RS57803B1 - Biocompatible micro emulsion systems with controlled release of ibuprofen, their preparation, and application thereof - Google Patents

Description

THE USE OF IL-18 INHIBITORS FOR THE TREATMENT AND/OR PREVENTION OF HEART DISEASE

DISEASES

TECHNICAL FIELD

This invention relates to the field of cardiovascular diseases. More specifically, it refers to the use of IL-18 inhibitors for the treatment and/or prevention of cardiovascular diseases, primarily ischemic heart disease.

STATE OF THE ART

The cytokine interleukin 18 (IL-18) was initially described as an interferon-γ (IFN-γ) inducing factor (Nakamura et al., 1989). It is an early signal in the development of T-lymphocyte helper cell type 1 (TH1) responses. IL-18 acts together with IL-12, IL-2, antigens, mitogens, and possibly other factors, to induce IFN-γ production. IL-18 also enhances GM-CSF and IL-2 production, enhances anti-CD3-induced T cell proliferation, and enhances Fas-mediated killing of natural killer cells.

Mature IL-18 is produced from its precursors by IL-1p converting enzyme (ICE, caspase-1). The IL-18 receptor consists of at least two components that cooperate in ligand binding. High- and low-affinity binding sites for IL-18 have been found in murine IL-12-stimulated T cells (Yoshimoto et al., 1998), suggesting a complex chain receptor complex. Two receptor subunits have been identified so far, both belonging to the IL-1 receptor family (Pamet et al., 1996; Kim et al., 2001). IL-18 signaling involves activation of NF-kB (DiDonato et al., 1997). The IL-18 receptor complex consists of two receptor chains: a ligand-binding chain called IL-18Ra and a signal transduction chain called IL-18Rfi chain. The IL-18R chain was initially isolated as a cell surface protein that binds to radiolabeled IL-18; This protein was purified and its amino acid sequence revealed identity with a previously reported "orphan" receptor called IL-1 R-related protein (IL-1 Rrp) (Torigoe et al., 1997).

More recently, a soluble protein with high affinity for IL-18 was isolated from human urine, as well as from human and mouse cDNA, and the human gene was cloned (Novick et al., 1999; WO 99/09063). This protein is designated IL-18 binding protein (IL-18BP).

IL-18BP is not the extracellular domain of one of the known IL18 receptors, but a secreted, naturally circulating protein. It belongs to a new family of secreted proteins, which also includes several Poxvirus-encoded proteins (Novick et al., 1999). Urinary as well as recombinant IL-18BP specifically bind IL-18 with high affinity and modulate the biological affinity of IL-18.

The IL-18BP gene was localized to human chromosomes 11 q 13, and no exon coding for the transmembrane domain was found in the 8.3kb genomic sequence. To date, four splice variants or isoforms of IL-18BP generated by alternative mRNA splicing have been found in humans. These have been named IL-18BP a, b, c and d, and all have the same N-terminus and different C-terminus (Novick et al, 1999). These isoforms differ in their ability to bind IL-18. Of these four, NL-18BP isoforms a and c are known to have neutralizing properties for IL-18. Human IL-18BP isoform cross-reacts with murine IL-18.

Heart diseases are defined as diseases affecting the heart muscle or blood vessels of the heart (The Merck Manual Home Edition, www.merck.com). A vascular disorder is a problem with blood vessels such as poor circulation caused by a blockage. Heart diseases are also called cardiovascular diseases.

Ischemic heart disease is a common cause of cardiac dysfunction and the most common cause of death in the Western world. It is usually caused by atheroma of the coronary arteries. Myocardial lesions include ischemic fibrosis and acute infarction. Under normal circumstances, blood flow in the coronary arteries is fully coordinated with the metabolic needs of the heart muscle. Ischemic heart disease occurs when the blood supply becomes insufficient, either because the blood supply is weakened or because the myocardium becomes hypertrophied and requires a better blood supply. Coronary flow is normally independent of aortic pressure. There is an efficient autoregulatory mechanism that controls blood flow through the coronary blood vessel.

When an obstruction develops in a major coronary artery, usually due to atherosclerosis or arteriosclerosis, coronary blood flow is initially maintained because peripheral resistance distal to the obstruction is reduced. When the vessel lumen is occluded by more than 75%, ischemia develops, especially if the coronary collateral circulation is poorly developed.

Heart muscle is metabolically extremely active, and mitochondria account for over 30% of the volume of individual fibers. Aerobic metabolism is crucial, because the reserves of high-energy phosphates are small. Cardiac muscle death occurs when tissue adenosine triphosphate (ATP) levels are very low and anaerobic glycolysis virtually ceases. As with other tissues, the exact cause of death is uncertain, but lethal cardiac muscle injuries are associated with membrane damage and sudden influx of calcium into the cell cytoplasm. After a short period of ischemia, blood flow through the heart can be restored (reperfusion). However, after a critical interval, reperfusion is no longer possible, probably as a result of capillary endothelial cell edema.

Atherosclerosis is the cause of the vast majority of coronary artery diseases. Ischemic heart disease can also result from low perfusion of the coronary arteries. A frequent cause is a stroke, especially as a result of hemorrhage.

As emphasized above, ischemic heart disease is caused by an imbalance between blood flow through the myocardium and the metabolic needs of the myocardium itself. Blood flow can be further reduced by superimposed events such as vasospasm, thrombosis, or circulatory changes leading to hypoperfusion.

Coronary artery perfusion depends on the pressure differential between the ostium (aortic diastolic pressure) and the coronary sinus (right atrial pressure). Coronary blood flow decreases in systole due to the Venturi effect on the coronary orifices and compression of the intramuscular arteries during ventricular contraction. Factors that reduce coronary blood flow include decreased aortic diastolic pressure, intraventricular pressure and myocardial contraction, coronary artery stenosis, aortic valve stenosis and regurgitation, and increased right atrial pressure.

Thrombolytic therapy with agents such as steptokinase or tissue plasminogen activator (TPA) are often used to lyse a recently formed thrombus. Such clot lysis therapy can restore blood flow in most cases. This helps prevent more severe damage to the myocardium, if administered early (meaning within an hour) during this event and can at least help reduce further damage.

Angina pectoris is a complex of symptoms of ischemic heart disease characterized by paroxysmal attacks of chest pain, usually substernal or precordial. It is caused by myocardial ischemia that is not yet large enough to cause a heart attack. Sudden cardiac death can occur, usually within an hour of such a cardiac event, with or without symptoms. Every year this affects 300,000 - 400,000 people.

Other forms of heart disease include alcoholic cardiomyopathy, aortic valve prolapse, aortic valve stenosis, arrhythmias, cardiogenic shock, congenital heart disease, dilated cardiomyopathy, heart attack, heart failure, heart tumor, cardiopulmonary valve stenosis, hypertrophic cardiomyopathy, idiopathic cardiomyopathy, ischemic heart disease, ischemic cardiomyopathy, mitral regurgitation, mitral valve prolapse, peripartum cladiomyopathy, stable angina.

Myocardial infarction is another form of ischemic heart disease. The pathogenesis may include occlusive intracoronary thrombus, i.e. a clot located on an ulcerated or ruptured stenotic plaque. Occlusive intracoronary tomb causes 90% of transmural acute myocardial infarctions. Vasospasm can be with or without atherosclerosis and possibly associated with platelet aggregation. Myocardial infarction can also be associated with embolism.

The macroscopic morphological appearance of myocardial infarction can vary. A transmural infarction involves the entire thickness of the left ventricular wall from the endocardium to the epicardium. Subendocardial infarction includes multifocal fields of necrosis limited to the inner 1/3-1/2 of the left ventricular wall. Complications of myocardial infarction may include arrhythmias and conduction disorders, with possible "sudden death", expansion of the infarction, or re-infarction, congestive heart failure (pulmonary edema), cardiogenic shock, pericarditis, mural thrombosis, with possible embolization, rupture of the myocardial wall, with possible tamponade, papillary muscle rupture, with possible valvular insufficiency, formation of ventricular aneurysm.

Myocardial infarction (MI) is defined as ischemic necrosis of the myocardium, usually caused by a sudden decrease in coronary blood flow to a segment of the myocardium.

In > 90% of patients with acute Ml, an acute thrombus, often followed by plaque rupture, occludes an artery (previously partially occluded by atherosclerotic plaque) supplying blood to the damaged area. Altered platelet function induced by endothelial change in atherosclerotic plaque reportedly contributes to thrombogenesis. Spontaneous thrombolysis occurs in about 2/3 of patients, so that after 24 hours thrombotic occlusion exists and persists in only about 30%.

