in patients with
premature atherosclerosis and normal fasting plasma lipids (40% of
all patients with myocardial infarction below 60 years of age in
males and beyond 65 years of age in females), chylomicron-remnant
clearance is decreased. It has been hypothesized and it is widely
accepted that this may be one of the important mechanisms
underlying atherosclerosis in these groups of patients.
Identification of the underlying defect(s) in these patients and
modulation and improvement of their chylomicron-remnant clearance
will contribute to a reduction of the risk for coronary artery
disease and therefore to decreased morbidity and mortality.
[0070] Free fatty acids (FFA) arising from the breakdown of
chylomicrons by the endothelial lipoprotein lipase (LPL) and their
uptake by the adipocytes stimulate these adipocytes to synthesize
complement component C3 and "alternative" pathway components
factors B and D (note that in healthy individuals, there is a
linear relationship between total body fat and C3 levels), and
according to the invention complement activation occurs.
[0071] The prior art discloses several important pathways involved
in lipid metabolism and remnant clearance. However, designing
optimal treatment and/or prophylactic measures for atherosclerosis
and underlying and/or related diseases have thus far been
impossible to achieve. It is now found by the present inventors
that the existence of a pathway that was hitherto unknown allows
for the first time the development of such measures based on a more
complete and physiological and immunological understanding of the
diseases. The surprisingly intricate relationship between the
complement system and the clearance of chylomicron remnants
unraveled by the inventors signifies the presence of such a
pathway, which is termed the lipid eliminating complement
activation pathway or complement/lipid pathway.
[0072] Due to this new finding the identification of additional
risk factors, novel therapeutic interventions and pharmaceuticals
and the treatment and prophylaxis of atherosclerosis have now
become available, which will result in a significant reduction in
occurrence and/or progression of this disease and other diseases
associated with this pathway. The novel pathway was revealed inter
alia by three independent findings. The first finding comprises
that chylomicrons can induce complement activation. The second
finding comprises that chylomicrons bind to erythrocytes which
binding comprises complement factors as a result of which lipid
transport through the blood is complement and erythrocyte mediated.
The third finding relates to the glycosylation of apolipoprotein B,
its kinship to MBL binding specificity and the insight that the
complement-mediated lipid transport may thus be modulated through
intervention in the complement/lipid pathway and its individual
elements or components. Such elements or components are understood
to comprise all molecules and complex substances that play a role
in the complement/lipid pathway.
[0073] It has now surprisingly been found that chylomicrons,
isolated from healthy individuals after an oral fat load, carry
complement components C3 (i.e., the opsonins C3b and/or C3bi) (FIG.
4A). Thus, these chylomicrons initiate complement activation. In
addition, it was also surprisingly found that chylomicrons,
isolated from healthy individuals after an oral fat load, also
carry the "lectin" pathway complement component mannose-binding
lectin (MBL), and the terminal-complement-pathway inhibitors
clusterin and vitronectin (FIGS. 4B-E). Thus, chylomicrons activate
the "lectin" pathway (MBL-binding) which may ultimately lead to
opsonization with C3b(i) (see "lectin" pathway)) and to binding to
the CR1 receptor of phagocytes and erythrocytes (see general
description of complement system). Furthermore, the presence of
clusterin and vitronectin indicates a capacity to inhibit the
"terminal" pathway of the human complement system.
[0074] It was indeed found that virtually all erythrocytes of
healthy volunteers carry chylomicrons and chylomicron remnants
(FIG. 5A), whereas erythrocytes in the "fasting" state carry
considerably less chylomicrons and chylomicron remnants (FIG. 5B).
This finding is in accordance with the new concept of an
erythrocyte-mediated elimination of triglyceride-rich particles
(and possibly also LDL particles) and complement-mediated lipid
transport, and can be interpreted in terms of immune adherence of
remnant particles and targeting of lipids to the liver and
spleen.
[0075] The pathway revealed by the present inventors provides an
explanation for the observed complement activation and for a more
complete physiological and immunological understanding of
atherosclerosis and/or underlying and/or related disease. The
present inventors disclose that the prominent glycosylation sites
of apolipoproteins B48 and B100, that are present as structural
proteins on plasma chylomicrons and VLDL, respectively, match fully
with the mannose, N-acetylglucosamine, and/or fucose binding
specificity of MBL. This means that triglyceride-rich particles
(LDL, chylomicrons, VLDL, etc.) in blood directly activate the
complement system's "lectin" pathway through binding of
apolipoprotein B to MBL.
[0076] As an intrinsic complement activator (of MBL), apo B is
potentially very harmful (note the existence of autoantibodies
against the C3 convertases F-42 and C3 nephritic factor in patients
with collagen diseases). In particular, the intrinsic complement
activatory nature of the structural apolipoprotein B molecules of
triglyceride-rich particles is now predicted to be harmful for
individuals with decreased serum levels of "terminal" pathway
inhibitors vitronectin and/or clusterin, since such a situation
will, subsequent to "lectin" pathway activation, allow "terminal"
pathway activation to occur. "Terminal" pathway activation on
triglyceride-rich particles may result in the release of
atherogenic lipid material, particularly in patients with a genetic
or acquired deficiency in the "terminal" pathway regulators
vitronectin or clusterin. The binding of the "terminal" pathway
inhibitors vitronectin and clusterin to chylomicrons can
teleologically be explained in terms of protection from
atherosclerosis.
