HK1055681B - Use of il-18 inhibitors for the treatment and/or prevention of atherosclerosis - Google Patents

Description

use of IL-18 inhibitors for the treatment and/or prevention of atherosclerosis

Technical Field

The present invention relates to the field of vascular diseases. More specifically, the invention relates to the use of IL-8 inhibitors in the treatment and/or prevention of atherosclerosis.

Background

Atherosclerosis is the most common and predominant vascular disease, although many others are possible. Atherosclerosis primarily affects the large and medium arteries, and its lesions include fatty streaks, fibrinolytic plaques (fibrolytics) and complications. Atherosclerosis is a chronic inflammation of the arterial wall characterized by the gradual accumulation of lipids, such as cholesterol, cells, such as macrophages, T lymphocytes or smooth muscle cells, and extracellular matrix (1). The massive accumulation is called atheroma or plaque, which usually contains calcium. Adipose tissue can erode the arterial wall, reducing the elasticity of the artery and affecting blood flow. Eventually, a blood clot forms around the plaque accumulation, further impeding blood flow, so as to completely occlude the blood vessel. Atherosclerosis is usually associated with high levels of Low Density Lipoprotein (LDL) -cholesterol, lp (a) fibrinogen and factor VII, and low levels of high density lipoprotein (LDL) -cholesterol. Risk factors include age, male gender, smoking, diabetes, obesity, hypercholesterolemia, a high fat diet, and a personal and family history of heart disease. Organ ischemia, such as myocardial infarction, is the leading cause.

Atheroma is the most common lesion of an artery and may further be complicated by thromboembolism. Atheromatous plaques tend to narrow the lumen of the artery, causing ischemia and sometimes tissue atrophy in the perfused area of the blood stream. Serious consequences include angina due to myocardial ischemia, heart failure due to ischemic or non-ischemic events, hypertension due to renal artery stenosis, and perfusion of the kidney in response to physiologically increased renin secretion.

Atherosclerosis and arteriosclerosis sometimes refer to different pathological conditions, with atherosclerosis in the present invention being defined as hardening or loss of elasticity of the arteries, in particular due to atheroma, and arteriosclerosis being hardening or loss of elasticity of the arteries due to any one of the causes.

Complications or consequences of atherosclerosis include coronary artery disease (coronary atherosclerosis), inadequate blood supply due to obstruction (ischemia/angina pectoris), acute myocardial infarction (myocardial infarction, heart attack), Transient Ischemic Attack (TIA) or stroke, and damage to blood vessels, muscles or body organs.

Aneurysms are permanent, abnormal dilatations of blood vessels and are also a common cause of atherosclerosis. Atherosclerotic abdominal aortic aneurysms commonly occur in elderly patients. They can rupture into the retroperitoneal cavity. In atherosclerotic aneurysms, due to ischemia of the muscle in the middle aorta, large amounts of elastic tissue are often lost and the media are fibrosed, followed by release of macrophage enzymes, which rupture the elastic fibers.

Recommended medications for treating or preventing atherosclerosis include reducing blood lipids/cholesterol. In particular, LDL-cholesterol lowering therapies are widely used. Currently, the most commonly used are statins, which are specific inhibitors of HMG CoA reductase. Other lipid lowering agents include drugs such as cholestyramine (cholestyramine), colestipol (colestipol), nicotinic acid, gemfibrozil (gemfibrozil), probucol, mevastatin (lovastatin), and the like.

Another approach is to minimize the risk of thrombosis in already established atherosclerotic lesions. Aspirin, which is useful for reducing the risk of blood clot formation, appears to be a specific inhibitor of thromboxane a2, which mediates platelet aggregation, or an anticoagulant.

Percutaneous "balloon angioplasty" uses a tip balloon catheter to flatten plaque and increase blood flow through the occlusion. This technique is similar to that used to open the arteries of the heart, but it can be applied to many other arteries of the body. Coronary stenosis can be shunted by suturing a vein to a portion of the saphenous vein in the proximal aorta or by cutting the internal mammary artery of the chest wall and anastomosing its distal end to the anterior artery of the heart.

In some cases (e.g., endarterectomy of the carotid artery) it is advisable to remove the accumulation surgically (endarterectomy).

However, it is also essential to treat or control risk factors such as maintaining a low-fat, low-cholesterol, and low-salt diet and follow expert advice to attend to health, treat and control hypertension, diabetes and other diseases, reduce weight and quit smoking, and perform normal exercise to improve heart function and enhance circulation.

Inflammatory processes occur in different stages of atherosclerosis (1). Endothelial activation by factors including low shear stress, modified lipoproteins, and proinflammatory cytokines is considered the first step in atherosclerosis and is controlled by inflammation (1). Many recent studies have shown that the interaction between blood vessels and inflammatory cells plays a key role in atherogenesis (1). In particular, inhibition of defined proinflammatory pathways reduces the development of atherosclerosis (1).

Inflammation also plays a dominant role in atherosclerotic plaque rupture and thrombosis (2-5), leading to the development of acute ischemic syndrome and its associated death (6). In fact, severe clinical manifestations of atherosclerosis include infarctions of the heart, brain and other organs affected by atherosclerosis, mainly due to the formation of thrombi blocking the vascular lumen at the interface of the ruptured atherosclerotic plaque (3, 4). Pathological studies have shown that vulnerable or unstable plaques, i.e. plaques that are prone to rupture or have ruptured, differ significantly in cellular and matrix composition compared to stable plaques that are not prone to rupture (7). Inflammatory cells (macrophages and T lymphocytes) contain a large number of vulnerable plaques, which comprise a thrombotic lipid core, characterized by a thin fibrous cap that loses a large amount of extracellular matrix (7).

Reduction of collagen synthesis mediated by the proinflammatory cytokine IFN γ, and increased activity of matrix degrading metalloproteinases and derived from macrophages, can both thin and embrittle the fibrous cap (7). Rupture of the fragile fibrous cap exposes the highly thrombotic lipid core to the circulating blood, resulting in obstructive thrombosis (1, 7). Therefore, the density of inflammatory cells within a given atherosclerotic lesion is considered a good indicator of its instability.

The clinical prognosis of atherosclerotic patients depends only in part on the size of the lesion (19, 20). It is now generally believed that the quality (composition of the plaque) rather than the size of the lesion is better predictive of the occurrence of an ischemic event. In fact, the severe clinical manifestations of atherosclerosis (cardiac and cerebral infarctions) are mainly due to the formation of thrombi blocking the vascular lumen at the interface of the ruptured atherosclerotic plaques (19). Pathological studies have demonstrated that vulnerable or unstable plaques, i.e. plaques that are either vulnerable or ruptured, are predominantly present in inflammatory cells and exhibit a large loss of smooth muscle cells and collagen levels (20, 21). In addition, such plaques dramatically increase the death rate of apoptotic cells, leading to the formation of a highly thrombotic lipid core (13, 22).

Proinflammatory cytokines are involved in the inflammatory process. The cytokine interleukin 18(IL-18) was originally referred to as interferon-gamma (IFN-gamma) inducing factor (8). It is an early signal generated in the development of T lymphocyte helper 1(Th1) responses. IL-18 together with IL-12, IL-2, antigen, mitogen, and some potential factors together induced IFN-gamma production. IL-18 also increases the production of GM-CSF and IL-2, enhances the proliferative capacity of anti-CD 3-induced T cells, and increases Fas-mediated killing of natural killer cells. Mature IL-18 is produced by L-1. beta. converting enzyme (ICE, caspase-1) from its precursor. The IL-18 receptor is composed of at least two components that cooperate in ligand binding. High and low affinity binding sites for IL-18 were found in mouse IL-12 stimulated T cells (9), which can be assumed to be a multi-chain receptor complex. To date, two receptor subunits have been identified, both belonging to the IL-1 receptor family (10). Signal transduction of IL-18 involves activation of NF-. kappa.B (11).

Recently, soluble proteins with high affinity for IL-18 were isolated from human urine and human and mouse cDNAs as well as human genes were cloned (12; WO 99/09063). This protein was designated as IL-18 binding protein (IL-18 BP).

IL-18BP is not an extracellular domain of one of the known IL-18 receptors, but a secreted native circulating protein. It belongs to a new family of secreted proteins, which also includes several poxvirus-encoded proteins (12). IL-18BP is constitutively expressed in the spleen (12). Urine as well as recombinant IL-18 specifically binds to IL-18 with high affinity and modulates the biological affinity of IL-18.

The IL-18BP gene is located on human chromosome 11q13, and no exon encoding a transmembrane region is found in the genomic sequence of 8.3 Kb. Four splice variants or isoforms of IL-18BP, designated IL-18BPa, b, C and d, are found in humans, all sharing the same N-terminus, but differing in C-terminus (12).

In various cDNA libraries, 4 human and 2 mouse IL-18BP isoforms were found to be produced by mRNA splicing, which have been expressed, purified and evaluated for their ability to bind and neutralize IL-18 bioactivity (23). Human IL-BP isoform a (IL-18BPa) exhibits the greatest affinity for IL-18, binds quickly, dissociates slowly, and has a dissociation constant (K (d)) of 399 pM. IL-18BPc shares the Ig domain of IL-18BPa in addition to the 29C-terminal amino acids. IL-18BPc has a K (d) 10 times smaller (2.94 nM). However, IL-18BPa and IL-18BPc neutralize more than 95% of IL-18 at concentrations in excess of 2 molar. IL-18BPb and IL-18BPd isoforms lack intact Ig domains and lack the ability to bind or neutralize IL-18. Mouse IL-18BPc and IL-18BPd isoforms have identical Ig domains and neutralize greater than 95% of mouse IL-18 at concentrations greater than 2 molar. And with human IL-18BPa common C terminal type main mouse IL-18BPd can also neutralize human IL-18. Molecular modeling recognizes a large mixed electrostatic and hydrophobic binding site in the Ig domain of IL-18BP, which explains its high affinity binding to ligands (23).