Myocardial infarction is sometimes caused by arterial embolization (eg, mitral or aortic stenosis, infective endocarditis, and marantic endocarditis). Myocardial infarction has been reported in patients with coronary spasm and otherwise normal coronary arteries. Cocaine causes intense coronary artery spasm, so users may present with cocaine-induced angina pectoris or myocardial infarction. Autopsy examinations and coronary angiography have shown that cocaine-induced coronary thrombosis can also occur in normal coronary arteries or be superimposed on an already existing atheroma.

Myocardial infarction is primarily a disease of the left ventricle, but the damage can also extend to the right ventricle (RV) or the atria. Right ventricular infarction usually results from occlusion of the right coronary or dominant left circumflex artery and is characterized by high right ventricular filling pressure. Often with more severe tricuspid regurgitation and decreased cardiac output. Some ventricular dysfunction occurs in about half of patients with inferior-posterior infarction, leading to hemodynamic abnormalities in 10 to 15%.

The ability of the heart to continue to function as a pump is directly related to the extent of myocardial damage.

Transmural infarctions involve the entire thickness of the myocardium from the epicardium to the endocardium and are usually characterized by abnormal Q waves on the ECG. Non-transmural or subendocardial infarcts do not extend through the ventricular wall and cause only ST segment and T wave abnormalities. Subendocardial infarctions usually involve the inner third of the myocardium, where the wall tension is the highest and the myocardial flow is the most sensitive to circulatory changes. They can also monitor prolonged hypotension. Since the transmural depth of necrosis cannot be determined with clinical precision, infarcts are better classified using ECG principles as Q wave and non-Q wave. The volume of the destroyed myocardium can be estimated by the extent and duration of the CK increase.

Ischemic cardiomyopathy is another disease within ischemic heart disease where there may be a previous myocardial infarction, but this disease is a consequence of severe coronary atherosclerosis that affects all the main branches. The result is inadequate vascular supply, leading to myocyte loss. The loss of myocytes in combination with fibrosis in the form of interstitial collagen deposits leads to a decrease in compliance, which with accompanying cardiac dilatation leads to an overload of the remaining myocytes. This maintains the process with the compensation of continuous myocyte hypertrophy. Compensatory hyperplasia as well as hypertrophy may occur, which explains the enormous size (2 to 3 times larger than normal) of such a heart. Eventually, the heart can no longer compensate, resulting in heart failure with arrhythmia and/or ischemic events. In this way, slow, progressive heart failure occurs with a history of previous myocardial infarction or anginal pain, or without these signs. Ischemic cardiomyopathy is responsible for as much as 40% of mortality in ischemic heart disease.

During ischemia, as well as reperfusion of the heart, numerous endogenous mediators are produced, such as small molecule second messengers, which affect myocardial function. Within a few minutes after an ischemic episode, the contractile force of the myocardium decreases and the total recovery of the contractile force largely depends on the duration of the ischemic period (Daemen et al., 1999). For example, during an ischemic event, Ca<2+>homeostasis is disrupted, oxygen free radicals are generated, and nitric oxide (NO) synthesis and release occurs. In addition, there is local production of cytokines, especially TNFα and IL-1β (Bolli, 1990). In the intact heart, these cytokines contribute to ischemia-induced myocardial dysfunction by inducing the gene expression of inducible NO synthase (iNOS) (Daemen et al., 1999), cyclooxygenase-2 (COX-2) and phopholipase A2 as well as vascular adhesion molecules and several chemokines. As a result, there is an immediate depression of myocardial contractile force mediated by small molecule mediators followed by cytokine-mediated infiltration of neutrophils, further damaging the heart muscle. Animal heart studies in the absence of blood and blood products process TNFα (Herskowitz et al., 1995) and IL-1β during an ischemic event. Cardiomyocytes also lose contractile force due to the action of these endogenous cytokines (Meldrum et al., 1998).

Most of the experimental data related to myocardial dysfunction mediated by TNFα and IL-13 have been obtained from animal studies. However, human myocardial tissue obtained from patients undergoing elective cardiopulmonary bypass has been studied under controlled, ex vivo conditions (Gurevitch et al., 1996; Cleveland et al., 1997). In this experimental model, human atrial trabeculae are suspended in a bloodless, physiologically oxygenated buffer bath and then exposed to an episode of simulated ischemia. During this time, the dramatically decreased contractile force when the tissues are re-exposed to oxygen is restored but to a reduced extent (60-70% reduction) and evidence of myocardial damage is observed through the release of creatine kinase (CK) (Gurevitch et al., 1996; Cleveland et al., 1997). When TNF bioactivity is specifically neutralized during ischemia/reperfusion (l/R), a greater recovery of contractile force is observed suggesting that endogenous myocardial TNF activity contributes to the contractile dysfunction induced by the ischemic event (Cain et al., 1999).

Daemen et al. (1999) examined tissue damage following ischemia followed by reperfusion using a mouse model of renal ischemia. They showed that the constitutive renal upregulation of IL-18 mRNA coincides with caspase-1 activation on the first day after ischemia. IFN-γ and IL-12 mRNA were then co-regulated on the sixth day after ischemia. Combined, but not separate, in vivo neutralization of the IFN-γ inducing cytokines IL-12 and IL-18 reduced IFN-gamma-dependent MHC class I and II upregulation to a similar extent as IFN-γ neutralization.

However, IL-18 has not been described to play a role in heart disease.

BRIEF SUMMARY OF THE INVENTION

This invention is based on the finding that an IL-18 inhibitor significantly improves cardiac contractile function in an ischemia/reperfusion model of suprafused human atrial myocardium. Inhibition of caspase-1 (ICE) also reduces the depression of contractile force after ischemia and reperfusion.

Furthermore, administration of an IL-18 inhibitor in a murine model of myocardial infarction resulted in increased survival and significant improvement in ventricular function.

These trials have shown that IL-18 inhibitors are suitable for the treatment or prevention of myocardial dysfunction.

Therefore, this invention relates to the use of IL-18 inhibitors for the production of drugs for the treatment and/or prevention of heart diseases, primarily ischemic heart disease and heart failure.

In order to apply a gene therapy approach and deliver an IL-18 inhibitor to a diseased tissue or cell, the present invention further relates to the use of an expression vector comprising the coding sequence of an IL-18 inhibitor for the treatment and/or prevention of heart disease.

SHORT DESCRIPTION OF THE DRAWINGS

SI. 1 shows the effect of IL-18BP on ischemia-induced contractile dysfunction

myocard..

(A) Kinetic response to ischemic injury. After equilibration (eq), control (Ctrl) trabeculae were perfused under normal oxygenation conditions throughout

experiment. Trabeculae were subjected to ischemia/reperfusion in the absence or presence of IL-18BP (5 µg/ml). The vertical axis shows the percentage of power developed compared to the start time of the experiment (zero point). These data are derived from the trabeculae of one patient and are representative of the methods used

used to calculate the mean change in developed power within 90 minutes.

(B) Post-ischemic force development upon IL-18 neutralization with 1 or 5 µg/ml IL-18BP. Results are expressed as the mean percentage change in developed power over the ratio

to the control at the end of reperfusion (90 minutes). Numbers in parentheses indicate IL-18BP in ug/ml. N=6.<*>p<0.01 compared to l/R (ischemia/reperfusion).

FIG 2: Shows myocardial IL-18 protein content. Trabeculae were homogenized after 90 minutes of suprafusion under conditions of normal oxygenation (control) or 45 minutes after 30 minutes of ischemia (l/R). Trabeculae were taken from the same subjects. IL-18 levels are expressed on the vertical axis in pg/ml. N=4.<*>p<0.01.

SI. 3 Shows steady state IL-18 and IL-18BP mRNA levels in control and ischemic atrial tissue. IL-18 and IL-18BP mRNA levels were determined by the RT-PCR method. The data of one of the two respondents are shown. A shows the ethidium bromide-stained agarose gel, in which the PCR products were separated, while B shows the results of quantification of the amount of PCT product as fold change relative to control (GAPDH).

Fig 4: shows the effect of ICE inhibition on post-ischemic developed force. Results are expressed as mean percentage change in developed force relative to control (Crti) after ischemia/reperfusion (l/R). The numbers in parentheses indicate the concentration of ICEi in the south/m I. N=7.<*>p<0.01 compared to l/R.