[0077] Combination of chylomicron (remnant)-induced complement
activation of the "lectin" pathway, the matching of glycosylation
sites of apolipoproteins B48 and B100, and the erythrocyte-mediated
elimination of triglyceride-rich particles predicts that increased
levels of triglyceride-rich particles in blood, as occurring in
FCHL and other disorders associated with atherogenic disturbances
of lipid metabolism, is due to sub optimal erythrocyte-dependent
clearance of chylomicrons and/or VLDL.
[0078] Also, disturbances in chylomicron- and/or VLDL- and/or
chylomicron-remnant- and/or VLDL-remnant-mediated complement
activation will lead to impaired lipid metabolism. Likewise,
disturbances in the complement cascade, albeit subtle and, e.g.,
acquired, may also lead to impaired lipid metabolism and, in the
long term, to atherosclerosis and CHD.
[0079] This bears considerable consequences for the treatment and
prophylaxis of all diseases related to the complement/lipid
pathway, specifically those relating to disturbances in lipid
metabolism. Such diseases are recognized to comprise
atherosclerosis and underlying or related disorders which include,
but are not limited to, ischemia, hyperlipidemia, such as familial
combined hyperlipemia (FCHL), postprandial hyperlipidemia and
hypertriglyceridemia with low levels of HDL cholesterol, insulin
resistance associated with type-II diabetes, obesity, coronary
heart disease and premature atherosclerosis.
[0080] Other diseases related to the disturbances in the
complement/lipid pathway are more immunological in appearance. The
similarity in their elimination pathways predicts that
triglyceride-rich particles have to compete with soluble immune
complexes and/or microbes for elimination sites on erythrocytes and
in the liver and spleen, which would explain the disturbed lipid
metabolism in, e.g., septic shock. This bears considerable
consequences for the treatment and prophylaxis of diseases such as,
but not limited to, the auto-immune disorders systemic lupus
erythematosus (SLE), rheumatoid arthritis (RA) and paroxysmal
nocturnal hemoglobinuria (PNH), virtually all infectious diseases
and related disorders such as AIDS-related (secondary)
lipodystrophy, septic shock, and multiple organ failure,
inflammatory diseases such as Crohn's disease, inflammatory bowel
syndrome (IBS), thermal injury including burns and frostbite,
uveitis, psoriasis, asthma and neoplastic diseases such as cancer.
This immunological aspect of the present invention holds
consequences for improving the effectiveness of vaccination
programs.
[0081] Disorders directly related to the complement/lipid pathway
comprise: [0082] disturbances in chylomicron-, chylomicron-remnant,
VLDL- and/or VLDL-remnant-mediated complement activation, [0083]
disturbances in the complement cascade itself, [0084] disturbances
in erythrocyte-dependent chylomicron remnant and/or VLDL-remnant
clearance, [0085] disturbances in the complement-mediated lipid
metabolism, disturbances in the regulation of lipid metabolism.
[0086] Such disorders are atherogenic and may lead to
atherosclerosis and/or an underlying and/or related disease or to a
disease directly related to disturbed lipid metabolism or to a
disease which may seem to be more related to an immunological
disorder or malfunction such as auto-immune diseases, infectious
diseases, neoplastic diseases and/or inflammatory diseases.
[0087] Treatment and/or prophylaxis can as a benefit of the present
invention occur by correction of disturbed complement function, in
case of impaired complement-mediated lipid metabolism and will lead
to an amelioration of lipid metabolism. By correcting the disturbed
complement function, in case of impaired complement-mediated lipid
metabolism, an amelioration of disorders associated with impaired
or disturbed chylomicron remnant clearance is achieved.
[0088] Further, correction of disturbed complement function, in
case of impaired complement-mediated lipid metabolism, will result
in an amelioration of atherosclerosis and underlying or otherwise
related diseases such as FCHL, insulin resistance in association
with type-2 diabetes and/or obesity, or coronary heart
disease/premature atherosclerosis.
[0089] Further, correction of disturbed complement function, in
case of impaired complement-mediated lipid metabolism, will result
in an amelioration of diseases of the immune system, as well as
concomitant infectious, autoimmune, neoplastic or hematological
diseases related to impaired complement-dependent lipid
metabolism.
