Use of IL-18 inhibitors

FIELD OF THE INVENTION

The present invention relates to the therapeutical use of IL-18 inhibitors in several pathological conditions. More specifically, the invention relates to the treatment and/or prevention of liver diseases.

BACKGROUND OF THE INVENTION

In 1989, an endotoxin-induced serum activity that induced interferon-γ (IFN-γ) obtained from mouse spleen cells was described (Micallef et al., 1996). This serum activity functioned not as a direct inducer of IFN-γ but rather as a co-stimulant together with IL-2 or mitogens. An attempt to purify the activity from post-endotoxin mouse serum revealed an apparently homogeneous 50-55 kDa protein. Since other cytokines can act as co-stimulants for IFN-γ production, the failure of neutralizing antibodies to IL-1, IL-4, IL-5, IL-6, or TNF to neutralize the serum activity suggested it was a distinct factor. In 1995, the same scientists demonstrated that the endotoxin-induced co-stimulant for IFN-γ production was present in extracts of livers from mice preconditioned with P. acnes (Novick et al., 1992). In this model, the hepatic macrophage population (Kupffer cells) expand and in these mice, a low dose of bacterial lipopolysaccharide (LPS), which in non-preconditioned mice is not lethal, becomes lethal. The factor, named IFN-γ -inducing factor (IGIF) and later designated interleukin-18 (IL-18), was purified to homogeneity from 1,200 grams of P. acnes-treated mouse livers. Degenerate oligonucleotides derived from amino acid sequences of purified IL-18 were used to clone a murine IL-18 cDNA (Novick et al., 1992). IL-18 is an 18-19 kDa protein of 157 amino acids, which has no obvious similarities to any peptide in the databases. Messenger RNAs for IL-18 and interleukin-12 (IL-12) are readily detected in Kupffer cells and activated macrophages. Recombinant IL-18 induces IFN-gamma more potently than does IL-12, apparently through a separate pathway (Novick et al., 1992). Similar to the endotoxin-induced serum activity, IL-18 does not induce IFN-γ by itself, but functions primarily as a co-stimulant with mitogens or IL-2. IL-18 enhances T cell proliferation, apparently through an IL-2-dependent pathway, and enhances Th1 cytokine production in vitro and exhibits synergism when combined with IL-12 in terms of enhanced IFN-γ production (Maliszewski et al., 1990).

Neutralizing antibodies to mouse IL-18 were shown to prevent the lethality of low-dose LPS in P. acnes pre-conditioned mice. Others had reported the importance of IFN-γ as a mediator of LPS lethality in pre-conditioned mice. For example, neutralizing anti-IFN-γ antibodies protected mice against Shwartzman-like shock (Fantuzzi et al., 1998), and galactosamine-treated mice deficient in the IFN-γ receptor were resistant to LPS-induced death (Byrn, 1990). Hence, it was not unexpected that neutralizing antibodies to murine IL-18 protected P. acnes-preconditioned mice against lethal LPS (Novick et al., 1992). Anti-murine IL-18 treatment also protected surviving mice against severe hepatic cytotoxicity.

After the murine form was cloned, the human cDNA sequence for IL-18 was reported in 1996 (Okamura et al., 1995). Recombinant human IL-18 exhibits natural IL-18 activity (Okamura et al., 1995). Human recombinant IL-18 is without direct IFN-γinducing activity on human T-cells, but acts as a co-stimulant for production of IFN-γ and other T-helper cell-1 (Th1) cytokines (Okamura et al., 1995). To date, IL-18 is thought of primarily as a co-stimulant for Th1 cytokine production (IFN-γ, IL-2 and granulocytemacrophage colony stimulating factor) (Izaki, 1978) and also as a co-stimulant for FAS ligand-mediated cytotoxicity of murine natural killer cell clones (Novick et al., 1989).

By cloning IL-18 from affected tissues and studying IL-18 gene expression, a close association of this cytokine with an autoimmune disease was found. The nonobese diabetic (NOD) mouse spontaneously develops autoimmune insulitis and diabetes, which can be accelerated and synchronized by a single injection of cyclophosphamide. IL-18 mRNA was demonstrated by reverse transcriptase PCR in NOD mouse pancreas during early stages of insulitis. Levels of IL-18 mRNA increased rapidly after cyclophosphamide treatment and preceded a rise in IFN-γ mRNA, and subsequently diabetes. Interestingly, these kinetics mimic that of IL-12-p40 mRNA, resulting in a close correlation of individual mRNA levels. Cloning of the IL-18 cDNA from pancreas RNA followed by sequencing revealed identity with the IL-18 sequence cloned from Kupffer cells and in vivo pre-activated macrophages. Also NOD mouse macrophages responded to cyclophosphamide with IL-18 gene expression while macrophages from Balb/c mice treated in parallel did not. Therefore, IL-18 expression is abnormally regulated in autoimmune NOD mice and closely associated with diabetes development (Novick et al., 1992).

IL-18 plays a potential role in immunoregulation or in inflammation by augmenting the functional activity of Fas ligand on Th1 cells (Conti et al., 1997). IL-18 is also expressed in the adrenal cortex and therefore might be a secreted neuroimmunomodulator, playing an important role in orchestrating the immune system following a stressful experience (Chater, 1986).

In vivo, IL-18 is formed by cleavage of pro-IL-18, and its endogenous activity appears to account for IFN-γ production in P. acnes and LPS-mediated lethality. Mature IL-18 is produced from its precursor by the IL-Ιβ converting enzyme (IL-1 betaconverting enzyme, ICE, caspase-1).

The IL-18 receptor consists of at least two components, co-operating in ligand binding. High- and low-affinity binding sites for IL-18 were found in murine IL-12 stimulated T cells (Yoshimoto et al., 1998), suggesting a multiple chain receptor complex. Two receptor subunits have been identified so far, both belonging to the IL-1 receptor family (Parnet et al., 1996). The signal transduction of IL-18 involves activation of NF-κΒ (DiDonato et al., 1997).

Several known cytokine binding proteins are soluble cytokine receptors and correspond to the extracellular ligand binding domains of their respective cell surface cytokine receptors. They are derived either by alternative splicing of a pre-mRNA, common to the cell surface receptor, or by proteolytic cleavage of the cell surface receptor. Such soluble receptors have been described in the past, including among others, the soluble receptors of IL-6 and IFN-γ (Nakamura et al., 1989), TNF (Dao et al., 1996; Engelmann et al., 1989), IL-1 and IL-4 (John, 1986), IFN-α/β (Mizushima and Nagata, 1990) and others. One cytokine-binding protein, named osteoprotegerin (OPG, also known as osteoclast inhibitory factor - OCIF), a member of the TNFR/Fas family, appears to be the first example of a soluble receptor that exists only as a secreted protein (Anderson, 1997; Bollon, 1980).

Recently, soluble protein having a high affinity for IL-18 has been isolated from human urine, and the human and mouse cDNAs were described (Novick et al., 1999; WO 99/09063). The protein has been 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 novel family of secreted proteins. The family further includes several Poxvirus-encoded proteins which have a high homology to IL-18BP (Novick et al., 1999). IL-18BP is constitutively expressed in the spleen, belongs to the immunoglobulin superfamily, and has limited homology to the IL-1 type II receptor. Its gene was localized on human chromosome 11q13, and no exon coding for a transmembrane domain was found in an 8.3 kb genomic sequence (Novick etal., 1999).