FIG 5: shows tissue creatine kinase (CK) activities after l/R. CK is expressed in units of activity per milligram of tissue wet weight. Experimental conditions are shown on the horizontal axis. Ctrl and l/R, N=6; IL-18BP (5 µg/ml), N=5; ICEi (10 and 20 p.g/ml), N=5 each;<*>p<0.05 compared to l/R.

SI. 6: shows the mean change in developed force per equilibration period, set at 100% (n=5), of trabeculae incubated with 10 µg/ml for 15 min, before addition of TNFα (1 ng/ml). TNFα and IL-18BP were added to each bath change.

FIG 7: shows the temporal response of human atrial trabeculae to IL-18 under conditions of normal oxygenation. Mature IL-18 (100 ng/ml) was added to the atrial trabeculae throughout the experimental period of 90 min. The vertical axis shows the mean percent change from baseline in developed power. The baseline was determined at the end of the equilibration period (not shown). (n=6).<*>P < 0.05,<**>P < 0.001 compared to the control in the same interval and for the remaining part of the experimental period.

SL 8: shows preservation of myocellular tissue creatine kinase activity after l/R exposure,

TNFα (1 ng/ml) and TNFα (10 ng/ml) + IL-18BP. CK activity is expressed in units of CK activity per milligram of wet tissue weight (n=6).

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the finding that IL-18 inhibitors have a beneficial effect in cases of heart disease, primarily in ischemic heart disease. As shown in the examples below, several different IL-18 inhibitors have been shown to exhibit remarkable beneficial effects on post-ischemic cardiac muscle strength.

In addition, an IL-18 inhibitor was tested in an in vivo model of myocardial infarction that resulted in improved survival and significant improvement in ventricular function.

Thus, the present invention relates to an IL-18 inhibitor for the production and treatment and/or prevention of heart disease.

In accordance with the present invention, the term "cardiac disease" includes diseases including cardiac dysfunction. These diseases are otherwise called cardiovascular disorders.

In a preferred embodiment of the present invention, the heart disease is ischemic heart disease.

The term "ischemic heart disease" as used herein includes all the various types of ischemic heart disease, including but not limited to those described in detail under the heading "Prior Art", as well as cardiovascular diseases or disorders related to ischemic heart disease.

Use in accordance with the present invention is well suited to long-term treatment, and thus is particularly useful for use in chronic heart disease. Therefore, in a preferred embodiment of the present invention, the ischemic heart disease is chronic. Angina, or angina pectoris is one of the most characteristic clinical manifestations in patients with a long history of ischemic heart disease. Weakened left ventricular function, after one or more previous episodes of myocardial infarction, can lead to left ventricular dysfunction and subsequently to congestive heart failure. Accordingly, the present invention further relates to the use of an IL-18 inhibitor for the treatment and/or prevention of angina pectoris.

In the additional recommended form, ischemic heart disease is acute and preferably myocardial infarction.

Acute myocardial heart disease or myocardial infarction usually involves necrosis of the heart muscle, most often the left ventricle. This is often due to atheroma of the coronary arteries with a superimposed thrombus or hemorrhagic plaque. Necrosis is followed by inflammatory infiltration and enzymes for fibrotic correction are released from the necrotic tissue into the blood, resulting in leukocytosis, which is usually diagnostically useful. Complications of acute myocardial infarction include arrhythmias, cardiac arrest, myocardial rupture leading to hemopericardium, mural thrombosis leading to embolism, and cardiac aneurysm. Additional complications include sudden cardiac death, arrhythmias, persistent pain, angina, heart failure with cessation of work, mitral incompetence, pericarditis, heart rupture (ventricular pain, septum or papillary muscle), mural thrombosis, ventricular aneurysm, Dressler's syndrome (chest pain, fever, effusions), pulmonary embolism. Medicines according to the present invention can also be used for the treatment and/or prevention of these complications of myocardial infarction.

In the additional recommended form, heart disease is the cessation of heart function or more severe heart failure. Heart failure is a disease state in which the heart is unable to pump blood at the rate required for normal metabolism. In almost all forms of heart failure, cardiac output is reduced, leading to a degree of hypoperfusion called arterial underfilling. The body compensates for this. by retaining an increased amount of blood. Heart failure can be acute or chronic. In the early stages, the clinical signs of heart failure may appear unilateral, but due to the inteventricular septum that divides the right and left ventricles, if one ventricle fails, the other will inevitably fail. Heart failure may be due to other causes, such as systemic hypertension, heart valve disease, or lung disease leading to congestive heart failure.

Heart failure can be congestive heart failure, which is symptomatic myocardial dysfunction that leads to a characteristic pattern of hemodynamic, renal, and neurohormonal responses. Clinical manifestations of heart failure can be left ventricular failure or right ventricular failure. Heart failure manifests as systolic or diastolic dysfunction, or both together. Common occurrence of systolic and diastolic abnormalities often occurs.

In another recommended form, heart disease is cardiomyopathy. Cardiomyopathy is any structural or functional abnormality of the ventricular myocardium.

The term "prevention" in the context of the present invention refers not only to the complete prevention of a particular effect, but also to any partial or substantial prevention, mitigation, reduction, decline or diminution of an effect before the onset or in the early stages 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 alleviating, reducing, abating or diminishing the pathological development after the onset of the disease.

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

An inhibitor of production can be any molecule that negatively affects the synthesis, processing or maturation of IL-18. Inhibitors referred to in this invention can be, for example, suppressors of interleukin IL-18 gene expression, antisense mRNAs that reduce or prevent IL-18 mRNA transcription or lead to mRNA degradation, proteins that impair correct folding, or partially or substantially prevent IL-18 secretion, proteases that degrade IL-18, once synthesized, protease inhibitors that cleave pro-IL-18 to generate mature IL-18, such as caspase-1 inhibitors, and the like.

An inhibitor of the action of IL-18 can be, for example, an antagonist of IL-18. Antagonists can either bind to or sequester IL-18 molecules themselves with sufficient affinity and specificity to partially or substantially neutralize the binding sites for IL-18 or IL-18 that are responsible for IL-18 binding to its ligands (eg, its receptors). The antagonist can also inhibit the IL-18 signaling pathway, which is activated in cells by IL-18/receptor binding.

Inhibitors of IL-18 action can be soluble receptors or molecules of IL-18 that stimulate receptors, or agents that block IL-18 receptors, or IL-18 antibodies, such as polyclonal or monoclonal antibodies, or any other agent or molecule that prevents the binding of IL-18 to its targets, thereby preventing or reducing intra- or extracellular reactions mediated by IL-18.

In a preferred embodiment of the present invention, the IL-18 inhibitor is selected from caspase-1 (ICE) inhibitors, antibodies directed against IL-18, antibodies directed against all IL-18 receptor subunits, IL-18 signaling pathway inhibitors, 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, tissue fractions or their circularly permuted derivatives that inhibit the biological activity of IL-18.

The term "IL-18 binding proteins" is used herein synonymously with "IL-18 binding protein" or "IL18BP". It includes IL-18 binding proteins as defined in WO 99/09063 or Novick et al., 1999, including "splice" variants and/or isoforms of IL-18 binding proteins, as defined in Kim et al., 2000, which bind to IL-18. In particular, human IL-18BP isoforms a and c are useful in accordance with the present invention. Proteins useful in accordance with the present invention may be glycolysed or non-glycosylated, may be obtained from natural sources, such as urine, or may preferably be obtained by recombinant techniques. Recombinant expression can be performed in prokaryotic E. coli expression systems, or in eukaryotic, and preferably mammalian, expression systems.

As used herein, the term "muteins" refers to analogs of IL-18BP, or analogs of viral IL-18BP, one or more amino acid residues of native IL-18BP or viral IL-18BP are replaced by different amino acid residues or deleted, or one or more amino acid residues are added to the native sequence of IL-18BP, or viral IL-18BP, without significantly altering the activity of the resulting product compared to wild type IL-18BP or viral IL-18BP. These muteins are prepared by known synthesis and/or site-directed mutagenesis techniques, or by any other suitable technique.