[0090] Disturbances of lipid metabolism due to delayed or disturbed
erythrocyte-dependent clearance of chylomicrons and/or VLDL may
have a number of possible causes, which will determine the nature
of the corrective measure. There may be: [0091] (i) congenital
defects in glycosylation of apo B48 and/or apo B100; or [0092] (ii)
absolute (homozygous), or relative or acquired deficiencies of
individual complement components of the "lectin" and "alternative"
pathways (such deficiencies are known to occur for MBL (9% of the
population), C4A (defective gene frequency 10 to 13% of the
population), C4B (defective gene frequency 7 to 18% of the
population), C2 (rare), C3 (rare), factor B (rare) and factor D
(rare)); or [0093] (iii) deficiencies of serum carboxypeptidases
(sCP) which exclude the conversion of C3a into C3adesArg (incidence
unknown); or [0094] (iv) absolute (rare) or relative (quite common)
deficiencies of complement receptor 1 (CR1) on erythrocytes as
occurring in some patients with systemic lupus erythematosus (SLE);
[0095] (v) deficiencies of terminal-pathway regulator vitronectin
(4% of the population), which may lead to the lysis of
triglyceride-rich particles resulting in unwanted deposition of
lipids; or [0096] (vi) decreased serum levels of clusterin in
association with exacerbations of SLE or with circulating immune
complexes accompanying neoplastic diseases (deficiencies of
clusterin are rare; <<1% of the population).
[0097] The incidence of serious cardiovascular disease (37% in
1997) in the Netherlands expressed as percentage of total numbers
of fatal cases per year, matches well with the combined figures for
MBL, C4A, C4B, vitronectin, and clusterin deficiencies, corrected
for the incidence of double and triple deficiencies.
[0098] It is one embodiment of the present invention to provide a
method for the treatment and/or prophylaxis of diseases associated
with disturbances in the complement/lipid pathway by modulating the
activity of one or more elements in the pathway.
[0099] In another embodiment according to the invention the
activity of one or more elements of the lectin pathway and/or the
alternative pathway of complement activation are modulated.
[0100] Modulating according to the present invention should be
understood as regulating, controlling, blocking, inhibiting,
stimulating, activating, mimicking, bypassing, correcting,
removing, washing, administering, adding, and/or substituting one
or more elements in the pathway or, in more general terms,
intervening in the pathway.
[0101] In one aspect of the invention, the elements in this pathway
comprise triglyceride-rich particles and/or their remnants and
their constitutive proteins, complement proteins, complement
activators, complement inhibitors, complement regulators and/or
complement receptors.
[0102] In one embodiment, the activity of one or more elements is
modulated through administration of a modulator.
[0103] Modulators according to the invention are substances that
can bring about a modulation in the complement/lipid pathway or the
complement system and may comprise triglyceride-rich particles
and/or remnants thereof and/or constitutive proteins thereof,
complement proteins, complement activators, complement inhibitors
such as serpines, factor H, factor I and/or C1INH, complement
regulators such as .alpha.2M, their metabolic precursors, encoding
genes and/or fragments thereof and they may be of physiologic
(human or primate-derived), natural (e.g., plant-derived),
recombinant, synthetic and/or semi-synthetic origin in enriched,
purified and/or chemically modified, complete and/or partial form,
as metabolic precursor, as biochemically functional analogue or as
functional equivalent of a (physiologic) modulator and/or
derivatives thereof used alone or in combination.
[0104] "Functional equivalents" as used herein are understood to
comprise molecules having at least one function of the original
compound, preferably all functions of the original compound
(although not necessarily to the same extent), more preferably
chemically similar compounds, most preferably compounds differing
by at most three groups not relevant for the relevant activity
and/or function of the original compound. In the context of the
present invention, functional equivalents of complement factors are
understood to comprise the split products of these factors.
[0105] In a preferred embodiment, modulators may be MBL-replacement
factors, which exhibit one or more functions of the mannose binding
lectin such as binding to C3b or a mimetic thereof, and/or binding
to the prominent apo B glycosylation sites or mimetics thereof.
Such an MBL-replacement factor may comprise lectins derived from
plants such as, e.g., concanavalin A, peanut lectin,
phytohemagglutinin or wheat-germ agglutinin, but they may also
comprise purified or enriched physiologic MBL or synthetic, or
semi-synthetic mimetics of MBL and/or functional equivalents of MBL
and may be used in an aspect of the invention relating to
substituting for MBL deficiencies in the complement/lipid pathway.
MBL replacement compounds also comprise lipid-C3 conjugates.
[0106] In another preferred embodiment, modulators may comprise apo
B-replacement factors, which may be functional equivalents of apo B
that, e.g., exhibit one or more functions of apolipoprotein B48 or
B100 such as binding to MBL or mimetics thereof and an ability to
form a constituent of a lipoprotein or a mimetic thereof. Such an
apo B-replacement factor may be chosen from the group comprising
physiologic apo B 48 or B100, natural lipo-oligosaccharides,
lipopolysaccharides, lipidated oligo- or polysaccharides,
glycoproteins, .beta.-glycyrrhetinic acid, chylomicron-bound sialic
acid, phytosterols (.beta.-sitosterol, campesterol, and/or
stigmasterol) and (an)other amphiphilic (=partially hydrophobic and
partially hydrophilic) complement activator(s) (e.g., mannosylated,
N-acetylglucosaminylated, and/or fucosylated phytosterols, or
mannosylated, N-acetylglucosaminylated, and/or fucosylated membrane
lipids, such as phosphoglycerides, glycolipids such as cerebroside
or ganglioside, or sphingomyelin, phosphatidyl choline,
phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl
inositol, diphosphatidyl glycerol or sphingosine), stanols
(glycosylated and non-glycosylated), lipidated dextran sulphate(s),
(lipo)glucan(s), lipidated tertiary or quaternary ammonium
compounds, sialylated glycolipids, combinations thereof and single
and/or combined related substances. In general, suitable apo
B-replacement factors comprise amphiphilic compounds or derivatives
thereof wherein the hydrophilic part comprises one or more
cationic, anionic and/or polar groups and wherein the hydrophobic
part comprises one or more fatty-acid ester moieties. The
fatty-acid ester moieties may comprise carbon chain lengths from 1
to 50 carbon atoms, they may be straight and/or branched and they
may comprise saturated and/or unsaturated fatty acids.