Disclosure of Invention

The present invention is based on the results of such studies: inhibitors of IL-18 have clear beneficial effects on plaque development, plaque development and plaque stability in mouse models of atherosclerosis. Inhibition of IL-18 not only prevents the formation of thoracic aortic lesions, but also induces the transformation of established atherosclerotic plaques into a stable plaque phenotype.

The invention therefore relates to the use of an inhibitor of IL-18 for the manufacture of a medicament for the prophylaxis and/or treatment of atherosclerosis. The invention also relates to therapeutic methods of gene therapy for the treatment and/or prevention of atherosclerosis.

Drawings

FIG. 1 is a histogram showing the survival rate of human umbilical vein endothelial cells incubated with oxidized lipoprotein alone or with oxidized lipoprotein and IL-18 antibody or IL-18BP, respectively.

FIG. 2 shows a Western Blot performed on protein extracts in atherosclerotic and controlled arteries. In Western Blot, antibodies against IL-18BP (hIL-18BP), IL-18 receptor alpha subunit (hIL18R alpha), IL-18(IL-18BP) and Caspase-1 (Caspase-1 p10) were used.

FIG. 3 shows ethidium bromide stained agarose gels showing the results of RT-PCT analysis of IL-18 and IL-18BP mDNA in atherosclerotic plaque cells.

FIG. 4 shows representative RT-PCT results for IL-18 and IL-18BP in atherosclerotic plaques compared to human α -actin (regulatory) expression in both symptomatic and asymptomatic plaques.

FIG. 5 shows a distribution diagram of expression vectors for intramuscular electrotransfer in mice.

FIG. 6 is a histogram showing the area of lipid staining in atherosclerotic arteries. Quantitative computer-assisted image analysis of lipid accumulation. Data are presented as mean ± standard deviation (empty plasmid n 19, IL-18BP plasmid n 14).^****Represents P < 0.0001.

FIG. 7 is a histogram comparing the lesion area of the aortic sinus after treatment with IL-18BP with controlled (empty body) lesion area. Quantitative computer-aided image analysis of lesion areas. Data are presented as mean ± standard deviation (empty plasmid n 19, IL-18BP plasmid n 14).^**Represents P < 0.0001.

FIG. 8 shows the effect of IL-18BP treatment on the amount of diseased inflammatory cells. The percentage of macrophage positive area (black squares) and the number of infiltrating T lymphocytes per square millimeter (grey squares) in mice treated with IL-18BP or under control of aortic sinus lesions (macrophage staining n-12, T lymphocyte staining n-15) were determined by quantitative computer-assisted image analysis. Data are presented as mean ± standard deviation.^***Represents P < 0.0001.

FIG. 9 shows the effect of IL-18BP treatment on pathological smooth muscle cell and collagen content. By means of a quantitative computerSecondary image analysis the percentage of smooth muscle cell positive areas (black squares) and collagen accumulation (grey squares) in controlled aortic sinus lesions (smooth muscle cells n 6, collagen n 11) and mice treated with IL-18BP (smooth muscle cells n 6, collagen n 13). Data are presented as mean ± standard deviation.^*Represents P < 0.05;^**represents P < 0.01.

Detailed Description

The present invention is based on the results of such studies: IL-18 production is increased by elevated circulating IL-18 levels in patients with acute coronary syndrome and unstable atherosclerotic plaques leading to stroke. In addition, it has been shown that in vivo electrotransfer of IL-18 BP-encoded expression plasmid DNA prevents fatty streak development in the thoracic aorta and slows the development of advanced atherosclerotic plaques in the aortic sinus in a well-established mouse model of atherosclerosis. More importantly, transfection with IL-18 plastids induced a major change in plaque composition (decreased macrophage, T cell, cell death and lipid content, and increased smooth muscle cell and collagen content) resulting in a stable plaque phenotype. These results show for the first time that inhibitors of IL-18 play an important role in reducing plaque production/development and promoting plaque stabilization.

The invention therefore relates to the use of an inhibitor of IL-18 for the manufacture of a medicament for the treatment and/or prevention of atherosclerosis.

The term "prevention" in the context of the present invention refers not only to the complete prevention of a certain effect, but also to any partial or substantial prevention, attenuation, reduction, diminution or elimination of such effect prior to or at an early stage of onset of the disease.

The term "treatment" in the context of the present invention refers to any beneficial effect on the progression of a disease, including diminishing, reducing, diminishing or eliminating the development of pathology after the onset of the disease.

The term "inhibitor of IL-18" in the context of the present invention refers to any molecule capable of modulating the production and/or action of IL-18 in such a way that the production and/or action of IL-18 is reduced, reduced or partially, substantially or completely prevented or blocked.

An inhibitor of production may be any molecule that negatively affects the synthesis, processing or maturation of IL-18. Inhibitors contemplated by the present invention may be, for example, suppressors of interleukin IL-18 gene expression, antisense mRNAs that reduce or prevent transcription of IL-18mRNA or cause degradation of such mRNA, proteins that impair proper folding or partially or substantially prevent maturation or secretion of IL-18, proteases that degrade IL-18 once synthesized, and the like. The produced inhibitor may be, for example, a Caspase-1 inhibitor or ICE inhibitor that prevents IL-18 maturation.

The inhibitor of IL-18 action can be, for example, an IL-18 antagonist. Antagonists can bind to or sequester the IL-18 molecule itself with sufficient affinity and specificity to partially or substantially neutralize the IL-18 or IL-18 binding site (e.g., its receptor) responsible for the binding of IL-18 to its ligand. Antagonists may also inhibit the IL-18 signaling pathway activated in cells upon IL-18/receptor binding.

Inhibitors of IL-18 action may also be soluble IL-18 receptors or molecules that mimic receptors, or agents that block IL-18 receptors, or IL-18 antibodies, such as monoclonal antibodies, or other agents or molecules that prevent IL-18 from binding to its target, thereby reducing or preventing the triggering of intracellular and extracellular responses mediated by IL-18.

Atherosclerosis is also known as arteriosclerosis. The term "atherosclerosis" in the context of the present invention includes all diseases or conditions that are manifested in arteries that generally describe atherosclerosis, in which fatty substances accumulate in the walls of the blood vessels, eventually leading to narrowing and impairment of blood flow, and formation of thrombi leading to rupture and/or erosion.

Atherosclerosis, according to the present invention, includes the hardening and loss of elasticity of arteries due to atheroma (atherosclerosis) and other causes (arteriosclerosis). The term "atherosclerosis" as used herein includes the pathological condition of atherosclerosis and the complications or sequelae of atherosclerosis, which have been described in detail above in the "background of the invention".

Atherosclerotic development includes the formation of atherosclerotic plaques and their progression to increasingly unstable forms. The invention therefore also relates to the use of an inhibitor of IL-18 for the manufacture of a medicament for attenuating or preventing the development of atherosclerosis.

The formation of thrombus on atherosclerotic plaques leading to vascular occlusion is a major cause of cardiac and cerebral infarction, one of the most serious sequelae of atherosclerosis. The invention therefore also relates to the use of an inhibitor of IL-18 for the manufacture of a medicament for the treatment and/or prevention of thrombosis of atherosclerotic plaques.

Plaque stability affects the progression of atherosclerotic plaques into harmful or vulnerable plaques that are prone to triggering thrombosis. The invention therefore further relates to the use of an inhibitor of IL-18 for the manufacture of a medicament for the prevention and/or treatment of atherosclerotic plaque instability.

Unstable plaques are prone to rupture, which in turn leads to thrombosis. Thus, the invention further relates to the use of an inhibitor of IL-18 in the manufacture of a medicament for the prevention of atherosclerotic plaque erosion or rupture.

Plaque instability and thrombosis may be caused by apoptosis, leading to increased activity of procoagulant agents, which may be the main cause of thrombosis and embolism in atherosclerotic plaques leading to rupture or erosion (13, 14). It has been demonstrated that oxidized lipoprotein (oxLDL) induces apoptosis of macrophages and endothelial cells in culture (15). As shown in the examples below, it has been found that inhibitors of IL-18 significantly reduce apoptosis induced by oxidized lipoproteins.

According to the present invention, it was unexpectedly found that IL-18 levels in the blood are significantly elevated in patients who have had a recurrent event, such as death, recurrent ischemia, repeated vascular occlusion, development of atherosclerosis, or frequent admission to hospital due to heart failure, as compared to patients who have not returned to hospital. This increased IL-18 levels are particularly evident in patients who subsequently die compared to those who survive. Both ischemic and non-ischemic patients have high levels of IL-18 in their blood circulation.

The invention therefore also relates to the use of an inhibitor of IL-18 for the manufacture of a medicament for the treatment and/or prevention of the recurrence of heart failure. The recurrent event can be any event following heart failure, such as death, recurrent ischemia, repeated vascular occlusion, development of atherosclerosis, or frequent admission to the hospital as a result of heart failure.

In a preferred embodiment of the invention, the heart failure is ischemia, i.e. heart failure due to myocardial ischemia.

In another preferred embodiment, heart failure is non-ischemic, such as due to systemic hypertension, cardiovascular disease, or pulmonary disease that returns to normal before congestive heart failure occurs.

In a preferred embodiment of the invention, the IL-18 inhibitor is selected from the group consisting of: ICE inhibitors, anti-IL-18 antibodies, antibodies against any one of the IL-18 receptor subunits, IL-18 receptor signaling channel inhibitors, antagonists capable of competing with IL-18 and blocking the IL-18 receptor, and IL-18 binding proteins, isoforms, muteins, fusion proteins, functional derivatives, active fragments or circularly permutated derivatives thereof having the same activity.