Four human and two mouse isoforms of IL-18BP, resulting from mRNA splicing and found in various cDNA libraries and have been expressed, purified, and assessed for binding and neutralization of IL-18 biological activities (Kim et al., 2000). Human IL18BP isoform a (IL-18BPa) exhibited the greatest affinity for IL-18 with a rapid on-rate, a slow off-rate, and a dissociation constant (K(d)) of 399 pM. IL-18BPc shares the Ig domain of IL-18BPa except for the 29 C-terminal amino acids; the K(d) of IL-18BPc is 10-fold less (2.94 nM). Nevertheless, IL-18BPa and IL-18BPc neutralize IL-18 >95% at a molar excess of two. IL-18BPb and IL-18BPd isoforms lack a complete Ig domain and lack the ability to bind or neutralize IL-18. Murine IL-18BPc and IL-18BPd isoforms, possessing the identical Ig domain, also neutralize >95% murine IL-18 at a molar excess of two. However, murine IL-18BPd, which shares a common C-terminal motif with human IL-18BPa, also neutralizes human IL-18. Molecular modelling identified a large mixed electrostatic and hydrophobic binding site in the Ig domain of IL-18BP, which could account for its high affinity binding to the ligand (Kim et al., 2000).

Recently, it has been suggested that the interleukin IL-18 is involved in the progression of pathogenicity in chronic inflammatory diseases, including endotoxin shock, hepatitis, and autoimmune-diabetes (Kahiwamura and Okamura, 1998). A further indication of a possible role of IL-18 in the development of liver injury resulted from experiments published by Tsuij et al. (Tsuij et al., 1999), showing an elevated level of IL18 in lipopolysaccharide-induced acute liver injury in a mouse model. However, the mechanism of the multi-functional factor IL-18 in the development of liver injury has not been elucidated so far.

Liver damage or injury may have diverse causes. It may be due to viral or bacterial infections, alcohol abuse, immunological disorders, or cancer, for example.

Viral hepatitis, due to Hepatitis B virus and Hepatitis C virus, for example, are poorly managed diseases that afflict large number of people world-wide. The number of known hepatitis viruses known is constantly increasing. Apart from Hepatitis B and C virus, at least four other viruses causing virus-associated hepatitis have been discovered so far, called Hepatitis A, D, E and G-Virus.

Alcoholic liver disease is another widespread disease associated with chronic consumption of alcohol. Immune hepatitis is a rare autoimmune disease that is poorly managed. Liver injury also includes damages of the bile ducts. Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by destruction of the intrahepatic bile ducts.

Several studies have demonstrated that damage to the liver in diseases such as alcoholic hepatitis, liver cirrhosis, viral hepatitis and primary biliary cirrhosis is associated with T-helper cell-1 (Th1) responses. In one study, a novel liver injury model was established in mice by targeting of ovalbumin-containing liposomes into the liver, followed by adoptive transfer of ovalbumin-specific Th1 cells. Combined treatment of mice with ovalbumin-containing liposomes and Th1 cell transfer caused an increase in serum transaminase activity that was paralleled with an elevation of serum IFN-γ levels. In sharp contrast, ovalbumin-specific Th2 cell transfer resulted in an increase of serum IL-4 levels but did not induce liver injury. The liver injury was blocked by anti-IFN-γ antibodies and anti-tumor necrosis factor (TNF)-a antibodies. These findings indicate that Th1 cells are the major effector cells in acute liver injury (Nishimura and Ohta, 1999) In another set of studies it was shown that mice over-expressing IFN-γ exhibit spontaneous hepatitis without any pathogen or any other stimulant (Okamoto et al., 1998).

Another study implicated Th1 responses in primary biliary cirrhosis (PBC). PBC is an autoimmune liver disease characterized by destruction of the intrahepatic bile ducts. It is generally believed that cellular immune mechanisms, particularly involving T cells, result in this bile duct damage. The relative strength of Th1 and Th2 responses has recently been proposed to be an important factor in the pathophysiology of various autoimmune diseases. In this study, the subset balance in PBC was evaluated by detection of cytokines specific to the two T-cell subsets, i.e., IFN-γ for Th1 cells and IL-4 for Th2 cells. IFN-γ and IL-4 messenger RNA (mRNA) positive cells were counted in liver sections from 18 patients with PBC and 35 disease controls including chronic active hepatitis C, extrahepatic biliary obstruction, and normal liver, using nonisotopic in situ hybridization and immunohistochemistry. Mononuclear cells expressing IFN-γ and IL-4 mRNA were aggregated in inflamed portal tracts in PBC livers, but were rarely present in extrahepatic biliary obstruction, alcoholic fibrosis, or normal liver sections. The IFN-γ and IL-4 mRNA positive cells in PBC livers were detected in significantly higher numbers than in control livers (P < 0,01). Moreover, IFN-γ mRNA expression was more commonly detected than IL-4 expression in PBC livers, and the levels of IFN-γ mRNA expression were highly correlated with the degree of portal inflammatory activity. IFN-γ mRNApositive cells were detected primarily around damaged bile ducts that were surrounded by lymphoid aggregates. The data indicate that Th1 cells are the more prominent T-cell subset in the lymphoid infiltrates in PBC (Harada et al., 1997).

The cytokine pattern on viral antigen recognition is also believed to exert a profound influence on the resolution of viral infections and viral clearance. One study investigated whether a cytokine imbalance oriented toward Th2 type response plays a role in chronic hepatitis B. Cytokine profiles of peripheral blood mononuclear cells associated with chronic hepatitis B were analyzed by RT-PCR. Upon hepatitis B surface antigen (HbsAg) stimulation, expression of IFN-γ, IL-2, IL-4, and IL-10 was detected in 41%, 8%, 41%, and 50% of the patients, respectively. Among these cytokines, the expression of the Th1 cytokine IFN-γ was associated with high levels of serum AST/ALT (Aspartate aminotransferase/Alanine aminotransferase), representing typical markers of liver damage. Th2 type cytokines were not shown to exert a protective effect on hepatocytes. In conclusion, production of a Th1 cytokine, IFN-γ, by HBsAg-reactive cells was associated with hepatocyte damage in chronic hepatitis B (Lee et al., 1999). High levels of the FAS ligand and its receptor (CD95) were reported in liver of hepatitis B patients (Luo et al., 1997). FAS ligand is considered to be one of the major cytotoxic agents leading to hepatocyte apoptosis.

Another study identified factors associated with the progression of liver injury in 30 hepatitis C virus/RNA (HCV/RNA)-positive untreated patients with chronic hepatitis. Necroinflammatory and architectural damage were evaluated using Ishak's score. Activated hepatic stellate cells (HSC) were visualized by immunohistochemistry for asmooth muscle actin (aSMA) and quantitated by morphometry. Plasma HCV/RNA was evaluated using a competitive RT-PCR method. To study the type of immune response involved in the progression of liver injury, IFN^-positive cells (as expression of a Th1like response) were evaluated by immunohistochemistry and quantitated by morphometry. It was found that HSC were mostly detected close to areas of lobular necroinflammation or lining fibrotic septa. The aSMA- and Sirius Red-positive parenchyma correlated significantly with necroinflammatory and architectural scores. IF^-positive cells were detected in periportal areas associated with the inflammatory infiltrates and significantly correlated with architectural damage. It was therefore concluded that HSC activation and progression of liver injury are associated with a Th1like response (Baroni et al, 1999). Similarly to the case of Hepatitis B, FAS ligand and its receptor were found in liver and sera of hepatitis C patients (Hiramatsu et al, 1994; Okazaki et al, 1996; Lio et al., 1998)