[0107] Preferred amphiphilic modulators additionally comprise one
or more sugar moieties, such as N-acetylgalactosamine, galactose
and/or sialic acid, which allow interaction with a lectin binding
site. In a most preferred embodiment according to the invention,
such one or more sugar moieties are mannose, N-acetylglucosamine,
and/or fucose moieties that allow interaction with the lectin
binding site of MBL. Other suitable apo B replacement factors may
comprise an IgA or IgD antibody, which is heavily mannosylated,
N-acetylglucosaminylated, and/or fucosylated of either polyclonal
or humanized monoclonal or combinatorial origin, directed towards
one of the apolipoproteins of chylomicrons or very low-density
lipoproteins (VLDL). Such antibodies may also be bi-specific
antibodies reactive towards both apoB and CR1, thereby being able
to, e.g., create bonds between its two antigens.
[0108] In another embodiment, modulators may be selected from the
group comprising MBL and MBL-replacement factors, C4A and
functional equivalents thereof, C4B and functional equivalents
thereof, C2 and functional equivalents thereof, C3 and functional
equivalents thereof, IgG- and IgM-antibodies raised against
triglyceride-rich particles and LDL or parts thereof, C3adesArg,
factor B and functional equivalents thereof, factor D and
functional equivalents thereof, factor P and functional equivalents
thereof, serum carboxypeptidases such as sCP-N and functional
equivalents thereof, erythrocyte-bound CR1 and functional
equivalents thereof, free CR1 and functional equivalents thereof,
CR1 mimetics such as C3b antibodies, vitronectin and functional
equivalents thereof, clusterin and functional equivalents thereof
and apo B (48 and 100) and apo B replacement factors and esterases
such as one of the MASP-proteins and functional equivalents
thereof.
[0109] In another preferred embodiment, modulators comprise
antibodies. In a more preferred embodiment, these antibodies for
the classical pathway are IgG and/or IgM antibodies.
[0110] In another embodiment, the group comprising apo B
replacement factors also comprises an IgA or IgD antibody directed
against an apo B lipoprotein, which antibody is heavily
mannosylated, and/or heavily N-acetylglucosaminylated and/or
heavily fucosylated.
[0111] In a more preferred embodiment, the modulator for the
classical pathway is selected from the group of antibodies wherein
the antibody comprises a polyclonal and/or humanized monoclonal
and/or combinatorial antibody and/or bi-specific antibodies
reactive towards both an apo B and CR1.
[0112] Administration of a modulator may comprise oral
administration, nasal administration, pulmonary administration,
inhalation, anal and/or rectal administration, intravenous
injection, intramuscular injection, intradermal injection,
subcutaneous injection, mucous membrane diffusion, skin absorption,
topical application, extracorporeal circulation-mediated
administration and/or any other suitable administration route,
single or in combination.
[0113] Modulators may be administered in pure form and/or diluted
form, they may be in solid, semi-solid, crystalline and/or fluidic
form, dissolved and/or dispersed single or as a constituent of a
fluid, a spray, a gel, an ointment, a tablet, a suppository, a
capsule (synthetic, natural or viral), a powder, a(n) (clinical)
intralipid, a (clinical) food product, a (clinical) food additive,
a lipidated vaccine for oral application, slow-release and/or
direct release carrier that contains the modulator and/or any other
suitable formulation for administration. Furthermore, modulators
may be unlabeled or labeled with signal molecules or groups such
as, e.g., dyes, fluorochromes, radioactive atoms or groups, enzymes
or luminescent molecules or groups.
[0114] Apo B replacement factors according to the invention may be
administered alone or in combination with other modulators in a
natural, artificial or synthetic lipid carrier compound comprising
lipoproteins, lipid micelles, lipid vesicles, artificial lipid
bilayer membranes, chylomicrons, liposomes and/or other suitable
and/or pharmaceutically accepted lipid substance. Clinical
intralipids (fat emulsions) used in relation to the invention as
parenteral feeding may comprise such a lipid carrier compound in
combination with one or more modulators. In a preferred embodiment
of such a parenteral feeding, the lipid carrier is selected from
the group comprising mineral oil and natural oils, such as soy oil,
sunflower oil, peanut oil, olive oil, palm oil and sesame oil and
processed (purified and/or modified) versions thereof. In a most
preferred embodiment of such a parenteral feeding, the lipid
carrier is (purified) olive oil.