Muteins according to the present invention include proteins encoded by nucleic acid, such as DNA or RNA, that hybridize to DNA or RNA, which encodes IL-18BP or encodes viral IL-18BP, according to the present invention, under stringent conditions. The term "stringent conditions" refers to the hybridization and subsequent washing conditions, which experts in this field conventionally call "stringent conditions". See Ausubel et al., Current Protocols in Molecular Biology, supra, Interscience, N.Y., §§6.3 and 6.4 (1987, 1992), and Sambrook et al., supra. Without limitation, examples of stringent conditions include washing conditions 12-20°C below the calculated Trn of the hybrid being tested in, eg, 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes; 0.1 x SSC and 0.5% SDS at 37°C for 30-60 minutes, and then, 0.1 x SSC and 0.5% SDS at 68°C for 30-60 minutes. Those of skill in the art will appreciate that the stringency of the conditions also depends on the length of the DNA sequence, oligonucleotide probes (such as 10-40 bases), or mixed oligonucleotide probes. If mixed samples are used, it is recommended to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.

Each such mutein preferably has an amino acid sequence that sufficiently supports that of IL-18BP, or sufficiently mimics viral IL-18BP, such that it would have IL-18BP-like activity. One activity of IL-18BP is its ability to bind IL-18. As long as the mutein has substantial IL-18 binding activity, it can be used to purify IL-18, for example by affinity chromatography, and thus can be considered to have substantially similar activity to IL-18BP. Thus, whether any given mutein has substantially the same activity as IL-18BP can be determined by routine experiments involving knockdown of such mutein, e.g. by a simple sandwich competition assay to determine whether it will bind to appropriately radiolabeled IL-18, such as a radioimmunoassay or ELISA assay.

In a preferred embodiment, each such mutein has at least 40% identity or homology to the sequence of either IL-18BP or a virally encoded IL-18BP homolog, as defined in WO 99/09063. More preferably, it has at least 50%, at least 60%), at least 70%, at least 80% or, preferably, at least 90% identity or homology.

IL-18BP polypeptide muteins or viral IL-18BPs muteins, which can be used in accordance with the present invention, or encoding nucleic acids include a final set of substantially suitable sequences as substitution peptides or polynucleotides which can be routinely obtained according to the rules of the art, without unnecessary experimentation, based on the instructions and guidelines provided in the description of the present invention.

Recommended mutein changes according to the present invention are what are termed "conservative" substitutions. Conservative amino acid substitutions of IL-18BP polypeptides or proteins or viral IL-18BPs can include synonymous amino acids in a group that has sufficiently similar physicochemical properties so that the replacement of group members preserves the biological function of the molecule (Grantham, 1974). It is clear that insertions and deletions of amino acids can be made in the sequences defined above without changing their function, especially if the insertions or deletions involve only a few amino acids, e.g. less than thirty and a half. preferably less than ten, and do not remove or dislocate amino acids that are critical for functional conformation, e.g. cysteine residues. Proteins and muteins produced by such insertions and/or deletions are within the scope of this invention.

Preferably, the groups of synonymous amino acids are those defined in Table 1. Even better, the groups of synonymous amino acids are those defined in Table 2, and most preferably, the groups of synonymous amino acids are those defined in Table 3.

Examples of amino acid substitution production in proteins that can be used to produce IL-18BP polypeptide or protein muteins, or viral IL-18BP muteins, for use in the present invention include all known method steps, such as those disclosed in the following US patents: US Patents 4,959,314, 4,588,585 and 4,737,462, to Mark et al; 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; and lysine-substituted pritenes disclosed in US Pat. No. 4,904,584 (Shaw et al).

The term "fusion protein" refers to a polypeptide comprising IL-18BP, or a viral IL-18BP, or a mutein or fragment thereof, fused to another protein, which, e.g. has a prolonged residence time in body fluids. IL-18BP or viral IL-18BP, can thus join (fuse) with another protein, polypeptide or the like, e.g. with some immunoglobulin or its fragment.

"Functional derivatives" are used herein to include derivatives of IL-18BP or viral IL-18BP, and their muteins and fusion proteins, which can be prepared from functional groups arising as side chains on residues or on N- or C-terminal groups, using methods known in the art, and are included in this invention as long as they are pharmaceutically acceptable, i.e. they do not destroy the activity of a protein that is substantially similar to the activity of IL-18BP, or viral IL-18BP, and has no toxic properties in the compositions in which it is included.

These derivatives can, for example, include polyethylene glycol side chains, which can mask antigenic sites and prolong the residence of IL-18BP or viral IL-18BP in body fluids. Other derivatives include aliphatic esters of carboxyl groups, amides of carboxyl groups by reaction with ammonia or primary or secondary amines, N-acyl derivatives of free amino groups of amino acid residues formed with acyl groups (e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (e.g. seryl or threonyl residues) formed with acyl groups.

As "active fractions" of IL-18BP, or viral IL-18BP, muteins and fusion proteins, this invention includes any fragment or precursors of the polypeptide chains of the protein molecules alone or together with companion molecules or linked residues, e.g. sugar or phosphate residues or aggregates of the protein molecule or the sugar residues themselves, provided that the fraction has substantially similar activity to IL-18BP.

In a further preferred embodiment of the present invention, the IL-18 inhibitor is an antibody against IL-18 or its receptor, IL-18R. Antibodies against any of the IL-18R subunits referred to as IL-18Ra and (3) can be used in accordance with the present invention.

Antibodies according to the present invention may be polyclonal or monoclonal, chimeric, humanized or fully human. Recombinant antibodies and their fragments are characterized by high binding affinity for IL-18 or IL-18Rin in vivo and low toxicity. Antibodies that can be used in the present invention are characterized by the ability to treat patients for a period sufficient to achieve good or excellent regression or amelioration of the pathogenic condition or any symptom or group of symptoms related to the pathogenic condition, and low toxicity.

Neutralizing antibodies are readily developed in animals such as rabbits, goats or mice by immunization with IL-18 or IL-18Ra or p. Immunized mice are particularly suitable for providing a source of B cells for the production of hybridomas, which in turn are cultured to produce large amounts of anti-IL-18 monoclonal antibodies.

Chimeric antibodies are immunoglobulin molecules characterized by two or more segments or parts derived from different animal species. Generally, the variable region of a chimeric antibody is derived from a non-human mammalian antibody such as a mouse monoclonal antibody, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. Preferably, both regions and combinations thereof have low immunogenicity as routinely determined (Elliott et al., 1994). Humanized antibodies are immunoglobulin molecules that have been created by a genetic engineering technique where the murine constant regions are replaced with human counterparts while retaining the murine antigen-binding regions. The resulting murine-human chimeric antibody preferably reduces immunogenicity and improves pharmacokinetics in humans (Knight et al., 1993).

Thus, in a further preferred embodiment, the IL-18 or IL-18R antibody is a humanized antibody. Recommended examples of humanized anti-IL-18 antibodies are described, for example, in European Patent Application EP 0 974 600.

In another preferred form, this antibody is fully human. The technology for the production of human antibodies is described in detail for example in WO00/76310, WO99/53049, US 6,162,963 or AU5336100.

One method for preparing fully human antibodies consists of "humanizing" the mouse humoral immune system, ie. production of mouse strains capable of producing human Ig (Xenomouse), by introducing a human immunoglobulin (Ig) locus into a mouse in which the endogenous Ig genes are inactivated. Ig loci are complex in terms of their physical structure and the gene rearrangement and expression process required to ultimately produce a broad immune response. Antibody diversity is primarily generated by combinatorial rearrangement between the various V, D, and J genes present in the Ig loci. These loci also contain interspersed regulatory elements, which control antibody expression, allelic exclusion, class switching, and affinity maturation. Introduction of rearranged human Ig transgenes into mice showed that the mouse recombination machinery is compatible with human genes. Moreover, hybridomas secreting antigen-specific hu-mAb of different isotypes can be obtained by immunization of xenomice with the antigen.

Fully human antibodies and methods for their production are already known in the art (Mendez et al (1997); Buggemann et al (1991); Tomizuka et al., (2000) Patent WO 98/24893).

In a highly preferred embodiment of the present invention, the IL-18 inhibitor is IL-18BP, or an isoform, mutein, protein, functional derivative, active fraction, or circularly permuted derivative thereof. These isoforms, muteins, fusion proteins or functional derivatives retain the biological activity of IL-18BP, primarily binding to IL-18, and preferably have at least IL-18BP-like activity. Ideally, such proteins have enhanced biological activity compared to unaltered IL-18BP. Recommended active fractions have activity superior to that of IL-18BP, or have other advantages, such as better stability or less toxicity or immunogenicity, or are easier to produce in larger quantities or easier to purify.