Th1 cytokines and other Th1 markers were found to be associated with alcoholic hepatitis and liver cirrhosis. Inflammatory stimuli and lipid peroxidation activate nuclear factor κ B (NF-κΒ) and upregulate proinflammatory cytokines and chemokines. In one study, the relationship between pathological liver injury, endotoxemia, lipid peroxidation, and NF-κΒ activation and imbalance between pro- and anti-inflammatory cytokines was evaluated. Rats (5 per group) were fed ethanol and a diet containing saturated fat, palm oil, corn oil, or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in control rats. Pathological analysis was performed and measurements of endotoxin were taken, lipid peroxidation, NF-κΒ, and messenger RNA (mRNA) levels of proinflammatory cytokines (TNFa, IL-1 beta, IFN-γ, and IL-12), C-C chemokines (regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein [MCPj-1, macrophage inflammatory protein [MIP]-1-a), C-X-C chemokines (cytokine induced neutrophil chemoattractant [CINC], MIP-2, IP-10, and epithelial neutrophil activating protein [ENAj-78), and anti-inflammatory cytokines (IL-10, IL-4, and IL-13). Activation of NF-κΒ and increased expression of proinflammatory cytokines C-C and C-X-C chemokines was seen in the rats exhibiting necroinflammatory injury (fish oilethanol and corn oil-ethanol). These groups also had the highest levels of endotoxin and lipid peroxidation. Levels of IL-10 and IL-4 mRNA were lower in the group exhibiting inflammatory liver injury. Thus, activation of NF-κΒ occurs in the presence of proinflammatory stimuli and results in increased expression of Th1 proinflammatory cytokines and chemokines (Naji et al., 1999). FAS ligand and its receptor are also elevated in alcoholic liver diseases, suggesting once again that Th1 cytokines are involved in the autoimmune processes induced in alcoholic hepatitis (Galle et al., 1995; Taieb et al, 1998; Fiore et al., 1999).

TNF -a has also emerged as a common pathway in the pathogenesis of alcoholrelated hepatic necro-inflammation. Increased levels of hepatic and serum TNF have been documented in animal models of alcoholic liver disease and in human alcoholic liver disease. This dysregulated TNF metabolism has been postulated to play a role in

many of the metabolic complications and the liver injury of alcoholic liver disease (Grove et al., 1997; McClain and Cohen, 1989). For instance it was found in one study that patients with alcoholic hepatitis had higher TNF-α levels (mean, 26.3 ng/L; 95% Cl, 21.7 to 30.9) than normal subjects (6.4 ng/L; Cl, 5.4 to 7.4). Patients who subsequently died had a higher TNF-α level (34.7 ng/L; Cl, 27.8 to 41.6) than survivors (16.6 ng/L; Cl, 14.0 to 19.2). In patients with alcoholic hepatitis, TNF-α levels correlated positively with serum bilirubin (r = 0.74; P = 0.0009) and serum creatinine (r = 0.81; P = 0.0003). Patients with alcoholic hepatitis had higher TNF-α levels than patients with inactive alcoholic cirrhosis (11.1 ng/L; Cl, 8.9 to 13.3) and severely alcoholic persons without liver disease (6.4 ng/L; Cl, 5.0 to 7.8). Patients with abnormal renal function had lower TNF-α levels (14.1 ng/L; Cl, 5.4 to 22.8) than patients with alcoholic hepatitis. It was therefore concluded that elevations in TNF-α in alcoholic hepatitis are most marked in severe cases, suggesting that TNF-α plays a role in the pathogenesis (Bird et al., 1990)

TNF mediates many of the biologic actions of endotoxin. Recent studies have shown that TNF administration may cause liver injury and that TNF may mediate the lethality of the hepatotoxin galactosamine. One of the most potent TNF inducers is endotoxin. Because patients with alcoholic liver disease frequently have endotoxemia and because many of the clinical manifestations of alcoholic hepatitis are known biologic actions of TNF, its activity was evaluated in patients with alcoholic hepatitis. Basal and lipopolysaccharide-stimulated TNF release from peripheral blood monocytes, a major source of TNF production, was determined in 16 patients with alcoholic hepatitis and 16 healthy volunteers. Eight of 16 alcoholic hepatitis patients and only two of 16 healthy volunteers had detectable spontaneous TNF activity (p less than 0.05). After lipopolysaccharide stimulation, mean monocyte TNF release from alcoholic hepatitis patients was significantly increased to over twice that of healthy controls (25.3 +/- 3.7 vs. 10.9 +/- 2.4 units per ml, p less than 0.005). It was therefore concluded that monocytes from alcoholic hepatitis patients have significantly increased spontaneous and lipopolysaccharide-stimulated TNF release compared to monocytes from healthy volunteers (McClain and Cohen, 1989.

Lipopolysaccharide (LPS)-binding protein (LBP) and CD14 play key intermediary roles in the activation of cells by endotoxin. Gut-derived LPS has been postulated to participate in promoting pathological liver injury in alcoholic liver disease. It was demonstrated that rats fed intragastrically with ethanol in oil for 4 weeks had elevated levels of CD14 and LBP in their Kupffer cells and hepatocytes, respectively. Expression of CD14 mRNA was also elevated in nonmyeloid cells. Enhanced LBP and CD14 expression rapidly increases the LPS-induced expression of various pro-inflammatory cytokines and correlates with the presence of pathological liver injury in alcoholic liver injury (Su et al., 1998; Lukkari et al., 1999).

SUMMARY OF THE INVENTION

The present invention is based on the finding that inhibitors of IL-18 are effective for treatment and/or prevention of different diseases or disorders.

It is an object of the present invention to provide for a novel means for treating and/or preventing certain liver injuries.

The invention provides a use of an IL-18 inhibitor selected from caspase-1 (ICE) inhibitors, inhibitors ofthe IL-18 signalling pathway, antagonists of IL-18 which compete with IL-18 and block the IL-18 receptor, and IL-18 binding proteins, isoforms, muteins, fused proteins, functional derivatives, active fractions or circularly permutated derivatives thereof having essentially the same activity as an IL-18 binding protein for the manufacture of a medicament for treatment and/or prevention of liver injury, wherein the liver injury is alcoholic hepatitis, viral hepatitis, immune hepatitis, liver cirrhosis, and primary biliary cirrhosis.

The use of combinations of an IL-18 inhibitor and/or an interferon and/or a TNF antagonist and/or a COX-2 inhibitor are also considered according to the invention. In order to apply gene therapeutical approaches to deliver the IL-18 inhibitor to diseased tissues or cells, further aspects of the invention relate to the use of expression vectors comprising the coding sequence of an IL-18 inhibitor for the treatment and/or prevention of the disease conditions. The invention further relates to the use of endogenous gene activation of IL-18 inhibitors and to the use of cells genetically engineered to express IL18 inhibitors for the prevention and/or treatment of the liver injuries.

BRIEF DESCRIPTION OF THE DRAWINGS Fig, 1 shows a histogram depicting the serum levels of IFN-γ (pg/ml) after injection of various amounts of recombinant IL18BP (0; 0,04; 0,4; 4 mg/kg) into mice 1h before the injection of LPS. Blood samples were taken 5 h after LPS injection and analyzed by ELISA for circulating IFN-γ.

Fig. 2 shows a histogram depicting the serum levels of Alanine aminotransferase (ALT). Mice were injected with increasing doses of recombinant human IL18BP (0; 0,04; 0,4; 4 mg/kg) before injection of LPS into P. acnes sensitized mice. Blood samples were taken 5h after LPS injection and serum levels of ALT were measured. SF = Sigma-Frankel: 1 SF Unit of AST/ALT will form 4,82x1 O'⁴ pmol glutamate/minute at pH 7.5 at 25°C.

Fig. 3 shows the survival time of the mice after LPS injection. Mice were injected with different doses of recombinant human IL18BP (0; 0,04; 0,004; 4 mg/kg) 20 min before injection of LPS into P. acnes sensitized mice. Triangles: 4 mg/kg; small diamond: 0,4; big diamond: 0,04; circles: no IL18BP (only LPS).