[0115] It is another embodiment to administer modulators in such a
manner that the modulator is generated in vivo, e.g., by gene
therapy and/or by local administration of enzymes (e.g., apo B
glycosylation enzymes) their encoding gene(s) and/or gene
fragments.
[0116] It is a further embodiment to use a method for modulating
the activity of one or more elements in the complement/lipid
pathway for the treatment and/or prophylaxis of diseases associated
with impaired complement-mediated lipid metabolism.
[0117] It is a further embodiment to use a method for modulating
the activity of one or more elements in the complement/lipid
pathway for the treatment and/or prophylaxis of concomitant
(infectious, autoimmune, or neoplastic) diseases that (partially)
occupy the lipid eliminating complement activation pathway.
[0118] It is a further embodiment to use a method for modulating
the activity of one or more elements in the complement/lipid
pathway to prevent atherogenic processes of concomitant
(infectious, autoimmune, or neoplastic) diseases that (partially)
occupy the lipid eliminating complement activation pathway.
[0119] It is a further embodiment to use a method for modulating
the activity of one or more elements in the complement/lipid
pathway to efficiently manipulate the immune system.
[0120] It is a further embodiment to use a method for modulating
the activity of one or more elements in the complement/lipid
pathway to achieve optimum systemic immunosuppression by lipophilic
immunosuppressants.
[0121] It is a further embodiment to use a method for modulating
the activity of one or more elements in the complement/lipid
pathway to achieve optimum oral immunization. In a preferred such
embodiment, a method for modulating the activity of one or more
elements in the complement/lipid pathway is used as a
lymph-targeting, oro-mucosal adjuvant to induce enhanced mucosal
antibody (IgA) responses, T-cell reactivity, and/or systemic T-cell
and/or B-cell (IgM and/or IgG) antibody responses.
[0122] It is an embodiment to provide prophylactic measures for
diseases associated with disturbances in the complement/lipid
pathway by providing improved methods for diagnosing such
diseases.
[0123] It is one embodiment to estimate the anti-atherogenic
potential of plant-derived, synthetic, or semisynthetic substances
by determining their complement activation and/or consumption
activity. Complement consumption should be understood as complement
entering the complement cascade thereby disappearing as free
component.
[0124] It is another embodiment to estimate one or more of the
complement components selected from the group comprising MBL, C4A,
C4B, C2, factor B, C3adesArg, serum carboxypeptidase N,
vitronectin, clusterin, and erythrocyte-bound complement receptor 1
(CR1), in blood, blood serum and/or blood plasma of a patient in
order to establish the underlying or related defect of his/her
atherosclerosis.
[0125] It is a further embodiment that concomitant (infectious,
autoimmune, or neoplastic) diseases that may (partially) occupy the
lipid eliminating complement activation pathway can be diagnosed
more adequately so that atherogenic processes are prevented.
[0126] It is another aspect that an individual's lipid profile can
be determined with greater accuracy by using whole blood rather
than blood plasma in a lipid profile test.
[0127] It is a further embodiment to provide compositions for the
treatment and/or prophylaxis of diseases associated with
disturbances in the complement/lipid pathway. Compositions
according to such an embodiment of the present invention may be
pharmaceutical compositions, additives for pharmaceutical
compositions, active substances for pharmaceutical compositions,
additives for clinical nutrition and/or regular food additives and
that comprise modulators according to the invention, metabolic
precursors of such modulators, biochemically functional analogues,
functional equivalents and/or derivatives of such modulators with
or without expedients such as fillers, binders, other complement
activators such as vitamin A, thickening agents, preservatives,
lubricants, emulgators, and/or stabilizers.
[0128] It is an embodiment that such compositions are used to
modulate the activity of one or more elements of the
complement/lipid pathway according to a method of the
invention.
EXAMPLES
Example 1
Complement Components Associated with Chylomicrons
[0129] Experimental procedure: Chylomicrons were isolated from
plasma by ultra centrifugation and purified by column
chromatography, in the following manner: For separation of
lipoproteins, plasma samples were subjected to a single
ultra-centrifugation step as described in detail..sup.(12)
Chylomicron (Sf >1000) and non-chylomicron (Sf <1000)
fractions were separated by flotation. The chylomicron fraction
contained chylomicrons and large VLDL. The non-chylomicron fraction
contained chylomicron remnants, small VLDL and its remnants, LDL,
HDL and the remainder of the plasma proteins. Aliquots were stored
at -20.degree. C. until use. In the fractions containing large
chylomicrons (large triglyceride-rich particles) complement
components C3, MBL, clusterin (exp. 1), clusterin (exp. 2) and
vitronectin were measured by competitive ELISA using the purified
proteins and MBL- and C3-specific polyclonal and clusterin-specific
monoclonal-antibodies G7* and EB-8* and vitronectin-specific
monoclonal antibody MO-24* as reagents. The presence of the
complement factors could consistently be demonstrated in fractions
13 through 20 (see FIGS. 4A-E). In addition, C3 and MBL were also
found in other lipoproteins isolated by one-step density gradient
ultra centrifugation (Redgrave gradient) (IDL, LDL, HDL) in
subjects fasting and postprandial after a fat challenge.