The term "mutein" as used herein refers to an analogue of an IL-18BP, or an analogue of a viral IL-18BP, wherein one or more amino acid residues of the native IL-18BP or the viral IL-18BP are replaced by, or deleted from, different amino acid residues, or one or more amino acid residues are added to the native sequence of the IL-18BP, the viral IL-18BP, but the activity of the resulting product is not significantly altered compared to the wild-type IL-18BP or the viral IL-18 BP. These muteins are prepared by known synthetic and/or site-directed mutagenesis techniques, or any other suitable known technique.

Any such mutein preferably has an amino acid sequence in which IL-18BP replicates sufficiently, or viral IL-18BP replicates sufficiently, to have substantially similar activity to IL-18 BP. One of the activities of IL-18BP is its ability to bind IL-18. Provided that the mutein has a large binding activity for IL-18. It can be used to purify IL-18, e.g., by affinity chromatography, and thus is believed to have substantially similar activity as IL-18 BP. Thus, one can determine whether a given mutein has substantially the same activity as IL-18BP by means of routine experimentation, including, for example, a simple sandwich competition assay (ELISA) performed on such a mutein to determine whether it binds to an appropriately labeled IL-18, such as a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA).

Muteins of IL-18BP polypeptides or muteins of viral IL-18BP, which are useful in the present invention, or nucleic acids encoding the same, comprise a limited set of sequences substantially corresponding to the substituted peptides or polypeptides, and can be obtained by routine methods, without undue experimentation, by one of ordinary skill in the art in light of the teachings and guidance presented herein.

Preferred changes in the muteins of the present invention are referred to as "conservative" substitutions. Conservative amino acid substitutions of an IL-18BP polypeptide or protein or a viral IL-18BP may include synonymous amino acids within a group that have sufficiently similar physiochemical properties that substitutions between atoms of the group will retain the biological function of the molecule (16). It is clear that amino acid insertions and deletions can be made without altering the function of the above-mentioned sequences, in particular if such insertions or deletions involve only a few amino acids, for example less than 30, preferably less than 10, and that amino acids critical for functional organization, such as cysteine residues, are not removed or substituted. Proteins and muteins produced by such deletions and/or insertions fall within the scope of the present invention.

Preferred groups of synonymous amino acids are those listed in Table 1; a better group of synonymous amino acids are those listed in table 2; the best groups of synonymous amino acids are those listed in Table 3.

TABLE 1 preferred synonymous amino acid groups

Amino acid SerAlgLeuProThrAlaValGlyIlePheTyrCys HisGlnAsnLysAspGluMet

Synonymous groups Ser, Thr, Gly, AsnArg, Gln, Lys, Glu, HisIle, Phe, Tyr, Met, Val, LeuGly, Ala, Thr, ProPro, Ser, Ala, Gly, His, Gln, ThrGly, Thr, Pro, AlaMet, Tyr, Phe, Ile, Leu, ValAla, Thr, Pro, Ser, GlyMet, Tyr, Phe, Val, Leu, IleTrp, Met, Tyr, Val, Leu, PheTrp, Met, Phe, Ile, Val, Leu, TyrSer, Thr, CysGlu, Lys, Gln, Thr, Arg, HisGlu, Lys, Asn, His, Thr, Arg, GlnGln, Asp, Ser, AsnGlu, Gln, His, Arg, LysGlu, Asn, Lys, Asn, Arg, Gln, His, Glu, Ile, Met, Ill, Leu, Ill, Leu

Trp

Trp

TABLE 2 better group of synonymous amino acids

Amino acid SerAlgLeuProThrAlaValGlyIlePheTyrCys HisGlnAsnLysAspGluMetTrp

Synonymous groups SerHis, Lys, ArgLeu, Ile, Phe, MetAla, ProThrPro, AlaVal, Met, IleGlyIle, Met, Phe, Val, LeuMet, TyrCys, SerHis, Gln, ArgGlu, Gln, HisAsp, AsnLys, ArgAsp, AsnGlu, GlnMet, Ile, Val, LeuTrp

TABLE 3 best synonymous amino acid groups

Amino acid serargLeuProThrAlaValGlyIlePhe

Synonymous group SerArgLeu, Ile, MetProThrAlaValGlyIle, Met, LeuPhe

TyrCysHisGlnAsnLysAspGluMetTrp

TyrCys，SerHisGlnAsnLysAspGluMet，Ile，LeuMet

Examples of amino acid substitutions made in proteins can be used to obtain muteins of IL-18BP polypeptides or proteins used in the present invention, or muteins of viral IL-18BP, including any known method steps, such as, for example, Mark et al, U.S. Pat. nos. 4,959,314, 4,588,585 and 4,737,462; 5,116,943 to Koths et al; 4,965,195 to Namen et al; 4,879,111 to Chong et al and 5,017,691 to Lee et al, as well as lysine substituted proteins as set forth in U.S. Pat. No.4,904,584 to Shaw et al.

The term "fusion protein" refers to a polypeptide comprising an IL-18BP or viral IL-18BP or muteins thereof fused to another protein, such as a protein with an extended residence time in body fluids. Thus, the IL-18BP or viral IL-18BP may be fused to another protein, polypeptide, etc., such as an immunoglobulin or fragment thereof.

"functional derivatives", as used herein, include derivatives of IL-18BP or viral IL-18BP and muteins and fusion proteins thereof, which may be prepared from functional groups present as side chains on the residues or N-or C-terminal groups by methods known in the art. They are included in the invention as long as they are still pharmaceutically acceptable, i.e., they do not disrupt protein activity substantially similar to that of IL-18BP or viral IL-18BP, and do not render the compositions containing them toxic. These derivatives may include, for example, polyethylene glycol side chains which mask the antigenic site and prolong the residence of the IL-18BP or viral IL-18BP in body fluids. Other derivatives include fatty esters of carboxyl groups, amides of carboxyl groups by reaction with ammonia or primary or secondary amines, N-acyl derivatives of the free amino group of an amino acid residue with an acyl moiety (e.g., alkanoyl or carbocyclic aroyl), or O-acyl derivatives of the free hydroxyl group (e.g., of a seryl or threonyl residue) with an acyl moiety.

"active portions" of the IL-18BP or viral IL-18BP, muteins and fusion proteins of the present invention include fragments or precursors of the polypeptide chain of the protein molecule alone, or together with the relevant molecule or residues associated therewith, such as sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues themselves, so long as the portion has substantially similar activity as IL-18 BP.

In another preferred embodiment of the invention, the inhibitor of IL-18 is an IL-18 antibody. The anti-IL-18 antibody may be a polyclonal or monoclonal, chimeric, humanized or even fully humanized antibody. Recombinant antibodies and fragments thereof are characterized by high affinity binding to IL-18 in vivo and low toxicity. Antibodies useful in the invention should be characterized by having the patient treated for a sufficient period of time that they provide good to excellent regression or remission of the condition or any symptom or group of symptoms associated with the condition, and low toxicity.

By immunizing with IL-18, neutralizing antibodies can be easily produced in animals such as rabbits, goats or mice. The immunized mice are particularly useful for providing a source of B cells to prepare hybridomas, which are then cultured to produce large quantities of anti-IL-18 monoclonal antibodies.

Chimeric antibodies are immunoglobulin molecules having two or more segments or portions from different animal species. Typically, the variable region of the chimeric antibody is derived from a non-human mammalian antibody, such as a murine monoclonal antibody, and the constant region of the immunoglobulin is derived from a human immunoglobulin molecule. The two regions and combinations thereof are preferably of low immunogenicity as determined by conventional methods (24). Humanized antibodies are immunoglobulin molecules constructed using genetic engineering techniques in which the murine stabilizing region is replaced with a human control, while the murine antigen binding region is retained. The resulting murine-human chimeric antibodies have better reduced immunogenicity and improved pharmacokinetics in humans (25).

Thus in another preferred embodiment the IL-18 antibody is a humanized IL-18 antibody, for example, European patent application EP0974600 describes preferred examples of humanized anti-IL-18 antibodies.

In another preferred embodiment, the IL-18 antibody is a fully human antibody. Techniques for producing human antibodies are described in detail in WO00/76310, WO99/53049, US6,162,963 or AU 5336100. The fully human antibody is preferably a recombinant antibody produced in a transgenic animal, such as a xenogeneic mouse, containing all or part of a functional human Ig locus.

In a more preferred embodiment of the invention, the IL-18 inhibitor is IL-18BP, or an isoform, mutein, fusion protein, functional derivative, active fraction or circularly permutated derivative thereof. These isoforms, muteins, fusion proteins or functional derivatives retain the biological activity of IL-18BP, in particular the activity of binding to IL-18, and preferably have at least one activity substantially similar to IL-18 BP. Ideally, these proteins have even higher biological activity than unmodified IL-18 BP. Preferably, the active moiety has a better or more advantageous activity than IL-18BP, e.g.better stability, lower toxicity or immunogenicity, or they are easier to produce in large quantities, or easier to purify.

The sequences of IL-18BP and its splice variants/isomers can be obtained from WO99/09063 or (12) and (23).

Functional derivatives of IL-18BP may be conjugated to polymers to improve properties of the protein, such as stability, half-life, bioavailability, tolerance by humans, or immunogenicity. To achieve this, IL-18BP may be conjugated to polyethylene glycol (PEG). Pegylation may be carried out, for example, as described in WO 92/13095.

Thus, in a preferred embodiment of the invention, the IL-18BP is pegylated.