Fig. 4 shows a histogram depicting serum levels of IFN-γ, measured 5 h after injection of different amounts of IL18BP (0; 0,4; 4 mg/kg), which was administered 20 min before LPS injection into P. acnes sensitized mice.

Fig. 5 shows the survival of mice injected either with polyclonal IL-18 antiserum or normal rabbit serum (NDS = control) 30 min before injection with 40 mg/ml (lethal dosis) of LPS derived from E. coli (Fig. 5 A) or S. thyphimurium (Fig. 5 B). Triangles: mice were injected with IL-18 antiserum; circles: mice were injected with NDS. On the x-axis, the days after LPS challenge are depicted. * p<0,05.

Fig. 6 shows a histogram, depicting the mean + SEM of five mice per group treated in the following way. Mice were injected intraperitoneally (i.p.) either anti-IL-18 antiserum, soluble TNF-α receptors (TNFsRp55) or vehicle (saline), immediately followed by the intravenous (i.v.) administration of Concanavalin A (Con A; Fig. 6 A) or PEA (Pseudomonas aeruginosa, Fig. 6 B). **p<0,01; ***p<0,001 vs ConA or PEA alone; # p<0,01 vs either TNFsRp55 or anti-IL-18 factorial ANOVA.

DESCRIPTION OF THE INVENTION

The present invention is based on the finding of a beneficial effect of an inhibitor of IL-18 in different diseases and disorders.

The term “inhibitor of iL-18” within the context of this invention refers to any molecule modulating IL-18 production and/or action in such a way that IL-18 production and/or action is attenuated, reduced, or partially, substantially or completely prevented or blocked. The term “IL-18 inhibitor” is meant to encompass inhibitors of IL-18 production as well as of inhibitors of IL-18 action.

An inhibitor of production can be any molecule negatively affecting the synthesis, processing or maturation of IL-18. The inhibitors considered according to the invention can be, for example, suppressors of gene expression of the interleukin IL-18, antisense mRNAs reducing or preventing the transcription of the IL-18 mRNA or leading to degradation of the mRNA, proteins impairing correct folding, or partially or substantially preventing secretion of IL-18, proteases degrading IL-18, once it has been synthesized, inhibitors of proteases cleaving pro-IL-18 in order to generate mature IL-18, such as inhibitors of caspase-1, and the like.

An inhibitor of IL-18 action can be an IL-18 antagonist, for example. Antagonists can either bind to or sequester the IL-18 molecule itself with sufficient affinity and specificity to partially or substantially neutralise the IL-18 or IL-18 binding site(s) responsible for IL-18 binding to its ligands (like, e.g. to its receptors). An antagonist may also inhibit the IL-18 signalling pathway, which is activated within the cells upon IL18/receptor binding.

Inhibitors of IL-18 action may be also soluble IL-18 receptors or molecules mimicking the receptors, or agents blocking the IL-18 receptors, or IL-18 antibodies, such as polyclonal or monoclonal antibodies, or any other agent or molecule preventing the binding of IL-18 to its targets, thus diminishing or preventing triggering of the intraor extracellular reactions mediated by IL-18.

Disclosed herein is the use of inhibitors or IL-18 are used for the manufacture of a medicament for treatment and/or prevention of liver injury. Also disclosed herein is the use of an IL-18 inhibitor for the manufacture of a medicament for treatment and/or prevention of acute and chronic liver diseases, and more preferably, alcoholic hepatitis, viral hepatitis, immune hepatitis, fulminant hepatitis, liver cirrhosis, and primary biliary cirrhosis.

The term liver injury, or liver disease, a used herein, comprises a variety of different pathological conditions. Several of the conditions contemplated in the present invention have been explained in detail in the “Background of the invention” above. Further liver diseases which can be treated and/or prevented according to the invention comprise, for example, pyrogenic liver absess. It is also called bacterial liver, and it is a pus-producing cavity within the liver. The causes of a liver abscess are multiple. It can develop from an abdominal infection such as appendicitis, diverticulitis, or a perforated bowel; an infection in the blood; an infection from the biliary (liver secretion) tract; or trauma when a bruised liver becomes infected. The most common organisms causing liver abscess are Escherichia coli, Proteus vulgaris, and Enterobacter aerogenes. The incidence is 1 out of 10,000 people.

Alcoholic liver diseases can be treated and/or prevented using IL-18 inhibitors according to the invention. They comprise acute or chronic inflammation of the liver induced by alcohol abuse. Alcoholic hepatitis usually occurs after years of excessive drinking. The longer the duration of alcohol use and the larger the consumption of alcohol, the greater the probability of developing liver disease. Malnutrition develops as a result of empty calories from alcohol, reduced appetite, and malabsorption (inadequate absorption of nutrients from the intestinal tract). Malnutrition contributes to liver disease. The toxicity of ethanol to the liver, individual susceptibility to alcoholinduced liver disease, and genetic factors also contribute to the development of alcoholic liver disease.

In accordance with the present invention, liver cirrhosis can be treated and/or prevented using the IL-18 inhibitors. Cirrhosis is a chronic liver disease which causes damage to liver tissue, scarring of the liver (fibrosis; nodular regeneration), progressive decrease in liver function, excessive fluid in the abdomen (ascites), bleeding disorders (coagulopathy), increased pressure in the blood vessels (portal hypertension), and brain function disorders (hepatic encephalopathy). The damaged and scarred liver becomes unable to adequately remove waste products (toxins) from the blood, and the formation of scar tissue leads to increased pressure (portal hypertension) in the veins between the intestines and spleen to the liver. Excessive alcohol use is the leading cause of cirrhosis. Other causes include infections (such as hepatitis), diseases and defects of the bile drainage system (such as biliary stenosis or obstruction), cystic fibrosis, and increased iron and copper absorption.

The type of cirrhosis depends on the cause of the disease. Complications of cirrhosis can be severe. In the U.S. cirrhosis is the 9th leading cause of death. Neurological problems (such as hepatic encephalopathy) can develop. Increased fluid collection in the abdominal cavity (ascites) is caused by decreased body protein, increased sodium, and increased pressure within the liver's blood vessels (portal hypertension). Portal hypertension can cause increased pressure, size, and fullness in the blood vessels in the esophagus (esophageal varices). Problems with bleeding and clotting can occur. The increased pressures within the blood vessels and the problems with blood clotting can increase the possibility of severe and life-threatening hemorrhage.

A further disorder meant to be encompassed by the term “liver injury” according to the present invention is autoimmune hepatitis. It is an inflammation of the liver caused by interaction with the immune system. Autoimmune hepatitis is a type of chronic active hepatitis. Cellular immune reactions may be a cause of chronic active hepatitis. A variety of circulating autoantibodies can be found in the blood of patients with chronic active hepatitis. Other autoimmune diseases may be associated with chronic active hepatitis, or may occur in the relatives of patients with chronic active hepatitis. These diseases are thyroiditis, diabetes mellitus, ulcerative colitis, Coombs-positive hemolytic anemia, proliferative glomerulonephritis, and Sjogren's syndrome. Risk factors may include these diseases, or risk factors associated with chronic active hepatitis. The incidence is 4 out of 10,000 people.

Biliary atresia is a further disorder within the scope of the term “liver injury”. It is an obstruction of the bile ducts caused by their failure to develop normally before birth (in utero). Biliary atresia is caused by the abnormal and inadequate development of the bile ducts inside or outside the liver. The purpose of the biliary system is to remove waste products from the liver, and to carry bile salts necessary for fat digestion to the small intestine. In this condition, bile flow from the liver to the gallbladder is blocked. This can lead to liver damage and cirrhosis of the liver, which, if not treated, is eventually fatal.