Example 2
Adherence of Triglyceride-Rich Particles to Erythrocytes in Whole
Blood
[0130] Experimental procedure: To observe adherence of
triglyceride-rich particles to erythrocytes in whole blood, Sudan
Black staining of erythrocytes in whole blood was performed. In
this procedure, blood smears were prepared and Sudan Black staining
was performed with a filtered and saturated solution of Sudan Black
in 80% ethanol (4 grams of Sudan Black B, Electran in 200 ml of 80%
ethanol) by the following procedure. The blood film on the glass
slide was fixed by heat fixation (three times through a flame). The
slide was soaked in Sudan Black solution for three minutes after
which the slide was rinsed with 80% ethanol. The preparation was
re-hydrated by a graded ethanol series (one minute 40% ethanol, one
minute 20% ethanol, one minute demineralized water). Excess water
was shaken off and the slides were dried to air. Microscopic
examination of the slides revealed that virtually all erythrocytes
of healthy volunteers carried chylomicrons and chylomicron remnants
four hours after an oral fat intake (FIG. 5A), whereas erythrocytes
in the "fasting" state carried considerably less such particles
(FIG. 5B).
Example 3
Measurement of Erythrocyte-Bound Apo B-Containing Lipoproteins by
Flow Cytometry
[0131] Experimental procedure: Full capillary blood was drawn from
non-fasting healthy subjects by capillary punction. The blood was
washed three times in 10 ml of VSB.degree. buffer (Veronal Saline
Buffer) by centrifugation (3,000 rpm, ten minutes, 20.degree. C.)
and the cell count was adjusted to 1.5.times.10.sup.8/ml with
VSB.degree. buffer. A volume of 50 .mu.l of the sample was pelleted
and the pellet was re-suspended in 50 .mu.l of a goat raised
anti-human apo B polyclonal antibody solution (Chemicon 1:25
diluted in VSB.degree. buffer). After a 30-minute incubation of the
sample at room temperature (RT), the cells were washed twice in 1
ml of VSB.degree. buffer. The cells were pelleted and resuspended
in 50 .mu.l of a FITC-labeled anti-goat antibody solution (Rabbit
anti-goat Ig FITC, DAKO 1:10 diluted in VSB.degree. buffer). After
a 30-minute incubation of the sample at RT, the cells were washed
twice in 1 ml of VSB.degree. buffer, pelleted, resuspended in 0.5
ml of VSB.degree. buffer and analyzed by flow cytometry (10,000
cells were counted). Erythrocytes were gated on forward and side
scatter. It could be demonstrated that the FITC-label was
associated with the side-scattering particles (erythrocytes) only
in the presence of the anti-apo B antibodies (FIG. 6, bottom
panel), whereas no erythrocytes-associated FITC fluorescence could
be detected in the case that incubation with anti-apo B antibodies
was omitted from the analysis (negative control sample, FIG. 6, top
panel). It was therefore concluded that apo B was associated with
the erythrocytes in whole blood.
Example 4
Binding and Internalization of Chylomicron Remnants by Leukocytes
in the Blood (in Vivo)
[0132] Experimental procedure: Fasting venous blood was drawn and
Sudan Black staining as described in Example 2 was carried out
(left panel of FIG. 7). In the right panel of FIG. 7, venous blood
of the same healthy volunteer was drawn four hours after
administration of a standardized oral fat load. In the oral RP-fat
loading test, cream is used as fat source; this is a 40% (w/v) fat
emulsion with a P/S ratio of 0.06, which contains 0.001% (w/v)
cholesterol and 2.8% (w/v) carbohydrates. After an overnight fast
of 12 hours, the subjects ingest the fresh cream, to which 120,000
U of aqueous RP (Retinyl palmitate=vitamin A) had been added 18
hours before the test, in a dose of 50 g per m.sup.2 body surface.
After the ingestion of the fat load, subjects were only allowed to
drink water or tea during the following 24 hours. Peripheral blood
samples were obtained in sodium EDTA (2 mg/ml) before (T=0), at
hourly intervals up to 10 hours and at 12 and 24 hours after the
meal. Tubes were protected against light by aluminum foil and
centrifuged immediately for 15 minutes at 800.times.g at 4.degree.
C. Blood samples for FFA measurement were chilled and a lipase
inhibitor (Orlistat) was added in order to block in vitro
lipolysis.
[0133] Increased leukocyte concentrations in the postprandial
situation are involved in the process of atherosclerosis (novel
finding by our own group).
[0134] After having taken up surface fragments from
triglyceride-rich particles or whole remnant particles,
neutrophilic granulocytes become activated and induce a
pro-inflammatory response which is the first step in the generation
of atherosclerosis and endothelial damage.
Example 5
Magnitude and Time-Dependency of Increase of Complement Component 3
(C3) in the Postprandial Period
[0135] Experimental procedure: Standardized oral fat loading tests
(oral RP fat loading test) were performed in volunteers and
patients and plasma C3 levels were determined nephelometrically at
regular intervals. Complement component 3 was measured by
nephelometry (Dade Behring Nephelometry type II). Maximal
postprandial C3 concentrations were in most cases found after two
hours (data not shown). This is consistent with the concept of
chylomicron-driven complement activation (MBL mediated) followed by
a compensatory C3 synthesis in vivo. We therefore conclude that
complement activation occurs in vivo during postprandial lipemia
(high blood lipid concentrations).