In another preferred embodiment of the invention, the inhibitor of IL-18 is a fusion protein comprising all or a portion of an IL-18 binding protein fused to all or a portion of an immunoglobulin. The person skilled in the art knows that the fusion proteins produced retain the biological activity of IL-18BP, in particular the activity of binding to IL-18. Such fusions may be direct, or via a short cross-linked peptide, as short as 1-3 amino acid residues in length or longer, such as 13 amino acid residues in length. The cross-linking agent may be an E-F-M (Glu-Phe-Met) sequence; or a 13 amino acid cross-linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met positioned between the IL-18BP sequence and the immunoglobulin sequence. The resulting fusion protein has improved properties, such as an increased residence time in body fluids (half-life), increased specific activity, increased expression levels or facilitated purification of the fusion protein.

In a preferred embodiment, the IL-18BP is fused to the stabilizing region of an Ig molecule. Preferably fused to heavy chain regions, e.g., the CH2 and CH3 regions of human IgGl. The specific fusion proteins produced include IL-18BP and a portion of an immunoglobulin as described in example 11 of WP 99/09063. Other isoforms of Ig molecules are also suitable for use in producing fusion proteins of the invention, e.g., isoform IgG₂Or IgG₄Or other Ig classes, such as IgM or IgA. The fusion protein can be monomeric or multimeric, heterogeneous or homogeneous.

Interferons are mainly recognized for their inhibitory effect on viral replication and cell proliferation. For example, interferon-gamma plays an important role in promoting immune and inflammatory responses. Interferon beta (IFN-. beta., type I interferon) is said to have anti-inflammatory effects.

The invention also relates to the use of a combination of an IL-18 inhibitor and an interferon for the manufacture of a medicament for the treatment of atherosclerosis.

Interferons may also be conjugated to polymers to improve the stability of the protein, for example, WO99/55377 describes conjugates between interferon beta and the polyol polyethylene glycol (PEG).

In another preferred embodiment of the invention, the interferon is interferon-beta (IFN- β), more preferably IFN- β 1 a.

Inhibitors of IL-18 production and/or action are preferably used simultaneously, sequentially or separately with interferon.

In another embodiment of the invention, the IL-18 inhibitor is used in combination with a TNF antagonist. TNF antagonists exert their activity in several ways. First, the antagonist is capable of binding to or sequestering the TNF molecule itself with sufficient affinity and specificity so as to partially or substantially neutralize the TNF epitope or epitopes responsible for binding to the TNF receptor (hereinafter "sequestering antagonist"). For example, a masked antagonist can be an anti-TNF antibody.

On the other hand, TNF antagonists inhibit TNF signaling pathways activated by cell surface receptors after TNF binding (hereinafter "signaling antagonists"). These two groups of antagonists can be used alone or together in combination with an IL-18 inhibitor to treat atherosclerosis.

TNF antagonists are readily identified and evaluated by screening in a routine manner by assessing the effect of candidate antagonists on native TNF activity in sensitive cell lines in vitro, such as human B cells, whose proliferation and immunoglobulin secretion is induced by TNF. The assay includes TNF formulations of candidate antagonists at various dilutions, e.g., 0.1-100 times the molar amount of TNF used in the assay, and modulation of no TNF or only antagonist (26).

Occlusion antagonists are preferred TNF antagonists for use in the present invention. Among the blocking antagonists, those polypeptides which bind TNF with high affinity and have low immunogenicity are preferably used. Particular preference is given to using soluble TNF receptor molecules and neutralizing antibodies against TNF. For example, soluble TNF-RI and TNF-RII can be used in the present invention. These truncated receptors are more preferred antagonists of the invention and include the extracellular domain of the receptor or a functional portion thereof, for example, EP914431 describes truncated soluble TNF-type I and TNF-type II receptors.

Truncated TNF receptors are soluble and TNF inhibitory binding proteins of 30kDa and 40kDa, termed TBPI and TBPII, respectively, were detected in urine and serum (27). According to the invention, the IL-18 inhibitor and the TNF antagonist and/or the interferon may be administered simultaneously, sequentially or separately.

According to the present invention, it is preferred that the TNF antagonists are TBPI and TBPII, used in combination with an IL-18 inhibitor. Derivatives, fragments, segments and biologically active portions of the receptor molecules are functionally assembled into receptor molecules for use in the present invention. Such biologically active equivalents or derivatives of the receptor molecules refer to polypeptide portions or sequence portions encoding the receptor molecules that are of sufficient size and capable of binding TNF with affinity such that interaction with membrane-bound TNF receptors is inhibited or blocked.

In another preferred embodiment of the invention, the soluble human TNF-RI (TBPI) is a TNF antagonist for use in the invention. European patents EP308378, EP398327 and EP433900 describe natural and recombinant soluble TNF receptor molecules and methods for their preparation.

The IL-18 inhibitor may be used simultaneously, sequentially or separately with the TNF inhibitor. Preferably, an IL-18 antibody or antiserum is used in combination with a soluble TNF receptor having TNF inhibitory activity.

In another preferred embodiment of the invention, the medicament further comprises a COX-inhibitor, preferably a COX-2 inhibitor. COX inhibitors are well known in the art. For example, WO01/00229 describes some specific COX-2 inhibitors.

Thromboxane (thromboxane), particularly a thromboxane a2 inhibitor, is currently widely used for the treatment of atherosclerosis. Therefore, in another preferred embodiment of the invention, the medicament further comprises a thromboxane inhibitor, in particular a thromboxane A2 inhibitor, for simultaneous, sequential or separate use. According to the invention, aspirin and an IL-18 inhibitor are particularly preferably used in combination.

One of the causes of atherosclerosis is an excessive concentration of lipids in the blood. Therefore, in another preferred embodiment of the invention, the medicament further comprises a lipid lowering agent, which may be administered simultaneously, sequentially or separately. The lipid-lowering agent known in the technical field comprises cholestyramine, colestipol, nicotinic acid, gemfibrozil, probucol and other medicaments. HMG CoA reductase inhibitors are particularly preferred, and reductase inhibitors known as statins are even more preferred. There are many statins reductase inhibitors known in the art, such as simvastatin or lovastatin.

For better prevention and/or treatment of atherosclerosis, a preferred embodiment of the invention relates to the use of an IL-18 inhibitor in combination with a low-fat and/or low-cholesterol and/or low-salt diet.

In a preferred embodiment of the invention, the IL-18 inhibitor is present in an amount of about 0.0001 to about 10mg/kg body weight, or about 0.01 to about 5mg/kg body weight, or about 0.1 to about 3mg/kg body weight, or about 1 to about 2mg/kg body weight. In another preferred embodiment, the IL-18 inhibitor is present in an amount of about 0.1 to about 1000. mu.g/kg body weight, or about 1 to about 100. mu.g/kg body weight, or about 10 to about 50. mu.g/kg body weight.

The invention also relates to the use of an expression vector comprising the coding sequence of an inhibitor of IL-18 for the preparation of a medicament for the prevention and/or treatment of atherosclerosis. I.e. the treatment and/or prevention of diseases using gene therapy. The advantage is that the inhibitor of IL-18 is expressed in situ, thereby effectively blocking IL-18 directly in the tissues or cells affected by the disease.

As explained in the examples below, it has been shown that a mouse model of disease can show efficient expression of IL-18BP following electrotransfer of an expression vector containing the coding sequence of IL-18 BP.

Thus, in a preferred embodiment, the expression vector is administered by electrotransfer, preferably by intramuscular injection.

The invention also contemplates the use of vectors that induce and/or enhance the endogenous production of an inhibitor of IL-18 in cells that do not normally express the inhibitor of IL-18 or that express an insufficient amount of the inhibitor. The vector may comprise regulatory sequences which function in the cell in which expression of the IL-18 inhibitor is desired. Such regulatory sequences may be, for example, promoters or enhancers. The regulatory sequences can then be introduced into the genome at the correct locus by homologous recombination, operably linked to the gene, and the desired expression induced or enhanced. This technique is commonly referred to as "endogenous gene activation" (EGA) and is described in WO 91/09955.

One skilled in the art will appreciate that the same technique may also be used to reduce IL-18 expression by introducing a negative regulatory element, such as a silencing element, into the IL-18 locus, thereby resulting in down-regulation or prevention of IL-18 expression. One skilled in the art will appreciate that this down-regulation or silencing of IL-18 expression is as effective as the use of an IL-18 inhibitor for the prevention and/or treatment of disease.

The invention also relates to the use of cells genetically modified to produce an inhibitor of IL-18 for the manufacture of a medicament for the treatment and/or prevention of atherosclerosis.

The invention also relates to a pharmaceutical composition, in particular for the prevention and/or treatment of atherosclerosis, comprising a therapeutically effective amount of an IL-18 inhibitor and a therapeutically effective amount of an interferon. As IL-18 inhibitors, the compositions may include caspase-1 inhibitors, anti-IL-18 antibodies, antibodies against all IL-18 receptor subunits, inhibitors of IL-18 signaling pathways, IL-18 antagonists that compete with IL-18 and block the IL-18 receptor, and IL-18 binding proteins, isoforms, muteins, fusion proteins, functional derivatives, active portions, or circularly permuted derivatives thereof, having the same activity.

The preferred active ingredients of the pharmaceutical composition are the above-mentioned IL-18BP and its isoforms, muteins, fusion proteins, functional derivatives, active fractions or circularly permutated derivatives.

The interferon of the pharmaceutical composition is preferably IFN-beta.