The sequences of IL-18BP and its "splice" variants/isoforms can be obtained from VVO99/09063 or from Novick et al., 1999, as well as from Kim et al., 2000.

Functional derivatives of IL-18BP can be conjugated to polymers to improve protein properties, such as stability, half-life, bioavailability, human tolerance, or immunogenicity. To achieve this goal, IL18-BP can associate with e.g. polyethylene glycol (PEG). PEGylation can be carried out by known methods, described for example in WO 92/13095.

Therefore, in a preferred embodiment of the present invention, IL-18 inhibitors, particularly IL-18BP, are PEGylated.

In another preferred embodiment of the present invention, the IL-18 inhibitor comprises an immunoglobulin fusion, i.e. an IL-18 inhibitor is a fusion protein comprising all or parts of the IL-18 binding protein, which is fused to all or part of an immunoglobulin. Methods for making immunoglobulin fusion proteins are well known in the art, such as, for example, those described in WO 01/03737. One skilled in the art will understand that the resulting fusion protein of the present invention retains the biological activity of IL-18BP, primarily binding to IL-18. This fusion can be direct or go via a short linker peptide that can be very short, ie have only 1 to 3 amino acid residues, or it can be longer, for example 13 to 20 amino acid residues. This linker can be a single tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker of the sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-ly-Gly-Gln-Phe-Met which is introduced between the IL-18BP sequence and the immunoglobulin sequence. The resulting fusion protein has improved properties, such as increased residence time in body fluids (half-life), improvement of specific activity, increased expression level, or purification of the fusion protein is facilitated.

In the recommended form, IL-18BP binds to the constant region of the Ig molecule. Preferably, it binds to a region of heavy chains, such as, for example, the CH2 and CH3 domains of human IgG1. The generation of specific fusion proteins comprising IL-18BP and an immunoglobulin part are described, inter alia, in example 11 of WO 99/09063. Other isoforms of Ig molecules are also suitable for generating fusion proteins according to the present invention, such as isoforms lgG2 or lgG4, or other classes of Ig, such as, for example, IgM or IgA. Fusion proteins can be monomeric or multimeric, hetero- or homomultimeric.

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

Alternatively, TNF antagonists can inhibit the TNF signaling pathway that is activated by cell surface receptors upon TNF binding (hereafter referred to as "signaling antagonists"). Both groups of antagonists are useful, either alone or together, in combination with an IL-18 inhibitor, in the treatment or prevention of heart disease.

TNF antagonists are easily identified and evaluated by routine screening of candidates for effects on the activity of native TNF on sensitive cell lines in vitro, for example human B cells, in which TNF causes proliferation and immunoglobulin secretion. This assay contains a formulation of TNF in various dilutions of candidate antagonists, e.g. from 0.1 to 100 times the molar amount of TNF used in the assay, and controls without TNF or antagonist alone (Tucci et al., 1992).

Sequestering antagonists are the recommended TNF antagonists to be used in accordance with this guideline. Among sequestering antagonists, preference is given to those polypeptides that bind TNF with high affinity and have low immunogenicity. Soluble TNF receptor molecules and neutralizing antibodies to TNF are particularly preferred. For example, soluble TNF-RI and TNF-RII are useful in the present invention. Branched forms of these receptors, which include the extracellular domains of the receptors or their functional parts are particularly preferred antagonists according to the present invention. Branched soluble TNF receptors type-1 and type-ll are described, for example in EP914431.

Branched forms of the TNF receptor are soluble in both urine and serum as 30 kDa and 40 kDa TNF inhibitory binding proteins, called TBPI and TBPII, respectively (Engelmann et al., 1990). In accordance with the present invention, the simultaneous, sequential or separate use of an IL-18 inhibitor with a TNF antagonist is preferred.

In another preferred embodiment, human soluble TNF-RI (TBPI) is a TNF antagonist used in accordance with the present invention. Natural and recombinant soluble TNF receptor molecules and methods for their production are described in European patents EP 308 378, EP 398 327 and EP 433 900.

Derivatives, fragments, regions and biologically active parts of receptor molecules functionally resemble receptor molecules that can also be used in the present invention. Such biologically active equivalents or derivatives of the receptor molecule refer to the part of the polypeptide, or to the sequence that encodes the receptor molecule, which is of sufficient size and which is able to bind TNF with such affinity that the interaction with the membrane-bound TNF receptor is inhibited or blocked. An IL-18 inhibitor can be used simultaneously, sequentially, or separately with a TNF inhibitor.

In accordance with the present invention, the drug may further include known agents used to treat heart disease such as nitrates, e.g. nitroglycerin, diuretics, ACE inhibitors, digitalis, beta-blockers or calcium blockers in combination with an IL-18 inhibitor. Active components can be used simultaneously, sequentially or separately.

In a still further preferred embodiment of the present invention, the IL-18 inhibitor is used in an amount of about 0.001 to 100 mg/kg or about 1 to 10 mg/kg or 2 to 5 mg/kg. An IL-18 inhibitor according to the present invention is preferably administered systemically, and preferably subcutaneously or intramuscularly.

The present invention further relates to the use of an expression vector comprising the coding sequence of an IL-18 inhibitor in the preparation of a drug for the prevention and/or treatment of heart diseases. Therefore, a gene therapy approach should be considered when delivering an IL-18 inhibitor to the site of need. In order to treat and/or prevent heart diseases, a gene therapy vector comprising an IL-18 inhibitor sequence can, for example, be injected directly into diseased tissue, thereby avoiding problems involved in systemic administration of gene therapy vectors such as vector dilution, reaching or targeting target cells and tissues, and side effects.

The use of vectors to induce and/or enhance the endogenous production of an IL-18 inhibitor in a cell that normally lacks the expression of an IL-18 inhibitor, or that expresses insufficient amounts of the inhibitor, is also contemplated in accordance with the present invention. This vector may include regulatory sequences functional in cells that we desire to express the inhibitor or IL-18. These can be, for example, promoters or enhancers. Regulatory sequences can then be introduced into the right locus of the genome by homologous recombination, thereby operatively linking the regulatory sequence to the gene whose expression is to be induced or enhanced. This technology is commonly referred to as "Endogenous Gene Activation" (EGA), and is described in, for example, WO 91/09955.

One of ordinary skill in the art will readily understand that it is also possible to directly shut down IL-18 expression, not using an IL-18 inhibitor, using the same technique. To do this, a negative regulatory element, such as a silencing element, can be introduced into the IL-18 gene locus, thereby leading to the down-regulation or prevention of IL-18 expression. One skilled in the art will readily understand that such down-regulation or prevention of IL-18 expression has the same effect as the use of an IL-18 inhibitor to prevent and/or treat disease.

The present invention further relates to the use of a cell genetically modified to produce an IL-18 inhibitor in the manufacture of a medicament for the treatment and/or prevention of heart disease.

The IL-18 inhibitor used in accordance with the present invention may, upon recommendation, be used within a single pharmaceutical composition, optionally in combination with a therapeutically effective amount of a TNF inhibitor. IL-18BP and its isoforms, muteins, fusion proteins, functional derivatives, active fractions or circularly permuted derivatives as described above are the recommended active ingredients of this pharmaceutical composition.

The definition of "pharmaceutically acceptable" should include any carrier, which does not affect the effectiveness of the biological activity of the active component and which is not toxic to the host to which it is administered. For example, for parenteral administration, the active protein(s) may be formulated in an injectable dosage unit in vehicles such as saline, dextrose solution, serum albumin, and Ringer's solution.

The active ingredients in the pharmaceutical composition according to the present invention can be administered in a number of different ways. Routes of administration include intradermal, transdermal (eg in slow-release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intracranial, epidural, topical, and intranasal routes. Any other therapeutically effective route of administration may be used, for example absorption through epithelial or endothelial tissue or by gene therapy where a DNA molecule encoding an active agent is administered to the patient (e.g. via a vector), causing the active agent to be expressed and secreted in vivo. In addition, the protein(s) according to the present invention may be administered together with other components of biologically active agents such as pharmaceutically active surfactants, excipients, carriers, diluents and additives.

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

The bioavailability of the active protein(s) according to the present invention can be improved by using a conjugation process that extends the half-life of the molecule in the human body, for example by linking the molecule to polyethylene glycol, as described in PCT application WO 92/13095.