According to the invention, the IL-18 inhibitors are also used for the manufacture of a medicament for treatment and/or prevention of chronic acitve hepatitis, also called chronic aggressive hepatitis. It is a continuing inflammation of the liver that damages the liver cells. Causes of chronic active hepatitis include viral infection, drug reaction/ingestion, metabolic disorders, or autoimmune diseases. There may also be no apparent cause. The disease is characterized by necrosis or death of liver cells, active inflammation, and fibrosis that may lead to liver failure, cirrhosis, and death. The incidence is 1 out of 10,000 people. Risk factors are autoimmune diseases, previous infection with hepatitis C, or a positive hepatitis A or hepatitis B antigen for over 6 months.

Chronic persistent hepatitis is a mild, nonprogressive form of liver inflammation, and it is also a disease encompassed by the term “liver injury” according to the present invention.

According to the invention, IL-18 inhibitors are also used for the manufacture of a medicament for treatment and/or prevention of primary biliary cirrhosis (PBC). PBC is an inflammatory condition resulting from obstruction of the flow of bile in the liver, causing damage to the liver cells. Bile ducts within the liver become inflamed due to unknown cause. The disease affects middle-aged women most frequently. The onset of symptoms is gradual, with itching skin as the first symptom. Inflammation of the bile ducts within the liver occurs. Eventually, liver cirrhosis develops. The disease may be associated with autoimmune disorders. The incidence is 8 out of 100,000 people. The IL-18 inhibitors contemplated herein may also be used for the treatment of acute hepatic poisoning, e.g. caused by a high amount of paracetamol. Such an acute hepatic poisoning may be due to an overdose of paracetamol, be it accidental or on purpose.

As shown in the examples below, the inventors of the present invention have surprisingly found that IL-18 inhibitors are particularly effective in the prevention and treatment of fulminant hepatitis (acute hepatitis). Therefore, the invention preferably relates to the prevention and/or or treatment of fulminant hepatitis.

According to the present invention, the inhibitor of IL-18 is selected from inhibitors of caspase-1 (ICE), antibodies directed against IL-18, antibodies directed against any of the IL-18 receptor subunits, inhibitors of the IL-18 signalling pathway, antagonists of IL-18 which compete with IL-18 and block the IL-18 receptor, and IL-18 binding proteins, isofomns, muteins, fused proteins, functional derivatives, active fractions or circularly permutated derivatives thereof having the same activity.

The term “IL-18 binding proteins” is used herein synonymously with “IL18BP”. It comprises IL-18 binding proteins as defined in WO 99/09063 or in Novick et al., 1999, including splice variants and/or isoforms of IL-18 binding proteins, as defined in Kim et al., 2000. In particular, human isoforms a and c of IL-18BP are useful in accordance with the presence invention. The proteins useful according to the present invention may be glycosylated or non-glycosylated, they may be derived from natural sources, such as urine, or they may preferably be produced recombinantly. Recombinant expression may be carried out in prokaryotic expression systems like E. coli, or in eukaryotic, and preferably in mammalian, expression systems.

As used herein the term muteins refers to analogs of an IL-18BP, or analogs of a viral IL-18BP, in which one or more of the amino acid residues of a natural IL-18BP or viral IL-18BP are replaced by different amino acid residues, or are deleted, or one or more amino acid residues are added to the natural sequence of an IL-18BP, or a viral IL-18BP, without changing considerably the activity of the resulting products as compared with the wild type IL-18BP or viral IL-18BP. These muteins are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable therefor.

Any such mutein preferably has a sequence of amino acids sufficiently duplicative of that of an IL-18BP, or sufficiently duplicative of a viral IL-18BP, such as to have substantially similar activity to IL-18BP. One activity of IL-18BP is its capability of binding IL-18. As long as the mutein has substantial binding activity to IL-18, it can be used in the purification of IL-18, such as by means of affinity chromatography, and thus can be considered to have substantially similar activity to IL-18BP. Thus, it can be determined whether any given mutein has substantially the same activity as IL-18BP by means of routine experimentation comprising subjecting such a mutein, e.g., to a simple sandwich competition assay to determine whether or not it binds to an appropriately labeled IL-18, such as radioimmunoassay or ELISA assay.

Muteins of IL-18BP polypeptides or muteins of viral IL-18BPs, which can be used in accordance with the present invention, or nucleic acid coding therefor, include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.

Preferred changes for muteins in accordance with the present invention are what are known as conservative substitutions. Conservative amino acid substitutions of IL-18BP polypeptides or proteins or viral IL-18BPs, may include synonymous amino acids within a group which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule (Grantham, 1974). It is clear that insertions and deletions of amino acids may also be made in the above-defined sequences without altering their function, particularly if the insertions or deletions only involve a few amino acids, e.g., under thirty, and preferably under ten, and do not remove or displace amino acids which are critical to a functional conformation, e.g., cysteine residues. Proteins and muteins produced by such deletions and/or insertions come within the purview of the present invention.

Preferably, the synonymous amino acid groups are those defined in Table i. More preferably, the synonymous amino acid groups are those defined in Table II; and most preferably the synonymous amino acid groups are those defined in Table III.