In another preferred embodiment, the pharmaceutical composition comprises a therapeutically effective amount of an IL-18 inhibitor, optionally an interferon and TNF antagonist. The TNF antagonist may be an antibody that neutralizes TNF activity, or a soluble truncated TNF receptor fragment, also known as TBPI and TBPII. The pharmaceutical compositions of the present invention may also include one or more COX inhibitors, preferably COX-2 inhibitors. The pharmaceutical compositions of the invention may also include thromboxane inhibitors, such as aspirin, and/or lipid lowering agents, such as statins.

Definitions "pharmaceutically acceptable" means any carrier that does not interfere with the biologically active effects of the active ingredient and is non-toxic to the host to which it is administered. For example, for administration to the gastrointestinal tract, these active proteins may be formulated in injectable dosage unit forms in vehicles such as saline, dextrose, serum albumin and Ringer's solution.

The active ingredients of the pharmaceutical compositions of the present invention may be administered to an individual in a variety of ways. Routes of administration include intradermal, transdermal (e.g., sustained release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural, topical, and intranasal routes. Other therapeutically effective routes of administration may also be used, for example, by absorption through epithelial or endothelial tissue or by gene therapy to administer to the patient (e.g., via a vector) a DNA molecule encoding an active agent, resulting in expression and secretion of the active agent in vivo. In addition, the protein of the present invention may be administered with other components of a biologically active drug, such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and carrier agents, and the like.

For parenteral (e.g., intravenous, subcutaneous, intramuscular) administration, the active protein may be formulated as a solution, suspension, emulsion, or lyophilized powder in combination with a pharmaceutically acceptable parenteral vehicle (e.g., water, saline, dextrose solution) and additives that maintain isotonicity (e.g., mannitol) or chemical stability (e.g., preservatives and buffers). The formulations are sterilized by conventional techniques.

The active protein of the present invention can also be conjugated to improve the bioavailability of the molecule by increasing the half-life of the molecule in the human body. For example, the molecule is crosslinked with polyethylene glycol as described in PCT patent application WO 92/13095.

The therapeutically effective amount of active protein is a function of many variables, including the type of antagonist, the affinity of the antagonist for IL-18, the residual toxicity exhibited by the antagonist, the route of administration, and the clinical state of the patient (including whether maintenance of non-toxic levels of endogenous IL-18 activity is desirable).

A "therapeutically effective amount" is an amount of an IL-18 inhibitor that inhibits the biological activity of IL-18 when administered. The dosage of a single dose or multiple doses administered to an individual will depend on a variety of factors including the pharmacokinetic properties of the IL-18 inhibitor, the route of administration, the condition and characteristics of the patient (sex, age, weight, health and size), the extent of symptoms, concurrent therapy, the frequency of treatment and the desired effect. Those skilled in the art will be familiar with the adjustment and manipulation of established dosage ranges and methods for determining IL-18 inhibition in an individual in vitro or in vivo.

According to the invention, the IL-18 inhibitor is used in an amount of about 0.0001 to about 10mg/kg body weight, or about 0.01 to about 5mg/kg body weight, or about 0.1 to about 3mg/kg body weight, or about 1 to about 2mg/kg body weight. More preferred amounts of the IL-18 inhibitor are about 0.1-1000. mu.g/kg body weight, or about 1-100. mu.g/kg body weight or about 10-50. mu.g/kg body weight.

The preferred route of administration of the present invention is subcutaneous, and more preferred of the present invention is intramuscular.

In another preferred embodiment, the IL-18 inhibitor is administered daily or every other day.

The daily dose is usually divided into several doses or administered in a slow release form effective to obtain the desired result. The second or subsequent administration may be at the same dose, less than or more than the dose first or previously administered to the individual. The second or subsequent administration may be performed at or before the onset of the disease.

In accordance with the present invention, an IL-18 inhibitor may be prophylactically or therapeutically administered to an individual prior to, concurrently with, or sequentially with other treatment regimens or drugs (e.g., multiple drug regimens) in an effective amount of treatment. The active agent may be administered simultaneously with the other therapeutic agent in the same or different composition.

The invention also relates to a method for treating and/or preventing atherosclerosis, which comprises administering to a host in need thereof an effective inhibitory amount of an IL-18 inhibitor.

The invention also relates to a method for treating and/or preventing atherosclerosis, which comprises applying an expression vector containing the coding sequence of an IL-18 inhibitor to a host in need thereof.

In a preferred embodiment of the invention, the expression vector is administered systemically, more preferably by intramuscular injection.

The invention further relates to the use of IL-18 as a diagnostic marker for the severe clinical prognosis of heart failure. Severe clinical prognosis includes any deterioration of the patient's condition, such as relapse after the first myocardial infarction, or even death.

IL-18 is preferably used as a diagnostic marker for recurrent events after first heart failure. Recurrent diseases include, but are not limited to, death, recurrent ischemia, repeated occlusion of blood vessels, development of atherosclerosis, or frequent hospitalization due to heart failure.

The contents of the present invention will be more readily understood by referring to the following examples, which are provided for the purpose of illustration and are not intended to limit the present invention.

Examples

Materials and methods

Specimen (variants)

41 human atherosclerotic plaques excised from 36 patients undergoing carotid endarterectomy were collected. Used for regulation were 2 thoracic and 3 intramammary arteries without atherosclerosis (of which 2 minimal fibromuscular thickenings) taken from autopsy or coronary artery bypass. These arteries were quickly immersed in liquid nitrogen and stored at-80 ℃. Plaques for protein and RNA extraction were rapidly washed and then immersed in liquid nitrogen before being stored at-80 ℃. Plaques for immunohistochemical studies were first placed in 4% fresh paraformaldehyde for 2 hours, then transferred to 30% sucrose-PBS solution, then snap frozen with liquid nitrogen in tissue treatment medium at optimal cutting temperature and stored at-80 ℃ for cryo-isothermal sectioning. Several sections of 8 to 10 μm were obtained per specimen for histological analysis and immunohistochemical studies.

Patient classification

To investigate the potential relationship between IL-18/IL-18BP expression and signs of plaque instability, clinical data was collected in a randomized fashion for 23 consecutive patients (36 patients) undergoing endarterectomy between 5 and 8 months in 2000. The physician performing the endarterectomy systematically reports whether there is an ulcer in the plaque with a visual inspection. Thus, we can classify the plaque as an ulcerative plaque or a non-ulcerative plaque. In addition, patients were divided into two different groups according to clinical signs. Patients with clinical signs of ischemic brain attacks associated with carotid stenosis are classified as symptomatic. These patients underwent endarterectomy 2-66 days (17.6 + -5.3 days) after the onset of clinical signs. Those patients who never had signs of cerebral ischemia in the carotid artery region were classified as asymptomatic. Asymptomatic carotid stenosis is detected based on systematic clinical examinations of patients with coronary or peripheral disease, and its severity is determined by an experienced medical practitioner using repeated Doppler echography (Doppler echography). Although Asymptomatic patients never had an ischemic attack in the Carotid stenosis, studies have shown that cerebral endarterectomy is beneficial for these patients, as revealed by Asymptomatic Atherosclerosis investigators (ACAS) (28).

Western Blot analysis

Proteins were extracted from 12 atherosclerotic plaques and 5 normal arteries. The frozen samples were ground to a powder under liquid nitrogen. The powder was then resuspended in a freezing and dissolving buffer [20mmol/L Tris-hydrochloric acid, 5mmol/L pH7.5 EGTA, 150mmol/L NaCl, 20mmol/L glycerophosphate, 10mmol/L Na-fluoride, 1mmol/L sodium orthovanadate, 1% trinitrotoluene X-100, 0.1% Tween 20, 1. mu.g/L aprotinin, 1mmol/L PMSF, 0.5mmol/L N-methylsulfonyl-L-phenylalanine chloromethyl ketone (TPCK), 0.5mmol/L N (a) -p-methylsulfonyl-L-lysine chloromethyl ketone (TLCK) ]. The extract was incubated on ice for 15 minutes and then centrifuged (12000g, 15 minutes, 4 ℃). The supernatant fraction soluble in detergent was retained and the protein concentration in the sample was equilibrated using the Bio-Rad protein assay.

To analyze IL-18 and IL-18R using a wersternblot, the protein extract was boiled for 5 minutes and loaded on a 7.5% or 15% SDS-polyacrylamide gel. For IL-18BP, recombinant human IL-18, rhIL-18 purified from E.coli (E.coli) (Serono Pharmaceutical Research Institute, Geneva) was coupled with Afffligel 15(Biorad) using 1mg/mL resin according to the manufacturer's protocol. The protein extract (60. mu.g) was incubated overnight at 4 ℃ on a roller, and adjusted to 500. mu.l of Tween containing 0.05% PBS using 20. mu.l of resin. To eliminate any non-specific binding, the resin was centrifuged and washed with 10mM Tris hydrochloric acid pH8, 140mM sodium chloride, 0.5% trinitrotoluene-X-100 (Fluka), 0.5% dehydrocholate, once with 50mM Tris pH8, 200mM sodium chloride, 0.05% trinitrotoluene-X-100, 0.05% P40 detergent (Fluka), 2mM CHAPS (Boehringer, Mannheim), and finally with 50mM Tris hydrochloric acid pH 8. The resin was then centrifuged, resuspended in sample buffer under reducing conditions, boiled for 5 minutes and finally mounted on a 10% SDS-polyacrylamide NuPAGE gel (Invitrogen).