Therapeutically effective amounts of active proteins will depend on many variables, including the type of antagonist, the affinity of that antagonist for IL-18, any residual cytotoxic activity exerted by those antagonists, the route of administration, the clinical condition of the patient (including the desirability of maintaining non-toxic levels of endogenous IL-18 activity).

A "therapeutically effective amount" is that which, when administered, of the IL-18 inhibitor results in inhibition of the biological activity of IL-18. The administered dose, in the form of a single or multiple doses, will depend on a whole series of factors for each individual, including the pharmacokinetic properties of the IL-18 inhibitor, the method of administration, the clinical condition of the patient and his characteristics (gender, age, body weight, health, size), the extent of symptoms, accompanying therapy, frequency of therapy, as well as the desired effect. Adjustment and manipulation of established dose ranges are within the skill of those skilled in the art, as are in vivo and in vitro methods for determining IL-18 inhibition in an individual.

According to the present invention, the IL-18 inhibitor is used in an amount of about 0.001 to 100 mg/kg or about 0.01 to 10 mg/kg body weight, or about 0.1 to 5 mg/kg body weight or about 1 to 3 mg/kg body weight or about 2 mg/kg body weight.

A preferred mode of administration in the present invention is the subcutaneous route of administration. Intramuscular administration is further recommended in accordance with the present invention. In order to administer the IL-18 inhibitor directly to the site of action, it is also recommended that it be administered topically.

In other recommended forms, the IL-18 inhibitor is administered once daily or every other day.

The daily dose is usually given in divided doses or in a controlled-release form to obtain the desired results. The second or subsequent administration can be done in a dose that is the same, lower or higher than the initial or previous dose given to the patient. The second or subsequent administration can be done during or before the onset of the disease.

In accordance with the present invention, an IL-18 inhibitor can be administered prophylactically, or therapeutically to subjects prior to, concurrently with, or sequentially with other therapeutic regimens or agents (eg, a multidrug regimen), in therapeutically effective amounts, particularly with a TNF inhibitor and/or other cardioprotective agent. Active agents administered simultaneously with other therapeutic agents may be administered in the same composition or differently.

The present invention further relates to a method for administering a pharmaceutical composition comprising mixing an effective amount of an IL-18 inhibitor and/or a TNF antagonist with a pharmaceutically acceptable carrier.

This product further relates to a method of treating heart disease, including administering a pharmaceutically effective IL-18 inhibitor, optionally in combination with a pharmaceutically effective amount of a TNF antagonist, to a patient in need thereof.

Having now described the present invention in detail, those skilled in the art will appreciate that it can be practiced over a wide range of different equivalence parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without undue experimentation.

While the present invention has been described in connection with specific embodiments thereof, it will be understood that other modifications may exist. This application is intended to cover all variations, applications or adaptations of this invention which generally follow the principles of the invention itself and such departures from this disclosure of the invention as are arrived at by known or common practice in the art to which the invention relates, and as may be applied to the basic features set forth herein and which are covered by the claims that follow.

All references cited herein, including journal articles or abstracts, published or unpublished U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references herein are fully incorporated by their mere citation, including all data, tables, figures, and text appearing in the cited references. In addition, the entire content of the cited references is also included here by their citation itself.

Reference to a stage of a known method, a conventional method is in no way an admission that any aspect, description, or form of the present invention has been disclosed, suggested, or recited in that method.

The descriptions of specific embodiments of the present invention that follow will fully disclose the general nature of the invention that others may, by applying the knowledge of the art (including the contents of the cited references), readily modify and/or adapt these various embodiments to various applications, without undue experimentation, and without departing from the general concept of the present invention. Accordingly, both such adaptations and modifications are encompassed within the range of equivalents of the described forms, based on the teachings and knowledge disclosed herein. It is understood that the phraseology and terminology used herein is for purposes of description and not of limitation, so that the phraseology and terminology of the specifications set forth below should be interpreted by one skilled in the art in light of the knowledge and guidance set forth, combined with knowledge already known in the art.

EXAMPLES

Example 1: IL-18 inhibition reduces ischemic myocardial dysfunction

vitro

Material and methods

Reagents The IL-18BPa isoform was expressed with an N-terminal (His)6 tag in Chinese hamster egg cells and purified to homogeneity. The ability of IL-18BPa-(His)6 to neutralize IL-18 has been previously described (Kim et al., 2000). The ICE inhibitor (ICEi) Ac-Try-Val-Ala-Asp-chloromethylketone (YVAD) was purchased from Alexis Biochemicals (San Diego) and dissolved in DMSO at a concentration of 10 mg/ml. ICEi was diluted in Tyrode's solution before use. On human peripheral blood mononuclear cells, ICEi reduced endotoxin-induced secretion of mature IL-1P by 92%, as measured by ELISA (Cistron Biotechnology, Pine Brook, NJ).

lysolated atrial trabeculae Patients undergoing elective coronary artery bypass grafting with an oxygenator pump require a right atrial cannula. At that point, a small segment of the right atrial appendage is excised routinely and discarded. Trabeculae were obtained from that discarded tissue. Human atrial tissue was placed in oxygenated modified buffered Tyrode's solution at 4°C. Modified Tyrode's solution was prepared daily with deionized distilled water and contained D-glucose 5.0 mmol/liter, CaCl22.0 mmol/liter, NaCl 118.0 mmol/liter, KCl 4.0 mmol/liter, MgScy7H20 1.2 mmol/liter, NaHC0325.0 mmol/liter, and NaH2PO41.2 mmol/liter. Tyrode's solution without substrate contained choline chloride at 7 mmol/liter to maintain osmolarity. Unless otherwise stated, chemicals and reagents were purchased from Sigma. Two of four trabeculae (4–7 mm long and <1.0 mm in diameter) were attached to a force transducer and immersed in a warmed (37°C) 30-ml bath of modified Tyrode's solution; 92.5% 02/7.5%) C02 mixture was infused during normoxemic conditions. This gas mixture provided an O2 partial pressure of >350 mmHg (1 mmHg = 133 Pa), a CO2 partial pressure of 36-40 mmHg, and a pH of 7.35-7.45. Every parameter is routinely checked

with an automatic blood gas analyzer. The temperature of the bath in which the organ was kept was maintained at 37°C throughout the experiment. During simulated ischemia, the gas mixture was changed to 92.5% N2/7.5% CO2. This mixture gave an O2 partial pressure of <50 mmHg. The buffer solution was changed every 20 min except during the 30-min period when ischemia was simulated.

Experimental Design Trabeculae were equilibrated for 90 min to increase the baseline tensile strength to 1,000 mg and allow stabilization of the developed strength. Trabeculae that failed to generate more than 250 mg of developed force were excluded from the study. During a 90-min equilibration period, platinum electrodes (Radnoti Glass, Monrovia, CA) were paced to stimulate the field. These electrodes were placed on both sides of the trabeculae, stimulated (Grass SD9 stimulator, VVarvick, Rl) in a 6-ms pulse at a voltage of 20% above threshold, with a rhythm of 1 Hz during normoxia and 3 Hz during ischemia. Contractions were monitored by force transducers (Grass FT03) and recorded by a computerized preamplifier and digitizer (MacLab Quad Bridge, MacLab/8e, AD Instruments, Milford, MA) and continuously monitored by a single Macintosh computer.

After equilibration, trabeculae from one patient were studied under three types of experimental conditions: control conditions consisting of 90 min of normoxic suprafusion; l/R which consisted of 30 min of simulated ischemia followed by 45 min of reperfusion; while the third condition consisted of anticytokine intervention. In the latter case, the anticytokine was added to the suprafusion bath immediately before the onset of ischemia and was present throughout the 45 min of reperfusion.

Preserved Trabecular CK Activity At the end of tissue reperfusion (90 min) CK activity was determined as described (Kaplan et al., 1993). Tissues were homogenized in 100 vol of ice-cold isotonic extraction buffer (Cleveland et al., 1997, Kaplan et al., 1993). This assay was performed with a CK kit (Sigma) using an automated spectrophotometer. Results are presented as units of CK activity per mg (tissue wet weight).