Example 6
Binding and Internalization of Chylomicron Remnants by Leukocytes
in the Blood (in Vitro)
[0136] Experimental procedure: In vitro incubations of chylomicron
remnants with isolated human leukocytes were performed by methods
described in Example 3. Internalization of remnants in leukocytes
was observed (data not shown).
Example 7
Assay for Complement-Activation Cq. Complement-Consumption by
Drugs/Food Components Intended for Application in Atherosclerosis
or Clinical Nutrition
[0137] Experimental procedure: In microtiter plates, one
"classical" pathway unit of serum or one "alternative" pathway unit
of serum was incubated for 0.5 hours with a dilution series of the
substance to be investigated. In order to do so, the substance of
interest is suspended in micellar form. After incubation, residual
classical and alternative complement activities are estimated by
conventional techniques (J. P. A. M. Klerx, C. J. Beukelman, H. Van
Dijk and J. M. N. Willers (1983), J. Immunol. Lett. 63:215-220; H.
Van Dijk, P. M. Rademaker and J. M. N. Willers (1985), J. Immunol.
Meth. 85: 233-244). The degree of complement consumption is a
measure of complement activation by the components. A large number
of compounds were identified by this in vitro assay for application
in atherosclerosis or clinical nutrition.
Example 8
MBL-Dependent Complement Activation by Chylomicrons in Human
Serum
[0138] Experimental procedure: Chylomicrons were isolated from
human serum by ultra centrifugation and purified by column
chromatography. The purified chylomicron fractions were added to
MBL-positive serum (from healthy human subjects) and MBL-negative
serum (from MBL-deficient human subjects) and purified heterologous
chicken erythrocytes were added. Complement activation was allowed
to occur at 37.degree. C. for 45 minutes after which the extent of
hemolysis was evaluated by spectrophotometrical determination of
hemoglobin levels in serum supernatants. It was found that
hemolysis of heterologous erythrocytes was extensive in the case
that an MBL-positive serum was used, whereas hemolysis was
virtually absent in the case of an MBL-negative serum (data not
shown). This demonstrated that chylomicrons can bring about
complement activation in human serum in an MBL-dependent
manner.
[0139] Using the MBL in-vitro assay, we identified the components
from olive oil and soy oil inducing Complement Lipid Pathway
(CliP)-activation.
[0140] The results are summarized in FIG. 9.
[0141] From these results, two compounds were selected which show
strong CLiP-induction and which are known to be safe to be
administered to human (thanks to other clinical use), specifically
(i) glycosylated plant sterols and (ii) vitamin A.
Example 9
Postprandial C3 Buildup
[0142] Experimental procedure: Full capillary blood was draw from
healthy subjects and the C3 levels were determined together with
the leukocyte count. Postprandial (situation in blood after a meal)
leukocyte increase and activation was associated with postprandial
complement C3 increase. In the early postprandial phase (<4
hours) predominantly neutrophilic granulocytes were observed,
whereas between four and ten hours into the postprandial period, an
increase of lymphocytes was observed. These findings were
consistent with the notion that leukocytes play a role in
atherosclerosis by the formation of foam cells.
Example 10
Effect of Glycosylated Plant Sterols on Fasting Plasma
Triglycerides and Cholesterol Levels in Two MBL-Deficient Patients
and in One MBL-Normal Patient with Heterozygous Familial
Hypercholesterolemia
[0143] Proof of principle has been reached in two MBL-deficient
patients.
[0144] These subjects were treated with a diet enriched in
glycosylated plant sterols during three weeks. The glycosylated
plant sterols were selected in the MBL in vitro test as described
in Example 8. This intervention resulted in a decrease of fasting
plasma triglycerides and cholesterol (Table 1).
TABLE-US-00001 TABLE 1 Effect of glycosylated plant sterols on
blood parameters of MBL-deficient patients. Plasma TG (mM)
Cholesterol (mM) apoB (g/L) Before After Before After Before After
MBL def1 3.66 2.27 4.8 4.2 0.85 0.89 MBL def2 0.88 0.79 4.8 3.6
0.62 0.56
[0145] Using a different intervention with glycosylated plant
stanols in a patient with heterozygous Familial
Hypercholesterolemia (with relatively normal MBL activity in
plasma), refractory to therapy with expanded dose statins in
combination with a lipid lowering diet and resins, significant
reductions of plasma cholesterol (from 10 to 7.8 mmol/L), fasting
plasma triglycerides (from 2.3 to 1.08 mmol/L) and plasma apoB
(from 1.90 tot 1.62 g/L) were achieved reaching the lowest
concentration ever experienced by this patient (FIG. 10). This
example provides in vivo support for the Complement Lipid Pathway
(CliP) concept developed by C-Tres, using a sub-optimal lead.