The sample was transferred from the polyacrylamide gel to nitrocellulose by electrophoresis. The nitrocellulose membrane was immersed in a TBST solution [50mmol/L pH7.5 trihydrochloride, 250mmol/L sodium chloride, and 0.1% Tween saline ] containing 5% fat-free anhydrous milk at room temperature for 2 hours. These membranes were then incubated with goat anti-human IL-18 and IL-18R (. alpha. -chain) polyclonal antibodies (1. mu.g/ml) (R & D system), mouse anti-human IL-18BP monoclonal antibody (5. mu.g/ml monoclonal antibody 657.27) (Corbaz et al, 2000manuscript Submitted), rabbit anti-human caspase-1 polyclonal antibodies (1. mu.g/ml) (A-19, Santa Cruz). A200-fold molar excess of rhIL-18BP-6his (purified from Chinese hamster ovary cells, Serono pharmaceutical research Institute, Geneva) was incubated with 5. mu.g/ml of monoclonal antibody 657.27 for 1 hour, and the specificity of monoclonal antibody 657.27 was analyzed on a strip membrane by competition. Following the protocol provided by the manufacturer, the cells were incubated with HRP conjugated to the corresponding antibody, and positive bands were revealed by chemiluminescence and were visualized upon contact with Hyperfilm ECL (Amersham).

Immunohistochemistry

Frozen sections from 6 atheromatous plaques were incubated with 1: 10 normal horse serum or 1: 10 normal goat serum for 30 minutes at room temperature, washed once with PBS, and incubated with anti-CD 68 master mouse monoclonal antibody to recognize macrophages (DAKO-CD68, KPl), or anti-human smooth muscle alpha-actin master mouse monoclonal antibody to recognize smooth muscle cells (1A4, DAKO). To recognize IL-18 and IL-18 receptors in atheromatous plaques, a specific goat polyclonal antibody (R & D system) was used at a dilution concentration of 5. mu.g/ml. IL-18BP was detected using a specific monoclonal antibody (Corbaz et al, 2000manuscript submitted) against recombinant human IL-18BP isoform a (H20). After washing with PBS, the sections were incubated with the following second biotinylated antibody: biotinylated horse anti-mouse IgG (Vector Laboratories, Inc.) diluted 1: 200 for detection of staining with anti-CD 68, smooth muscle alpha-actin and IL-18BP antibodies, and biotinylated horse anti-goat IgG (Vector) diluted 1: 200 for detection of anti-IL-18 and anti-IL-18 receptor antibodies. Immunostaining was visualized using an avidin-biotin HRP imaging system (Vectastain ABC Kit PK-6100 Vector). For negative control, adjacent sections were stained with isotype-matched independent antibodies, but not primary antibodies.

RNA preparation

Total RNA was extracted from 29 atherosclerotic plaques in guanidinium thiocyanate acid solution followed by phenol and chloroform extraction as described by Chomczynski and Sacchi (29). Purified RNA was dissolved in water and its concentration was determined by absorbance at 260 nm. RNA integrity was assessed by electrophoresis on a 1% agarose gel. cDNA was synthesized from 1. mu.g total RNA using the Promega reverse transcription system according to the manufacturer's protocol.

Semi-quantitative and real-time (RT) -PCR of human IL-18 and IL-18BP in human atheromatous plaques

A semi-quantitative PCR reaction was performed in a total volume of 50. mu.l containing 1U of AmpliTag DNA polymerase (Perkin Elmer Roche, USA), 2.5mM dNTP (Amersham, USA) and 50pmole of forward and reverse PCR primers. The reaction was incubated in a PTC-200 Peltier Effect thermal cycler (MJResearch, USA) under the following conditions: denaturation at 94 ℃ for 1 min, annealing at 55 ℃ for 1 min, and extension at 72 ℃ for 1 min. To ensure that the amount of PCR product was comparable to that of the linear phase of the PCR reaction, IL-18BP, IL-18 and β -actin were analyzed after 25, 28 and 31 cycles. The optimal number of cycles for IL-18BP, IL-18 and β -actin before saturation of the bands was determined (31, 28 and 25 rounds, respectively). The following PCR primers were designed based on the published sequences (AF110799, D49950, X00351): IL-18: reverse 5'-GCGTCACTACACTCAGCTAA-3', forward 5'-GCCTAGAGGTATGGCTGTAA-3'; IL-18 BP: forward 5'-ACCTGTCTACCTGGAGTGAA-3', reverse 5'-GCACGAAGATAGGAAGTCTG-3'; β -actin: a reverse direction 5'-GGAGGAGCAATGATCTTGATCTTC-3'; positive direction 5'-GCTCACCATGGATGATGATATCGC-3'. To exclude amplification of genomic DNA that may contaminate the sample, a PCR reaction was performed in the absence of cDNA template. PCR products (10. mu.l) were analyzed electrophoretically on a 1% agarose gel in 1 XTAE buffer. The PCR product size was verified after gel staining compared to a 1kb ladder (Gibco). Relative quantification of ethidium bromide stained bands was performed under UV light using Kodak digital scientific analysis software and the results expressed as the ratio of target genes (hIL-18BP, hIL-18) to housekeeping gene (h β -actin).

The following SYBR Green RT-PCR primers for IL-18BP, IL-18 and glyceraldehyde phosphate dehydrogenase (GAPDH) (housekeeping gene regulation) were designed using primer expression software from PE biosystems according to published sequences (AF110799, D49950, NM 002046): IL-18: reverse 5'-CAGCCGCTTTAGCAGCCA-3', forward 5'-CAAGGAATTGTCTCCCAGTGC-3'; IL-18 BP: reverse 5'-AACCAGGCTTGAGCGTTCC-3', forward 5'-TCCCATGTCTCTGCTCATTTAGTC-3'; GAPDH: reverse 5'-GATGGGATTTCCATTGATGACA-3', forward 5'-CCACCCATGGCAAATTCC-3'; intron GAPDH: reverse 5'-CCTAGTCCCAGGGCTTTGATT-3', forward 5'-CTGTGCTCCCACTCCTGATTTC-3'. Specificity and optimal primer concentration were tested. PCR and specific intron GAPDH primers were reacted to exclude possible genomic DNA contamination. The PCR products were electrophoretically analyzed on a 3.5% agarose gel to confirm the absence of non-specific amplification. SYBR Green RT-PCR was performed using 5. mu.l/well of RT product (0.5ng total DNA), 25. mu.l/well of SYBR Green PCR master mix (PE Biosystem, CA, USA) containing Amperase Uracil Glycosylase (UNG) (0.5 units/well) and 20. mu.l of primers (300 nM). PCR was performed at 50 ℃ for 2 minutes (for Amperase UNG incubation to remove any uracil introduced into the cDNA), at 90 ℃ for 10 minutes (for activation of Amplitaq Gold), and then cycled on an ABI PRISM 7700 detection system at 95 ℃ for 15 seconds and 60 ℃ for 1 minute. Thus, the reverse-transcribed cDNA was amplified, and the Ct (cycle threshold) value thereof was determined. All Ct values were normalized to housekeeping gene GAPDH. Monospecific DNA bands were observed for IL-18, IL-18BP and GAPDH by gel electrophoresis analysis.

The principle of real-time detection with the "SYBR Green PCR master mix" is based on the enhancement of fluorescence due to the binding of SYBR Green to double-stranded DNA, thus allowing direct detection of PCR products.

Statistical analysis

Data are expressed as mean ± standard deviation (mean ± SEM). The Mann-Whitney test was used to compare IL-18 levels between groups, and P < 0.05 was considered statistically significant.

Example 1: IL-18 inhibitors protect endothelial cell death due to oxidized lipoprotein (oxLDL)

Cultured Human Umbilical Vein Endothelial Cells (HUVECs) were contacted with oxidized lipoprotein in the presence or absence of IL-18 binding protein or anti-IL-18 antibody for 16 hours. As shown in figure 1, 83% HUVEC died after contact with oxidized lipoprotein. Whereas incubation with IL-18BP or anti-IL-18 antibody allowed almost all cells to survive. No cell death was observed in culture with IL-18 BP. While the survival rate of the cells cultured with the anti-IL-18 antibody was 89%.

This experiment clearly demonstrates the protective effect of two different inhibitors of IL-18 on cell death due to apoptosis in atherosclerotic plaques.

Example 2: expression of IL-18 protein in atherosclerotic plaques and its endogenous inhibitor IL-18BP

Protein extracts from 12 carotid atherosclerotic arteries and 5 normally regulated arteries were analyzed by Western blot. IL-18 protein, including the activated form, was highly expressed in all atherosclerotic plaques, while little or no expression was detected in normal arteries (FIG. 2). Lanes 1 to 4 are samples from atherosclerotic plaques and lanes 5 to 7 are samples from normal arteries. Interestingly, the results of the detection of the activated form of IL-18 appeared to correlate with the expression of the activated form of caspase-1, which is involved in IL-18 processing (FIG. 2 front row). Significant expression of the IL-18 receptor protein (i.e., the alpha chain) was also detected in all atherosclerotic plaques, in contrast to normal arteries (second row in FIG. 2) which were expressed at very low levels. In addition, most atherosclerotic plaques expressed IL-18BP, although the expression levels were heterologous (FIG. 2, first row).

Example 3: cellular localization of IL-18 protein and its endogenous inhibitor IL-18BP in atherosclerotic plaques

To determine the cellular localization of IL-18 protein and IL-18BP, immunohistochemical studies were performed on 6 atherosclerotic plaques of the carotid artery. As shown in FIG. 2, IL-18 is expressed predominantly in macrophages, which may be the major source of IL-18 in plaques (not shown). These regions also had a high number of CD 3-positive lymphocytes. However, T lymphocytes do not appear to be directly involved in IL-18 production. Some intimal smooth muscle cells and adventitious endothelial cells also express IL-18. In contrast, although all vessels were not expressed, significant expression of IL-18BP was detected in plaque microvasculature and luminal surface endothelial cells. In some macrophage-rich regions, mainly extracellular, relatively low and more heterogeneous IL-18BP expression was also detected.