TABLE I

Preferred Groups of Synonymous Amino Acids

<td> Amino Acid</td><td> Synonymous Group</td>

<td> Ser</td><td> Ser, Thr, Gly, Asn</td>

<td> Arg</td><td> Arg, Gin, Lys, Glu, His</td>

<td> Leu</td><td> lie, Phe, Tyr, Met, Val, Leu</td>

<td> Pro</td><td> Gly, Ala, Thr, Pro</td>

<td> Thr</td><td> Pro, Ser, Ala, Gly, His, Gin, Thr</td>

<td> Ala</td><td> Gly, Thr, Pro, Ala</td>

<td> Val</td><td> Met, Tyr, Phe, lie, Leu, Val</td>

<td> Gly</td><td> Ala, Thr, Pro, Ser, Gly</td>

<td> lie</td><td> Met, Tyr, Phe, Val, Leu, lie</td>

<td> Phe</td><td> Trp, Met, Tyr, lie, Val, Leu, Phe</td>

<td> Tyr</td><td> Trp, Met, Phe, lie, Val, Leu, Tyr</td>

<td> Cys</td><td> Ser, Thr, Cys</td>

<td> His</td><td> Glu, Lys, Gin, Thr, Arg, His</td>

<td> Gin</td><td> Glu, Lys, Asn, His, Thr, Arg, Gin</td>

<td> Asn</td><td> Gin, Asp, Ser, Asn</td>

<td> Lys</td><td> Glu, Gin, His, Arg, Lys</td>

<td> Asp</td><td> Glu, Asn, Asp</td>

<td> Glu</td><td> Asp, Lys, Asn, Gin, His, Arg, Glu</td>

<td> Met</td><td> Phe, lie, Val, Leu, Met</td>

<td> Trp</td><td> Trp</td>

<td> TABLE II</td><td></td>

<td colspan="2"> More Preferred Groups of Synonymous Amino Acids</td>

<td> Amino Acid</td><td> Synonymous Group</td>

<td> Ser</td><td> Ser</td>

<td> Arg</td><td> His, Lys, Arg</td>

<td> Leu</td><td> Leu, lie, Phe, Met</td>

<td> Pro</td><td> Ala, Pro</td>

<td> Thr</td><td> Thr</td>

<td> Ala</td><td> Pro, Ala</td>

<td> Val</td><td> Val, Met, lie</td>

<td> Gly</td><td> Gly</td>

<td> lie</td><td> lie, Met, Phe, Val, Leu</td>

<td> Phe</td><td> Met, Tyr, lie, Leu, Phe</td>

<td> Tyr</td><td> Phe, Tyr</td>

<td> Cys</td><td> Cys, Ser</td>

<td> His</td><td> His, Gin, Arg</td>

<td> Gin</td><td> Glu, Gin, His</td>

<td> Asn</td><td> Asp, Asn</td>

<td> Lys</td><td> Lys, Arg</td>

<td> Asp</td><td> Asp, Asn</td>

<td> Glu</td><td> Glu, Gin</td>

<td> Met</td><td> Met, Phe, lie, Val, Leu</td>

<td> Trp</td><td> Trp</td>

<td> TABLE III</td><td></td>

<td colspan="2"> Most Preferred Groups of Synonymous Amino Acids</td>

<td> Amino Acid</td><td> Synonymous Group</td>

<td> Ser</td><td> Ser</td>

<td> Arg</td><td> Arg</td>

<td> Leu</td><td> Leu, Ile, Met</td>

<td> Pro</td><td> Pro</td>

<td> Thr</td><td> Thr</td>

<td> Ala</td><td> Ala</td>

<td> Val</td><td> Val</td>

<td> Gly</td><td> Gly</td>

<td> lie</td><td> lie, Met, Leu</td>

<td> Phe</td><td> Phe</td>

<td> Tyr</td><td> Tyr</td>

<td> Cys</td><td> Cys, Ser</td>

<td> His</td><td> His</td>

<td> Gin</td><td> Gin</td>

<td> Asn</td><td> Asn</td>

<td> Lys</td><td> Lys</td>

<td> Asp</td><td> Asp</td>

<td> Glu</td><td> Glu</td>

<td> Met</td><td> Met, lie, Leu</td>

<td> Trp</td><td> Met</td>

Examples of production of amino acid substitutions in proteins which can be used for obtaining muteins of IL-18BP polypeptides or proteins, or muteins of viral IL-18BPs, for use in the present invention include any known method steps, such as presented in US patents RE 33,653, 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 proteins presented in US patent No. 4,904,584 (Shaw et al).

The term fused protein refers to a polypeptide comprising an IL-18BP, or a viral IL-18BP, or a mutein or fragment thereof, fused with another protein, which, e.g., has an extended residence time in body fluids. An IL-18BP or a viral IL-18BP, may thus be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or a fragment thereof.

Functional derivatives as used herein cover derivatives of IL-18BPs or a viral IL-18BP, and their muteins and fused proteins, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the protein which is substantially similar to the activity of IL-18BP, or viral IL-18BPs, and do not confer toxic properties on compositions containing it.

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

As active fractions of an IL-18BP, or a viral IL-18BP, muteins and fused proteins, the present invention covers any fragment or precursors of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, e.g., sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has substantially similar activity to IL-18BP.

In a further preferred embodiment of the invention, the inhibitor of IL-18 is an IL18 antibody. Anti-IL-18 antibodies may be polyclonal or monoclonal, chimeric, humanised, or even fully human. Recombinant antibodies and fragments thereof are characterised by high affinity binding to IL-18 in vivo and low toxicity. The antibodies which can be used in the invention are characterised by their ability to treat patients for a period sufficient to have good to excellent regression or alleviation of the pathogenic condition or any symptom or group of symptoms related to a pathogenic condition, and a low toxicity.

Neutralising antibodies are readily raised in animals such as rabbits, goat or mice by immunisation with IL-18. Immunised mice are particularly useful for providing sources of B cells for the manufacture of hybridomas, which in turn are cultured to produce large quantities of anti-IL-18 monoclonal antibodies.

Chimeric antibodies are immunoglobulin molecules characterised by two or more segments or portions derived from different animal species. Generally, the variable region of the chimeric antibody is derived from a non-human mammalian antibody, such as murine monoclonal antibody, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. Preferably, both regions and the combination have low immunogenicity as routinely determined (Elliott et al., 1994). Humanised antibodies are immunoglobulin molecules created by genetic engineering techniques in which the murine constant regions are replaced with human counterparts while retaining the murine antigen binding regions. The resulting mouse-human chimeric antibody preferably have reduced immunogenicity and improved pharmacokinetics in humans (Knight et al., 1993).

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

In yet a further preferred embodiment, the IL-18 antibody is fully human. The technology for producing human antibodies is described in detail e.g. in WO00/76310, WO99/53049, US 6,162,963 or AU5336100. Fully human antibodies are preferably recombinant antibodies, produced in transgenic animals, e.g. xenomice, comprising all or parts of functional human lg loci.

In a highly preferred embodiment of the present invention, the inhibitor of IL-18 is a IL-18BP, or an isoform, a mutein, fused protein, functional derivative, active fraction or circularly permutated derivative thereof. These isoforms, muteins, fused proteins or functional derivatives retain the biological activity of IL-18BP, in particular the binding to IL-18, and preferably have essentially at least an activity similar to IL-18BP. Ideally, such proteins have a biological activity which is even increased in comparison to unmodified IL-18BP. Preferred active fractions have an activity which is better than the activity of IL18BP, or which have further advantages, like a better stability or a 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/isoforms can be taken from W099/09063 or from Novick et al., 1999, as well as from Kim et al., 2000.

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

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

In a further preferred embodiment of the invention, the inhibitor of IL-18 is a fused protein comprising all or part of an IL-18 binding protein, which is fused to all or part of an immunoglobulin. The person skilled in the art will understand that the resulting fusion protein retains the biological activity of IL-18BP, in particular the binding to IL-18. The fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length. Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-GlyGln-Phe-Met introduced between the IL-18BP sequence and the immunoglobulin sequence. The resulting fusion protein has improved properties, such as an extended residence time in body fluids (half-life), increased specific activity, increased expression level, or the purification ofthe fusion protein is facilitated.

In a preferred embodiment, IL-18BP is fused to the constant region of an Ig molecule. Preferably, it is fused to heavy chain regions, like the CH2 and CH3 domains of human lgG1, for example. The generation of specific fusion proteins comprising IL18BP and a portion of an immunoglobulin are described in example 11 of WP99/09063, for example. Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms lgG₂ or lgG₄, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomeric or multimeric, hetero- or homomultimeric.

Interferons are predominantly known for inhibitory effects on viral replication and cellular proliferation. Interferon-γ, for example, plays an important role in promoting immune and inflammatory responses. Interferon β (IFN-β, an interferon type I), is said to play an anti-inflammatory role. Studies published by Triantaphyllopoulos et al (1999) indicated that IFN-β has a beneficial effect in the therapy of rheumatoid arthritis, as shown in a mouse model of the disease, the collagen-induced arthritis (CIA) model. This beneficial effect of IFN-β was confirmed in the examples below.

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

Interferons may also be conjugated to polymers in order to improve the stability of the proteins. A conjugate between Interferon β and the polyol Polyethlyenglycol (PEG) has been described in WO99/55377, for instance.

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

The inhibitor of IL-18 production and/or action is preferably used simultaneously, sequentially, or separately with the interferon.

In yet a further embodiment of the invention, an inhibitor of IL-18 is used in combination with a TNF antagonist. TNF antagonists exert their activity in several ways. First, antagonists can bind to or sequester the TNF molecule itself with sufficient affinity and specificity to partially or substantially neutralise the TNF epitope or epitopes responsible for TNF receptor binding (hereinafter termed “sequestering antagonists”). A sequestering antagonist may be, for example, an antibody directed against TNF.

Alternatively, TNF antagonists can inhibit the TNF signalling pathway activated by the cell surface receptor after TNF binding (hereinafter termed “signalling antagonists”). TNF antagonists are easily identified and evaluated by routine screening of candidates for their effect on the activity of native TNF on susceptible cell lines in vitro, for example human B cells, in which TNF causes proliferation and immunoglobulin secretion. The assay contains TNF formulation at varying dilutions of candidate antagonist, e.g. from 0,1 to 100 times the molar amount of TNF used in the assay, and controls with no TNF or only antagonist (Tucci et al., 1992).