Example 11
Effect of Vitamin a on Post-Prandial CliP Stimulation
[0146] Another series of lead compounds, namely vitamin
A-analogues, were tested in 20 healthy volunteers in order to
determine the CLiP stimulating potency of these leads that had
shown CLiP stimulation in vitro (Example 8). Twenty healthy
volunteers were tested on two different occasions. Blood was drawn
before and after ingestion of a standardized oral fat load with and
without vitamin A (as representative for these series of leads)
given to the participants in random order. Addition of vitamin A to
the oral fat load resulted in a significantly higher postprandial
plasma C3 increase, whereas the same amount of fat was ingested in
both situations (FIG. 11a).
[0147] The levels of plasma trygliceride increase two hours after
acute oral fat load also showed a reduction in the volunteers if
the fat load was given with vitamin A (FIG. 11b).
[0148] This is in line with the CLiP concept that by activating the
Complement system, plasma triglycerides will be reduced even in
healthy normolipidemic subjects. Note: it should be stressed that
the C3 increase in this group of young, healthy, lean subjects was
expected to be lower due to the characteristics of the subjects. In
older, insulin-insensitive subjects the postprandial C3 response is
much higher.
[0149] These experiments in human gave the expected results upon
administration: increase of C3-titers and decrease of
triglycerides. It is therefore reasonable to assume that also other
compounds, active in the in vitro assay, show CLiP-activities in
human.
Other Analytical Methods:
[0150] Triglyceride-rich particles in plasma, chylomicrons and
non-chylomicrons were determined by HPLC as described.sup.(14) TG
and cholesterol were measured in duplicate by commercial
calorimetric assay (GPO-PAP, and Monotest Cholesterol kit,
Boehringer Mannheim) as described..sup.(14, 23) Plasma apo B and
apo AI were determined by immunoturbidimetry..sup.(23) Apo E
genotype was determined as described..sup.(47-49) HDL2 and HDL3
cholesterol concentrations were determined by precipitation
procedures as described..sup.(50) Complement factor 3 was measured
immunoturbidimetry or nephelometrically. Acylation stimulating
protein was determined by ELISA, as were factor B and D. Ketone
bodies were measured by HPLC.
REFERENCES
[0151] 1. Ross R. The pathogenesis of atherosclerosis: a
perspective for the 1990's. Nature 1993; 362:801-809. [0152] 2.
Willeit J., S. Kiechl, F. Oberhollenzer, G. Rungger, G. Egger, E.
Bonora, M. Mitterer, and M. Muggeo. Distinct risk profiles of early
and advanced atheroclerosis. Prospective results from the Brunneck
Study. Arterioscl. Thromb. Vasc. Biol. 2000; 20:529-537. [0153] 3.
Bucher H. C., L. E. Griffith, and G. H. Guyatt. Systematic review
on the risk and benefit of different cholesterol-lowering
interventions. Arterioscl. Thromb. Vasc. Biol. 1999; 19:187-195.
[0154] 4. The Heart Outcomes prevention Evaluation Study
Investigators. Effects of an angiotensin-converting-enzyme
inhibitor, ramipril, on cardiovascular events in high-risk
patients. N. Engl. J. Med. 2000; 342:145-53. [0155] 5. The Diabetes
Control and Complication Trial Research Group. The effect of
intensive treatment of diabetes on the development and progression
of long-term complications in insulin-dependent diabetes mellitus.
N. Engl. J. Med. 1993; 329:977-86. [0156] 6. UK Prospective
Diabetes Study (UKPDS) Group. Intensive blood-glucose control with
sulphonylureas or insulin compared with conventional treatment and
risk of complications in patients with type 2 diabetes (UKPDS 33).
Lancet 1998; 352:837-53. [0157] 7. Haffner S. M., S. Lehto, T.
Ronnemaa, K. Pyorala, and M. Laakso. Mortality from coronary heart
disease in subjects with type 2 diabetes and in nondiabetic
subjects with and without prior myocardial infarction. N. Engl. J.
Med. 1998; 339:229-34. [0158] 8. Ross R. Atherosclerosis: an
inflammatory disease. N. Engl. J. Med. 1999; 340:115-126. [0159] 9.
Law S. K. A. and K. B. M. Reid: Complement in focus, 2nd edition,
1995, Oxford University Press, Oxford, G.B. [0160] 10. Rosenberg M.
E. and J. Silkensen: Clusterin: Physiologic and pathophysiologic
considerations. Int. J. Biochem. Cell Biol. 1995; 27(7):633-645.
[0161] 11. Ishikawa Y., Y. Akasaka, T. Ishii, K. Komiyama, S.
Masuda, N. Asuwa, N.-H. Choi-Miura, and M. Tomita: Distribution and
synthesis of apolipoprotein J in the atherosclerotic aorta.
Artherioscler. Thromb. Vasc. Biol. 1998; 18:665-672. [0162] 12.
Madsen H. O., V. Videm, A. Svejgaard, J. L. Svennevig, and P.
Garred: Association of mannose-binding-lectin deficiency with
severe atherosclerosis. The Lancet 1998; 352:959-960.
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