Example 4: expression of IL-18 and IL-18BP mRNA transcripts in atherosclerotic plaques and correlation with plaque instability

To determine whether human carotid atherosclerotic plaques (FIG. 3) express hIL-18 and IL-18BP mRNA, semi-quantitative RT-PCR was performed on 6 atherosclerotic plaques. Although IL-18 and IL-18BP mRNA were not present in uniform amounts, IL-18 and IL-18BP mRNA were detected in all atherosclerotic plaques. Therefore, in order to accurately quantify the expression levels of IL-18 and IL-18BP mRNA, the 23 atherosclerotic plaques were further analyzed by SYBR Green RT-PCR (FIG. 4). These plaques are characterized by being clinically and pathologically divided into symptomatic (unstable) and asymptomatic (stable) plaques, with or without ulcers being visually observed. Table 4 briefly lists the clinical characteristics of these patients. The percentage of carotid diameter reduction (60% -95%) and risk factors including age, diabetes, high cholesterol, hypertension and smoking were not differentiated between the two groups.

TABLE 4

Hypertension of characteristic age and sex of patient2High cholesterol3Diabetes mellitus is smoking coronary artery disease	No symptoms	Has symptoms
			Patient (n ═ 9)66.9 ± 4.0 men (8)84375	Patient's health1(n-14) 70.2 ± 3.9 men (9)98184

¹These patients had transient or persistent ischemic attacks 2-66 days prior to endarterectomy

²Number of patients with clinical hypertension treated with antihypertensive agents

³Treatment of high cholesterol clinical numbers with lipid lowering agents

The amount of IL-18 was found to be upregulated in symptomatic atherosclerotic plaques (0.67. + -. 0.17 and 2.03. + -. 0.5, respectively) compared to asymptomatic atherosclerotic plaques (FIG. 4A). Statistical analysis showed that IL-18 production was significantly increased in symptomatic plaques (p < 0.0074), while no increase in IL-18BP was found in both symptomatic and asymptomatic plaques (4.64. + -. 0.98 and 2.5. + -. 0.92, respectively) (FIG. 4B). In other words, although both symptomatic and asymptomatic groups showed a positive functional relationship between IL-18 and IL-18BP mRNA, the slopes between the two groups were very different (symptomatic group: slope 1.16[0.19-2.14 ]]，r²0.36, and asymptomatic group: the slope is 4.79[2.39-7.20 ]]，r²0.76; p < 0.05). Therefore, the relative increase in IL-18BP expression in the symptomatic group does not appear to be sufficient to compensate for the increase in IL-18 expression. Furthermore, since ulceration is also believed to be one of the causes of plaque instability, further statistical analysis on ulcerated or non-ulcerated plaques revealed a significant up-regulation of the amount of IL-18 within the ulcerated plaques (p < 0.018) (FIG. 4C).

These data indicate that increased IL-18 expression in atherosclerotic plaques is associated with plaque instability.

Example 5: IL-18BP regulates the development and stability of atherosclerotic plaque lesions in vivo models of disease

Method

Characteristics of the patient

Plasma samples were taken within 7 days after onset of acute ischemic coronary syndrome (unstable angina and myocardial infarction) patients. Unstable angina is defined as a typical chest pain associated with ischemic changes in the electrocardiogram or with coronary artery disease. Myocardial infarction is a diagnosis based on ischemic changes on the electrocardiogram associated with a significant rise in cardiac myozymes (phosphocreatine kinase and troponin) in circulating blood. Non-ischemic patients who were treated in the same cardiology department had no evidence of ischemia at all. Plasma levels of hIL-18 were determined using a commercially available kit (MBL, Japan).

Intramuscular electrotransfer of mouse IL-18 BP-expressing plastids in vivo

14-week-old male C57BL/6apoE KO mice were injected 3 times with IL-18 BP-expressing plastids and pcDNA3IL-8BP every 3 weeks. Control mice (n ═ 19) were injected with control null plasmids. The mouse IL-18BP isoform d cDNA was isolated according to known methods (accession # Q9ZOM9) (23) and subcloned into the mammalian cell expression vector pcDNA3 at position EcoR1/Not1 under the control of cytomegalovirus activator (Invitrogen). FIG. 5 shows a configuration referred to as 334. yh. Regulatory plasmids have a similar structure, but no therapeutic cDNA. IL-18BP or regulatory expression plasmid (60 μ g) was injected intramuscularly in both tibiocranial bones of anesthetized mice as described previously (13). Briefly, two stainless steel electrodes spaced 4.2 to 5.3mm apart were placed on either side of the leg and a transcutaneous electrical pulse (8 square wave electrical pulse at 200V/cm, 200 milliseconds duration at 2 Hz) was delivered via a PS-15 electrical pulser.

Enzyme-linked immunosorbent assay (ELISA) for detecting IL-18BP (mIL-18BP) of mice

The well plates were covered overnight with recombinant mouse IL-18BP d affinity purified from rabbit polyclonal antibody (5. mu.g/well). Soluble mIL-18BP was detected with rabbit polyclonal antibody (0.3. mu.g/ml) against coliform biotinylated recombinant mouse IL-18BP (Peprotec) followed by exoavidin peroxidase (1/1000) (Sigma). To confirm the specificity of mIL-18BP, the capture rabbit polyclonal antibody was tested by Western Blot. Recombinant mouse IL-18BP d produced by HEK 293 cells was used as a standard. The ELISA sensitivity was 5 ng/ml.

Analysis of mice

Cryostat sections (8 μm) were obtained from the aortic sinus for detection of lipid accumulation with oil Red, collagen with Sirius Red and immunohistochemical analysis as previously described (13). These sections were stained with specific primary antibodies, such as anti-mouse macrophages, clone MOMA2(BioSource), anti-smooth muscle cell alpha-actin conjugated with phosphatase base, and anti-T lymphocyte CD3(Dako), as previously described (13). Cell mortality was measured using the TUNEL technique (13). CD3 positive cells were counted randomly with the naked eye. Atherosclerotic plaques staining positive for macrophages, smooth muscle cells, collagen or TUNEL in the aortic sinus and aortic region were determined by computer-assisted image quantification (NS15000 Microvision) as previously described (13). The specificity of the immunostaining was assessed by staining with immunoglobulins matching the non-immune isotype. Deletion of the enzyme terminal deoxynucleotidyl transferase was used to assess the specificity of TUNEL. The thoracic aorta spanning the left subclavian and renal arteries was coagulated with 10% buffered formaldehyde and stained for lipid accumulation with oil red. Then, these aortas were opened in the longitudinal direction, and the percentage of lipid accumulation was calculated using computer-assisted image quantification (NS15000 Microvision).

Results

This study test demonstrates that the hypothesis of IL-18/IL-18BP regulation plays a key role in both atherosclerosis and plaque stability. Plasma levels of IL-18 were measured in patients with acute coronary syndrome (20 males, 18 females, with a mean age of 66.2 + -1.8 years, 14 of which had unstable angina and 34 had myocardial infarction) and in non-ischemic-regulated patients (10 males, 3 females, with a mean age of 60.0 + -5.2 years) who were treated at the same department of cardiology. The plasma levels of IL-18 in patients with acute coronary syndrome were significantly elevated compared to those in the control patients (73.0 ± 12.2 and 146.9 ± 17.1pg/ml, respectively, with p < 0.05), whereas circulating levels of IL-18BP were just as slightly elevated (7.5 ± 2.5 and 20.1 ± 2.7ng/ml, respectively, with p ═ 0.06). In addition, IL-18 levels were also found to correlate with disease severity in patients with clinical signs of severe ischemic heart dysfunction and pulmonary edema (224.03. + -. 39.1pg/ml, p < 0.001 compared to control), with higher IL-18 levels at higher disease levels. These results, together with the known high IL-18 levels in atherosclerotic plaques of stroke patients, in patients with acute coronary artery disease suggest that IL-18/IL-18BP regulation may play an important role in the atherosclerotic process.

To this end, we tested this hypothesis using apoE comatose (KO) mice that spontaneously develop human-like atherosclerotic lesions. 14 male mice, 14 weeks old, received IL-18BP supplementation by intramuscular electrotransfer of expression plasmid DNA encoding mouse IL-18BPd in vivo, while 19 age-matched regulatory mice received empty plastids. Plastid electrotransfer was repeated every 3 weeks and after 9 weeks of treatment, mice were sacrificed at 23 weeks of age. In KO mice injected with empty plastids, plasma levels of mouse IL-18BP were below the detection limit (5 ng/ml). However, two days after injection of a single IL-18BP plasmid, the blood IL-BP level reached the highest (323.5 + -100.9 ng/ml), and after two weeks, 127.4 + -35.4 ng/ml was also measured. Total cholesterol (489.4 + -34.6 and 480.8 + -36.3 mg/dl, respectively) and high density lipoprotein serum (52.3 + -9.4 and 48.8 + -5.1 mg/dl, respectively) did not differ much between the 2 patients after 9 weeks of IL-18BP or empty body treatment. The body weight of animals treated with IL-18BP was moderately but significantly increased (28.6. + -. 0.8g and 31.8. + -. 0.9g, respectively, p < 0.05) compared to the control group.