RNA isolation and reverse transcription-coupled PCR Fresh trabeculae were homogenized in Tri-Reagent (Molecular Research Center, Cincinnati), and total RNA was isolated by chloroform extraction and isopropanol precipitation. RNA was dissolved in diethylpyrocarbonate-treated water, treated with DNase, and quantified using GeneOuant (Amersham Pharmacia Biotech). cDNA methods have already been described (Reznikov et al., 2000). For each PCR, the following sequence was used: preheating at 95°C for 15 min, then cycles at 94°C for 40 s, 55°C for 45 s, and 72°C for 1 min, with a final extension phase at 72°C for 10 min. The optimal number of cycles was determined to be 35. Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and human IL-18 (Reznikov et al., 2000) and for human IL-18BPa (Kim et al., 2000) have already been published. These PCR products were separated on a 1.5% agarose gel containing 0.5x TBE (50 mM Tris/45 mM boric acid/0.5 mM EDTA, pH 8.3) with ethidium bromide at 0.5 mg/ml, visualized by UV illumination and photographed. Densitometry was performed on the negative (IMAGEQUANT software, Molecular Dynamics), and the relative absorbance of IL-18 and IL-18BP PCR products was corrected with respect to the absorbance obtained for GAPDH.

IL-18 determinations Fresh trabeculae were homogenized as described above for CK measurements. IL-18 was analyzed by liquid phase electrochemiluminescence (ECL, Igen, Gaithersburg, MD). Mouse anti-human IL-18 mAb (R & D Systems) was labeled with ruthenium (Igen). In addition, affinity-purified goat anti-human IL-18 antibody (R & D) was labeled with biotin (Igen). This biotinylated antibody was diluted to a final concentration of ug/ml in PBS (pH 7.4) containing 0.25%> BSA, 0.5% Tween-20, and 0.01% azide (ECL buffer). Assay tubes, 25 µl of biotinylated antibody were preincubated at room temperature with 25 µl of streptavidin-coated paramagnetic beads (Dynal, Great Neck, NY) at 1 µg/µl for 30 min with vigorous shaking. Samples to be tested (25 µl) or standards were added to the tubes followed by the addition of 25 µl of ruthenylated antibody (final concentration, 1 µg/µl, diluted in ECL buffer). These test tubes were then shaken for the next 24 hours. The reaction was quenched by adding PBS at a concentration of 200 ul per test tube, and the amount of chemiluminescence was determined by the Origen Analyzer (Igen). The limit of detection for IL-18 is 16 pg/ml.

Confocal Microscopy Human atrial tissue obtained during oxygenation pump cannula insertion was placed in a 1 cm plastic holder (Meldrum et al., 1998), molded, and frozen in tissue freezing medium (Triangle Biomedical Sciences, Durham, NC) on isopentane cooled with dry ice. Frozen sections (5 µm) were cut with a Leica CM 1850 cryostat (Leica, Deerfield, IL). Slides were fixed for 10 min in 4% paraformaldehyde, air dried, and incubated for 20 min in PBS supplemented with 10% normal goat serum. Sections were incubated in a 1:100 dilution of rabbit anti-human IL-18 antibody (Peprotech, Rocky Hill, NJ) or non-immune rabbit IgG at a concentration of 1 ng/ml as a negative control. Antibodies were diluted in PBS containing 1% BSA. After overnight incubation at 4°C, sections were washed three times with 0.5% BSA in PBS. Sections were then incubated with secondary goat anti-rabbit antibody conjugated to Alexa488 (Molecular Probes) for 60 min at room temperature in the dark. Sails were stained blue with bisbenzimide (Sigma) at a concentration of 1 µg/100 ml. After staining, they were washed and examined using a Leica DM RXA (Leica) confocal laser scanning system and analyzed by SLIDEBOOK software for Macintosh (Intelligent Imaging Innovations, Denver).

Statistical analysis Data are expressed as mean ± SEM. Average changes in developed force were calculated relative to the control value after 90 min for each patient's tissue. Statistical significance of differences between groups was determined by factorial ANOVA with Bonferroni-Dunn post hoc analysis. Statistical analysis was performed with STAT-VIEW4.51 software (Abacus Concepts, Calabasas, CA).

Results

Effect of neutralization of endogenous IL-18 with IL-18BP on the postischemic development

strength

SI.1/Shows the kinetic response of trabeculae to l/R-induced damage. The final 15 min equilibration and normalization to 100% at the beginning of the experimental period is shown. Control trabeculae underwent suprafusion under normoxic conditions throughout the experiment. As shown, there is a reduction (10%) in the force developed in the control trabeculae. Trabeculae subjected to ischemia show a rapid decline in contractile function; at reperfusion, the contractile force returns to about 25% of the force developed in the control. In contrast, trabeculae exposed to ischemia but in the presence of IL-18BP recovered 55% of the force developed in the control. To assess the response to l/R heart tissue obtained from several patients, the levels of developed force in control trabeculae after 90 min were set as 100% of the value for each patient's sample, and the percentage of relative change in developed force was calculated for the experimental groups.

As shown in SI. 1S, postischemic developed force in untreated trabeculae (l/R) was reduced to an average of 35% of control. However, in the presence of IL-18BP, this reduction was attenuated to an average of 66.2% of control at a concentration of 1 µg/ml and to 76% at 5 µg/ml. These results suggest that l/R leads to the release of biologically active IL-18 upon processing of the endogenous IL-18 precursor by ICE. Therefore, IL-18 was measured in freshly obtained atrial tissue. As shown in SI. 2, basal IL-18 was present in trabeculae obtained before insertion of the oxygenator pump cannula into the right atrium. After 90 min of equilibration, 30 min of ischemia, and 45 min of reoxygenation, trabeculae were homogenized, and IL-18 levels were determined. A 4.5-fold increase of IL-18 in the tissue after l/R was noted (SI. 2).

Steady-state mRNA levels of IL-18 and IL-18BP were also determined in these tissues. We recorded basal gene expression for IL-18 and IL-18BP in freshly obtained homogenates of the preischemic atrium (SI. 3 A, B). Similar to the increase in IL-18 protein, l/R led to an additional increase in steady-state IL-18 mRNA levels (4.7-fold increase). IL-18BP gene expression was also noted in freshly obtained atrial tissue and only moderately increased (1.3-fold) after l/R.

Location of IL-18 in Human Myocardium Since IL-18 protein, as measured by ECL, and IL-18 mRNA are present in freshly obtained myocardial myocardium, histochemical staining is used to determine the location of IL-18. Atrial tissue was obtained immediately before insertion of the oxygenator pump cannula and immediately snap-frozen (not shown). IL-18 was observed in resident myocardial macrophages and in vascular endothelial cells. IL-18 in macrophages and in endothelial cells is present before surgery-related ischemia occurs and is present in the absence of contact with any foreign surface. Localization of IL-18 in resident macrophages and in vascular endothelial cells is consistent with previous studies of constitutive preformed IL-18 in freshly obtained human peripheral monocytes from healthy subjects (Puren et al., 1999). Therefore, it can be concluded that a preformed IL-18 precursor exists in the myocardium of patients scheduled for coronary artery bypass grafting due to ischemic heart disease.

Effect of ICE inhibition on postischemic developed force

Because IL-18BP effectively attenuates ischemia-induced myocardial dysfunction, we hypothesized that inhibition of the conversion of preformed IL-18 precursor to mature IL-18 would also attenuate ischemia-induced myocardial dysfunction. Therefore, the specific ICE inhibitor YVAD was added to the suprafusion bath before the onset of ischemia. ICE inhibition by addition of YVAD continued throughout the period of ischemia and during reperfusion. YVAD-mediated inhibition of ICE resulted in amelioration of ischemia-induced myocardial dysfunction, as demonstrated by an improvement in contractile function from 35% of control in l/R to 60% at 10 µg/ml and 75.8% at 20 µg/ml (SI. 4). These results confirm that the biologically active IL-18 in the human myocardium is the result of the cleavage of a previously formed IL-18 precursor by ICE. Additionally, these results suggest that myocardial ischemia can activate latent ICE.

Preservation of cell viability

Intracellular CK levels were used to assess the degree of cell viability after l/R. In this assay, the higher the CK value, the higher the number of viable cells. Each of the anticytokine interventions leads to preservation of cell viability. As shown in SI. 5, IL-18BP and ICE inhibition (10 and 20 ug/ml), increased CK levels after l/R from 1,399 to, 5,921, 5,675, 6,624, and 4,662 units of CK activity per mg (wet tissue). These observations suggest that inhibition of I/R-induced IL-18 activation preserves muscle cell viability in this in vivo model.