Sequestering antagonists are the preferred TNF antagonists to be used according to the present invention. Amongst sequestering antagonists, those polypeptides that bind TNF with high affinity and possess low immunogenicity are preferred. Soluble TNF receptor molecules and neutralising antibodies to TNF are particularly preferred. For example, soluble TNF-RI and TNF-RII are useful in the present invention. Truncated forms of these receptors, comprising the extracellular domains of the receptors or functional portions thereof, are more particularly preferred antagonists according to the present invention. Truncated soluble TNF type-l and type-ll receptors are described in EP914431, for example.

Truncated forms of the TNF receptors are soluble and have been detected in urine and serum as 30 kDa and 40 kDa TNF inhibitory binding proteins, which are called TBPI and TBPII, respectively (Engelmann et al., 1990). The simultaneous, sequential, or separate use of the IL-18 inhibitor with the TNF antagonist and /or an Interferon is preferred, according to the invention.

According to the invention, TBP I and TBPII are preferred TNF antagonists to be used in combination with an IL-18 inhibitor. Derivatives, fragments, regions and biologically active portions of the receptor molecules functionally resemble the receptor molecules that can also be used in the present invention. Such biologically active equivalent or derivative of the receptor molecule refers to the portion of the polypeptide, or of the sequence encoding the receptor molecule, that is of sufficient size and able to bind TNF with such an affinity that the interaction with the membrane-bound TNF receptor is inhibited or blocked.

In a further preferred embodiment, human soluble TNF-RI (TBPI) is the TNF antagonist to be used according to the invention. The natural and recombinant soluble TNF receptor molecules and methods of their production have been described in the European Patents EP 308 378, EP 398 327 and EP 433 900.

The IL-18 inhibitor can be used simultaneously, sequentially or separately with the TNF inhibitor. Advantageously, a combination of an IL-18 antibody or antiserum and a soluble receptor of TNF, having TNF inhibiting activity, is used.

In a further preferred embodiment of the invention, the medicament further comprises a COX-inhibitor, preferably a COX-2 inhibitor. COX inhibitors are known in the art. Specific COX-2 inhibitors are disclosed in WO 01/00229, for example.

The invention further relates to the use of a combination of IL-18 inhibitors and/or interferons and/or TNF antagonists and/or COX-2 inhibitors. The combination is suitable for the treatment and/or prevention of liver injury. The active components may be used simultaneously, sequentially, or separately.

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

The invention further relates to the use of an expression vector comprising the coding sequence of an inhibitor of IL-18 in the preparation of a medicament for the treatment of liver injury. A gene therapeutical approach is thus used for treating and/or preventing the disease. Advantageously, the expression of the IL-18 inhibitor will then be in situ, thus efficiently blocking IL-18 directly in the tissue(s) or cells affected by the disease.

The use of a vector for inducing and/or enhancing the endogenous production of an inhibitor of IL-18 in a cell normally silent for expression of an IL-18 inhibitor, or which expresses amounts of the inhibitor which are not sufficient, are also contemplated according to the invention. The vector may comprise regulatory sequences functional in the cells desired to express the inhibitor or IL-18. Such regulatory sequences may be promoters or enhancers, for example. The regulatory sequence may then be introduced into the right locus of the genome by homologous recombination, thus operably linking the regulatory sequence with the gene, the expression of which is required to be induced or enhanced. The technology is usually referred to as “endogenous gene activation” (EGA), and it is described e.g. in WO 91/09955.

It will be understood by the person skilled in the art that it is also possible to shut down IL-18 expression using the same technique, i.e. by introducing a negative regulation element, like e.g. a silencing element, into the gene locus of IL-18, thus leading to down-regulation or prevention of IL-18 expression. The person skilled in the art will understand that such down-regulation or silencing of IL-18 expression has the same effect as the use of an IL-18 inhibitor in order to prevent and/or treat disease.

The invention further relates to the use of a cell that has been genetically modified to produce an inhibitor of IL-18 in the manufacture of a medicament for the treatment and/or prevention of liver injury.

The bioavailability of the active protein(s) according to the invention can also be ameliorated by using conjugation procedures which increase the half-life of the molecule in the human body, for example linking the molecule to polyethylenglycol, as described in the PCT Patent Application WO 92/13095.

The therapeutically effective amounts of the active protein(s) will be a function of many variables, including the type of antagonist, the affinity of the antagonist for IL-18, any residual cytotoxic activity exhibited by the antagonists, the route of administration, the clinical condition of the patient (including the desirability of maintaining a non-toxic level of endogenous IL-18 activity

A therapeutically effective amount is such that when administered, the IL-18 inhibitor results in inhibition of the biological activity of IL-18. The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including IL-18 inhibitor pharmacokinetic properties, the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired. Adjustment and manipulation of established dosage ranges are well within the ability of those skilled in the art, as well as in vitro and in vivo methods of determining the inhibition of IL-18 in an individual.

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

The route of administration which is preferred according to the invention is administration by subcutaneous route. Intramuscular administration is further preferred according to the invention.

In further preferred embodiments, the inhibitor of IL-18 is administered daily or every other day.

The daily doses are usually given in divided doses or in sustained release form effective to obtain the desired results. Second or subsequent administrations can be performed at a dosage which is the same, less than or greater than the initial or previous dose administered to the individual. A second or subsequent administration can be administered during or prior to onset of the disease.

According to the invention, the IL-18 inhibitor can be administered prophylactically or therapeutically to an individual prior to, simultaneously or sequentially with other therapeutic regimens or agents (e.g. multiple drug regimens), in a therapeutically effective amount, in particular with an interferon and/or a TNF antagonist and/or a COX inhibitor. Active agents that are administered simultaneously with other therapeutic agents can be administered in the same or different compositions.

The invention further relates to a method for the preparation of a pharmaceutical composition comprising admixing an effective amount of an IL-18 inhibitor and/or an interferon and/or a TNF antagonist and/or a COX inhibitor with a pharmaceutically acceptable carrier.

Having now described the invention, it will be more readily understood by reference to the following examples that are provided by way of illustration and are not intended to be limiting ofthe present invention.

EXAMPLES

PART I: Examples 1 to 8, relating to the use of IL-18 inhibitors in liver injury

EXAMPLE 1: Production of IL-18BP-His taq

Purified recombinant human IL18BP containing a his-tag (r-hlL-18BP-His tag) was produced in CHO cells. The production of recombinant proteins in eukaryotic cells is known by the person skilled in the art. Well known methods are available for constructing appropriate vectors, carrying DNA that codes for IL-18BP and suitable for transfection of eukaryotic cells in order to produce recombinant IL-18BP. For expression in cells, the DNA coding for IL-18BP (see, e.g. (Novick et al., 1999) is cut out and inserted into expression vectors suitable for transfection of cells. Alternatively, such DNA can be prepared by PCR with suitable sense and antisense primers. The resulting cDNA constructs are then inserted into appropriately constructed eukaryotic expression vectors by techniques well known in the art (Manaitis, 1982). The recombinant protein was purified to over 95% purity and found to be biologically active in-vitro and in-vivo with a high affinity to its ligand.

EXAMPLE 2: Protective effect of IL18BP against endotoxin-induced death in the murine model

A murine model was used to test whether IL18BP, an inhibitor of IL-18, would protect mice against a high dose of lipopolysaccharides (LPS). LPS elicits acute liver injury, followed by rapid death of the mice.

mg/kg of recombinant, human IL-18BP (rhlL18BPhis) containing a his-tag (resulting from recombinant production ofthe protein) was injected intraperitoneally (i.p).

into C57BL/6 mice. 1 h later, 60 mg/kg LPS were injected (lethal doses). The survival of mice was compared to a group of animals who received LPS alone (no IL18BP).