The effect of IL-18 supplementation on atherosclerosis, i.e., the lower thoracic aorta and the aortic sinus, was examined at two different locations. The thoracic aorta was selected to determine the effect of IL-18BP on fatty streak development (atherogenesis) because the thoracic atherosclerotic lesions were barely visible in mice 14 weeks old (data not shown) at the time IL-18BP began transfection. Aortic sinus atherosclerotic lesions were found in 14-week old mice (data not shown) and were used to examine the development and complications of advanced plaque, an important determinant of plaque stability. IL-18BP treatment had a significant effect on the development and progression of atherosclerotic lesions in apoE KO mice. The results of the thoracic aorta examination showed that lipid accumulation was more reduced in mice treated with IL-18BP lipid compared to empty body (FIG. 6). Quantitative computer-assisted image analysis showed a 69% reduction in the extent of atherosclerotic lesions (p < 0.0001) (FIG. 6), demonstrating a therapeutic effect of IL-18 on atherosclerosis. In addition, treatment with IL-18BP plastids significantly limited the development of atherosclerotic aortic sinus plaques (24% reduction in plaque area, with p ═ 0.01) for only 9 weeks compared to empty plastid treatment (fig. 7).

More importantly, the composition of the advanced lesions is a major determinant of plaque instability, and is significantly improved by treatment with IL-18 BP. Treatment of atherosclerotic lesions in mice with IL-18BP plastids resulted in a dramatic reduction of macrophage infiltration by 50% (p < 0.0001) (FIG. 8), a reduction of T lymphocytes by 67% (p < 0.005) (FIG. 8), and a 2-fold increase in smooth muscle cell accumulation (p < 0.05) (FIG. 9). Furthermore, these important changes in the composition of diseased cells were associated with a large increase in collagen content of 85% and a decrease in total lipid content, as determined by sirius red staining.

Therefore, IL-18 treatment significantly attenuates the inflammatory process of atherosclerotic lesions and stabilizes atherosclerotic plaques. Moreover, a significant reduction in the inflammatory component of the lesions also resulted in a significant reduction in the rate of cell death within the plaques in mice treated with IL-18BP (2.9. + -. 0.9% in IL-18 BP-treated mice versus 10.5. + -. 3.6% in control mice, p < 0.05), thus limiting the enlargement of the acellular lipid core and thrombosis (Mallat, 1999).

Conclusion

From well-developed mouse models of human atherosclerosis, the above results clearly show that IL-18 and IL-18BP must play a key role in the generation, development and stabilization of atherosclerotic plaques. Inhibition of IL-18 activity by IL-18BP supplementation also had a significant effect on the composition of advanced aortic sinus lesions, including the transition to the stable plaque phenotype, in order to prevent the development of early thoracic aortic lesions.

The clinical prognosis of atherosclerotic patients will only depend in part on the area of the lesion. It is now widely recognized that the quality (plaque composition) rather than area of the lesion better predicts the onset of ischemic disease. In fact, the severe clinical manifestations of atherosclerosis (cardiac and cerebral infarctions) are mainly the formation of thrombi on the interface of the ruptured atherosclerotic plaques blocking the blood vessels (19). Pathological studies have demonstrated that vulnerable and unstable plaques are easily ruptured or have ruptured, mainly in inflammatory cells, where they lose a large amount of smooth muscle cells and collagen (20, 21). In addition, these plaques dramatically increase the death rate of apoptotic cells, leading to the formation of a highly thrombotic lipid core (13, 22). Notably, all of these signs of plaque instability were significantly diminished in mice treated with IL-18BP, suggesting that IL-18 signaling is a major determinant of plaque instability.

We have found that increased levels of circulating IL-18 in patients with acute coronary syndrome also increased IL-18 production in unstable carotid atherosclerotic plaques leading to stroke, thereby further confirming the relationship of the results obtained with apoE KO mice to human disease. Taken together with these findings, it was determined that the activity of IL-18 inhibitors could serve as an important new therapeutic tool for the prevention and treatment of atherosclerotic plaque development and for limiting plaque complications.

Example 6: high levels of IL-18 have been associated with relapse in patients with heart failure

IL-18 levels in the serum of patients are measured by ELSIA assay using IL-18 specific antibodies.

A total of 56 ischemic or non-ischemic patients with or without heart failure were tested.

In later-deceased patients, IL-18 levels were 216.0 ± 41.5pg/ml, while in surviving patients, IL-18 levels were 112.2 ± 12.2pg/ml (p ═ 0.0018).

In patients with recurrent events, such as death, recurrent ischemia, recurrent vascular occlusion, atherosclerotic development or frequent admission for heart failure, IL-1 levels were determined to be 165.8. + -. 23.8pg/ml, whereas in any non-recurrent patient IL-18 levels were 107.7. + -. 14.6pg/ml (p ═ 0.03).

These results indicate that patients with a severe clinical prognosis, such as relapse or even death, have high IL-18 levels.

IL-18 levels in blood samples were determined in 16 non-ischemic patients with or without heart failure. In later-deceased patients, IL-18 levels were 199.0 ± 34.8pg/ml, while in still-living patients IL-18 levels were 95.3 ± 20.4pg/ml (p ═ 0.09).

The IL-18 levels in patients with relapse and patients without relapse were 146.6 ± 34.4pg/ml and 95.4 ± 23.9pg/ml, respectively (p ═ 0.03). Although the differences in IL-18 levels did not reach the statistical data due to the smaller number of patients, the trend of increased IL-18 levels was clearly seen.

In ischemic patients, the levels of IL-18 in dead patients and in surviving patients were 214.2 ± 45.9pg/ml and 118.4 ± 12.8pg/ml, respectively (p ═ 0.007).

IL-18 levels in patients with and without relapse were 162.8. + -. 24.7pg/ml and 116.2. + -. 16.0pg/ml, respectively.

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Claims (35)

1. Use of an inhibitor of IL-18 in the manufacture of a medicament for the treatment and/or prophylaxis of atherosclerosis.

2. Use of an inhibitor of IL-18 in the manufacture of a medicament for the treatment and/or prophylaxis of atherosclerotic plaque thrombosis.

3. Use of an inhibitor of IL-18 in the manufacture of a medicament for the treatment and/or prophylaxis of atherosclerotic plaque ulcer.

4. Use of an inhibitor of IL-18 in the manufacture of a medicament for the treatment and/or prevention of destabilisation of atherosclerotic plaques.

5. Use of an inhibitor of IL-18 in the manufacture of a medicament for the prevention and/or treatment of ischemic syndrome caused by the loss of stability of atherosclerotic plaques.

6. Use of an inhibitor of IL-18 in the manufacture of a medicament for the treatment and/or prevention of atherosclerotic plaque rupture.

7. Use of an inhibitor of IL-18 in the manufacture of a medicament for the treatment and/or prevention of a heart failure recurrence event caused by atherosclerosis.

8. The use of claim 7, wherein: heart failure is ischemic.

9. The use of claim 7, wherein: heart failure is non-ischemic.

10. The use according to any one of claims 1-9, wherein: the IL-18 inhibitor is selected from the group consisting of ICE-inhibitors, anti-IL-18 antibodies, antibodies to any one of the IL-18 receptor subunits, inhibitors of IL-18 receptor signaling pathways, antagonists capable of competing with IL-18 and blocking the IL-18 receptor, and IL-18 binding proteins and isoforms or fusion proteins thereof comprising all or a portion of an IL-18 binding protein fused to all or a portion of an immunoglobulin, which isoforms or fusion proteins bind to IL-18.

11. The use of claim 10, wherein: the IL-18 inhibitor is an anti-IL-18 antibody.

12. The use of claim 11, wherein: the antibody is a humanized antibody.

13. The use of claim 11, wherein: the antibody is a human antibody.

14. The use of claim 10, wherein: the IL-18 binding protein is pegylated.

15. The use of claim 10, wherein: the fusion protein comprises all or part of an immunoglobulin stabilizing region.

16. The use of claim 15, wherein: the immunoglobulin is of the IgG1 or IgG2 isotype.

17. The use of claim 10, wherein: the medicament also comprises interferon, and the medicament and the interferon can be used simultaneously, sequentially or separately.

18. The use of claim 17, wherein: the interferon is interferon-beta.

19. The use of claim 10, wherein: the medicament also comprises a tumor necrosis factor antagonist, and the medicament and the TNF antagonist can be used simultaneously, sequentially or separately.

20. The use of claim 19, wherein: the TNF antagonist is TBPI and/or TBPII.

21. Use according to claim 10, characterized in that: the medicament may further comprise a COX-inhibitor, and the medicament and COX-inhibitor may be for simultaneous, sequential or separate use.

22. The use of claim 21, wherein: the COX-inhibitor is a COX-2 inhibitor.

23. The use of claim 10, wherein: the medicament also comprises a thromboxane inhibitor, and the medicament and the thromboxane inhibitor can be used simultaneously, sequentially or separately.

24. The use of claim 23, wherein: the thromboxane inhibitor is aspirin.

25. The use of claim 10, wherein: the medicine also comprises a lipid-lowering agent, and the medicine and the lipid-lowering agent can be used simultaneously, sequentially or separately.

26. The use of claim 25, wherein: the lipid lowering agent is an HMG CoA inhibitor.

27. The use of claim 26, wherein: the HMG CoA inhibitor is statin.

28. The use of claim 10, wherein: the medicament is in a subcutaneous injection form.

29. The use of claim 10, wherein: the medicament is in an intramuscular injection form.

30. The use of claim 10, wherein: the drug is in a 24 hour dosage form.

31. Use according to any one of the preceding claims, wherein: the drug is a 48 hour dosage form.

32. Use of an expression vector comprising the coding sequence of an inhibitor of IL-18 in the manufacture of a medicament for the treatment and/or prevention of atherosclerosis.

33. The use of claim 32, wherein: the medicament is in an intramuscular injection form.

34. Use of a vector for inducing and/or enhancing the endogenous production of an inhibitor of IL-18 in a cell in the manufacture of a medicament for the treatment and/or prevention of atherosclerosis.

35. Use of a cell genetically modified to produce an inhibitor of IL-18 in the manufacture of a medicament for the treatment and/or prevention of atherosclerosis.