Effect of neuralization of TNFa induced myocardial function

As shown in SI. 6, the developed force (DF) of trabeculae was reduced by 18%) after 90 min exposure to exogenous TNFα. Neuralization of endogenous IL-18 on contractile function in human myocardium exposed to exogenous TNFα by incubation with IL-18BP for ten minutes before addition of TNFα reduced the magnitude of the drop in developed force (DF), see SI. 6. After 90 minutes, the developed force in the control group was reduced by 18%), while in the tabeculae exposed to TNFa it was increased by 58% compared to the control. However, in trabeculae exposed to TNFα with IL-18BP, the force developed fell by only 30% compared to control. These data indicate that the direct effects of TNFa on depression of myocardial contractility are mediated, at least in part, by biologically active endogenous IL-I8.

Effect of exogenous IL-18 on developed strength

Furthermore, the direct effect of exogenous IL-I8 on myocardial contractile function was determined. IL-I8 was added to the suprafused trabeculae after 90 min of equilibration and with each bath change. As shown in Figure 7, IL-I8 leads to a slow but progressive decline in the developed force during the experimental period. After 90 minutes of continuous exposure to IL-18, the developed power decreased by 42%. These data show that exogenous IL-18, similar to TNFα, has a depressant effect on myocardial function.

Interestingly, IL-18 is not as potent a depressant agent for myocardial function as TNFα.

Preservation of cell viability

Casases are often associated with apoptosis. To assess cell viability in TNFα-induced trabeculae, we measured tissue intracellular creatine kinase (CK). In this assay, high CK levels indicate viable cells. As shown in SI. 8, control trabeculae subjected to normoxic suprafusion for 90 minutes contained 6801+276 units of CK activity per milligram of wet tissue weight. In contrast, trabeculae exposed to 30/45 minute l/R damage or 90 minute exposure to TNFα showed reduced levels of preserved CK expressed as 1774+181 and 3246+217 units/mg. Trabeculae exposed to TNFα in the presence of IL-18BP contained 5605+212 units/mg tissue. Interestingly, trabeculae treated with TNFα had higher levels of preserved CK compared to trabeculae exposed to l/R. This was an unexpected finding because the magnitude of the force developed at the end of the experimental period was similar for l/R and TNFα.

Example 3: IL-18BP protects against myocardial infarction IL-18BPin vivo

Method

In vivo intramuscular electrotransfer of mouse IL-18BP expression plasmid

C57BL/6 mice received at 3-week intervals 3 injections with an expression plasmid containing cDNA for IL-18BP (called pcDNA3-IL18BP, described in WO 01/85201). Control mice were injected with a control empty plasmid. Murine IL-18BP isoform d cDNA isolated as described (accession # Q9ZOM9) (Kim et al., 2000) was subcloned into the EcoR1/Not1 sites of the mammalian cell expression vector pcDNA3 under the control of the cytomegalovirus promoter (Invitrogen). The control plasmid was a similar construct devoid of the therapeutic cDNA. The control group had 31 mice, while the experimental group receiving IL-18BP had 27 mice.

IL-18BP or control expression plasmid (60 µg) was injected into both tibialis cranial muscles of anesthetized mice as previously described (Mallat et al., 1999). Briefly, transcutaneous electrical pulses (8 square-wave electrical pulses of 200 V/cm each, 20 msec duration at 2 Hz) were delivered via a PS-15 electropulsator (Genetronics, France) using two stainless steel plate electrodes placed 4.2 to 5.3 mm apart, on each side of the leg.

Induction of infarction in the left ventricle

Twenty-four hours after administration of IL-18BP plasmid or empty plasmid, mice were anesthetized by IP injection of xylazine and ketamine, ventilated, and underwent thoracotomy. The left main coronary artery was then permanently ligated using 8-0 prolene thread to induce myocardial infarction, after which the chest was closed and the animals allowed to recover from anesthesia. Perioperative mortality was less than 20%. Postoperative mortality was 48% in the control group and 26% in the experimental group and almost as a rule occurred 4-5 days after ligation.

Seven days after ligation, mice were again anesthetized and left ventricular (LV) dimensions were assessed by echocardiography while the chest was closed using an ATL HDI 5000 echocardiograph. LV fractional shortening was calculated from end-diastolic and end-systolic diameter measurements. At the end of the electrocardiographic measurement, the heart was removed, fixed, and later cut into sections. Histological sections were stained with Sirius red to assess infarct size.

Results

Left ventricular diastolic diameter seven days after ligation in surviving mice was: 0.53+0.01 mm (n=20) in IL-18BP treated mice compared to 0.59+0.01 mm in control mice (n=16), p < 0.01.

Left atrial systolic diameter seven days after ligation in surviving mice was: 0.45+0.02) in IL-18BP treated mice compared to 0.52+0.02 in control mice, p < 0.01

Left ventricular fractional shortening: 15+1% in IL-18BP treated mice compared to 11+1% in control mice p < 0.01.

Conclusion: IL-18BP reduces mortality in mice after myocardial infarction induced by total coronary artery ligation in the left ventricle by 50%. In addition, the function of the left ventricle is significantly improved, which is shown by reduced diameters of the left ventricle in systole and diastole.

Claims (25)

1. Use of an IL-18 inhibitor for the production of a drug for the treatment and/or prevention of heart disease. 2. Use according to claim 1, characterized in that the heart disease is actually ischemic heart disease. 3. Use according to claim 1 or 2, characterized in that the heart disease is chronic. 4. Use in accordance with all previous requirements, indicated by the fact that it is a heart disease angina pectoris. 5. Use according to claim 1 or 2, characterized in that the heart disease is chronic. 6. Use according to claim 5, characterized in that the heart disease is myocardial infarction. 7. Use in accordance with all of the preceding claims, wherein the heart disease is heart failure. 8. Use in accordance with all previous claims, indicated that the heart disease is cardiomyopathy. 9. Use according to claims 1 to 8, characterized in that the IL-18 inhibitor is selected from caspase-1 (ICE) inhibitors, antibodies against IL-18, antibodies against all IL-18 receptor subunits, IL-18 signaling pathway inhibitors, IL-18 antagonists that compete with IL-18 and block the IL-18 receptor, and IL-18 binding proteins, or isoforms, muteins, fusion proteins, functional derivatives, active fractions or circulatory permuted derivatives thereof that inhibit the biological activity of IL-18. 10. Use according to claim 9, characterized in that the IL-18 inhibitor is an antibody against IL-18. 11. Use according to claim 9, characterized in that the IL-18 inhibitor is an antibody against IL-18 receptor a. 12. Use according to claim 9, characterized in that the IL-18 inhibitor is an antibody against IL-18 receptor B. 13. Use according to claims 9 to 12, characterized in that the IL-18 antibody is a humanized or human antibody. 14. Use according to claim 9, characterized in that the IL-18 inhibitor is an IL-18 binding protein, or an isoform, mutein, fusion protein, functional derivative, active fraction or circulatory permuted derivative thereof that inhibits the biological activity of IL-18. 15. Use according to all previous claims, characterized in that the IL-18 inhibitor is glycosylated at one or more sites. 16. Use according to claim 14, characterized in that the fusion protein comprises an immunoglobulin (Ig) fusion. 17. Use according to claim 9 or 14, characterized in that the functional derivative includes at least one group attached to one or more functional groups, which is formed on one or more side chains of amino acid residues. 18. Use according to claim 17, characterized in that this group is a polyethylene group. 19. Use in accordance with all previous requirements, indicated by the fact that this drug additionally includes a tumor necrosis factor (TNF) antagonist. 20. Use according to claim 19, characterized in that the IL-18 inhibitor is used simultaneously, sequentially or separately from the TNF antagonist. 21. Use according to claim 19 or 20, characterized in that the TNF antagonist is TBPI and/or TBPII. 22. Use according to all preceding claims, characterized in that the IL-18 inhibitor is in concentrations ranging from about 0.001 to 100 mg/kg or about 1 to 10 mg/kg or 2 to 5 mg/kg. 23. Use of an expression vector comprising the coding sequence of an IL-18 inhibitor for the preparation of a drug for the treatment and/or prevention of heart diseases. 24. Using an expression vector to induce and/or enhance the endogenous production of IL-18 inhibitors in a cell for the preparation of a drug for the prevention and/or treatment of heart diseases. 25. A method for treating heart disease comprising administering an effective inhibitory amount of an IL-18 inhibitor to a host in need thereof.