Five out of 7 mice injected with rhlL-18BP-his survived the LPS injection in contrast to the control mice, in which all animals died within 3 days.

Blood samples were taken 5 h after the LPS injection in the absence or presence of increasing doses of rhlL-18BP-his and analyzed by ELISA for circulating IFN-γ (Fig. 1). 0,4 and 4 mg/kg rhlL-18BP induced a 2 fold reduction in serum IFN-γ. This inhibition was lost at lower doses of rhlL-18BP (0,004 and 0,4 mg/kg).

EXAMPLE 3: IL18BP has a protective effect against liver injury in a murine model of disease

A mouse model of fulminant hepatitis was used to test the effect of IL18BP. Mice develop acute liver injury when subjected to a sequential administration of Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS).

Mice were injected with increasing doses of rhlL-18BP-his (4; 0,4 ; 0,04; 0 mg/kg) at various times (1 h, 20 min, simultaneously) before the injection of LPS in C57BL/6 P. acnes sensitized mice. When rhlL-18BP-his was given i.p. at the same time as LPS, none of the mice survived and levels of circulating IFN-γ and TNF-α were unaffected. Surprisingly, rhlL-18BP (4 and 0.4 mg/kg) induced a 70% reduction of circulating Alanine aminotransferase (a marker of liver injury), as shown in Fig. 2.

In addition to this, the survival of mice was monitored (Fig. 3): When rhlL-18BP was given i.p. 20 minutes before LPS, the two highest doses of 11-18BP (4 and 0,4 mg/kg) delayed the death of the mice by 10 h as compared to the control mice who received NaCl instead of IL-18BP.

The results of the measurement of serum IFN-γ levels are shown in Fig. 4. rhlL18BP (4 mg/kg) inhibited 90 % of circulating IFN-γ levels and 80% of circulating Alanine aminotransferase (not shown).

When rhlL-18BP-his was given 1 h before LPS, survival curves and levels of circulating IFN-γ were similar to what was observed when rhlL-18BP-his was given 20 min before LPS, but levels of circulating Alanine aminotransferase were unaffected (not shown).

In addition to this, murine liver tissue was analyzed by hematoxilin-eosine staining, as well as by tunnel microscopy. The livers of mice, in which severe hepatitis had been induced before, showed severe necrosis as compared to normal liver tissue. In contrast to this, liver tissue of mice treated with IL-18BP showed significantly less necrotic foci than untreated mice.

EXAMPLE 4: Anti-IL-18 antibodies protect against lethal endotoxemia

In order to evaluate, whether blockade of IL-18 with IL-18 antibodies would protect mice against lethal doses of bacterial lipopolysaccharides, C57BL/6J mice were first injected with a neutralizing rabbit anti-mouse IL-18 antibody (polyclonal) or normal rabbit serum (NDS) as a control. 30 min after antibody treatment, a lethal dosis of LPS derived either from E. coli (Fig. 5 A) or S. thyphimurium (Fig. 5 B) was injected. Experiments involved 10-12 mice/group, and were performed twice on two different occasions.

As shown in Fig. 5 A, treatment of the mice with the anti-IL-18 antiserum prevented the mortality induced by 40 mg/kg E. coli LPS. 100 % of the mice survived after anti-IL-18 treatment vs. 10 % survival in mice treated with normal rabbit serum (p &lt; 0,005).

Fig. 5 B shows that the antibody treated mice were also protected against S. typhimurium lethal effects (50 % vs. 0 % survival; p &lt; 0,05).

EXAMPLE 5: Blockade of IL-18 and TNF-α protects mice from ConA- and PEAinduced hepatotoxicity

Two experimental models of hepatotoxicity were used to evaluate the role of IL18 and TNF-α in liver injury. Injection of Concanavalin A (Con A) and Pseudomonas aeruginosa (PEA) into mice both induce liver injury, and are models of T cell mediated hepatitis.

C57BL/6J mice were pretreated with an anti-IL-18 antiserum or a soluble TNF-a receptor, TNFsRp55. Serum Alanine aminotransferase (ALT) levels were measured as indicators of hepatic injury (Fig. 6).

As shown in Fig. 6 A, both IL-18 antiserum and soluble TNF-receptors significantly reduced ConA-induced serum ALT levels, as compared to a control injection of the vehicle alone (pyrogen free saline). A co-adminstration of soluble TNF receptor and IL-18 antiserum led to a complete inhibition of Con-A induced liver injury.

As shown in Fig. 6 B, in PEA-injected mice, neutralization of either TNF-a inhibitors or anti-IL-18 antibodies resulted in 93 % and 83 % inhibition of serum ALT levels, respectively. A combined blockade of both resulted in 99 % protection.

EXAMPLE 6: Plasma levels of IL-18-bindinq protein are elevated in patients with chronic liver disease

IL-18 BP plasma levels were measured in 133 patients with chronic liver disease (CLD) of varying etiologies and 31 healthy controls by a specific ELISA, using an IL18BP monoclonal antibody.

Plasma levels of IL-18 BP were significantly higher in CLD patients (12.91 ± 0.89 ng/ml; average ± SEM) than in healthy subjects (4.96 ± 0.43 ng/ml, p&lt;0.001). Cirrhotic patients had signifcantly higher levels than patients with non-cirrhotic CLD (19.23 ± 1.28 ng/ml, n=67, vs. 6.49 ± 0.51 ng/ml, n=66, p&lt;0.001). Patients with stage B ofthe ChildPugh classification had higher levels of IL-18 BP than those with stage A (22.48 ± 2.44 ng/ml vs. 9.57 ± 1.25 ng/ml, p&lt;0.001). However, there was no significant difference between Child B and C (22.48 ± 2.44 ng/ml vs. 20.62 ± 4.75 ng/ml, p=0.7). Plasma levels of IL-18 BP correlated positively with GOT, bilirubin and erythrocyte sedimentation rate. Negative correlation was found with prothrombin time.

In conclusion, the results show that IL-18 BP plasma levels are elevated in CLD and correlate with the severity of disease independent of the etiology of disease. Although an endogenous antagonist ofthe proinflammatory IL-18, increased levels of IL30

BP seem not to be sufficient to counteract the overwhelming proinflammatory mediators in CLD.

EXAMPLE 7: Inhibition of alcoholic hepatitis bv IL-18BP

Four groups of rats (5 per group) are fed ethanol and a diet containing corn oil by intragastric infusion for 4 weeks. Dextrose isocalorically replaces ethanol in control rats. The rats are injected daily with mouse IL-18BP (1 mg/kg), or saline. Pathological analysis is performed on liver sections and measurements of liver enzymes in serum, TNF-α, Fas ligand and IFN-γ are taken. Necroinflammatory injury and expression of liver enzymes, TNF-α, Fas ligand, and IFN-γ are seen in the ethanol-fed rats that were injected with saline.

Rats injected with mouse IL-18BP are protected from necroinflammatory injury and the levels of liver enzymes, TNF-α, Fas ligand and IFN-γ are significantly reduced (&gt;90%).

EXAMPLE 8: Inhibition of Concanavalin A-induced hepatitis bv IL-18BP

Balb/c mice are injected with 12 mg/kg Concanavalin A (Con A) with or without injection of murine IL-18BP (1 mg/kg), 2 h prior to Con A administration and then daily. Liver damage is evaluated by determining serum levels of liver enzymes, TNF-α, Fas ligand and IFN-γ. Hepatic histopathology is compared to mice treated with Concanavalin A only.

Pretreatment with IL-18BP significantly reduces serum levels of liver enzymes and TNF -a with no evidence of inflammation in histopathologic examination compared to control mice treated with Con A.

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