UA76416C2 - Use of il-18 inhibitor for preparing medicine intended for treating and/or preventing destruction of cartilage and use of il-18 inhibitor for preparing medicine intended for treating and/or preventing psoriatic arthritis - Google Patents

Abstract

The invention relates to the use of IL-18 inhibitor for preparing the medicine intended for treating and/or preventing the destruction of the cartilage and the use of the expressing vector comprising the coding sequence of IL-18 inhibitor for this purpose. The invention also relates to the use of IL-18 inhibitor for preparing the medicine intended for treating and/or preventing the psoriatic arthritis.

Description

Description of the invention

This invention relates to the therapeutic use of II-18 inhibitors in some pathological conditions.

More specifically, the invention relates to the treatment and/or prevention of arthritis, to the treatment and/or prevention of liver diseases, and to the treatment and/or prevention of inflammatory bowel disease (IBD).

In 1989, endotoxin-induced serum activity was described, which induces interferon-y (IEM-y), obtained from cells of the spleen of a mouse (Misailii ei ai!., 1996). Such serum activity does not act as a direct inducer of IEM, but as a co-stimulator together with 1/-2 or mitogens. When trying to isolate the activity in its pure form from the postendotoxin mouse serum, a clearly homogeneous protein of 50-55 kDa was found. Since other cytokines can act as co-stimulators for the production of IEM-y, the ineffectiveness of neutralizing antibodies to 1-1, 1I--4, 1-5,

I/-6 or TME to neutralize the serum activity suggested that it is a special factor.

In 1995, the same scientists showed that an endoxin-induced co-stimulator for the production of IRM- is present in extracts of the liver of mice conditioned in advance with R. aspes (MomiskKei a!., 1992). In this model, a population of hepatic macrophages (Kupffer cells) grows, and in such mice a low dose of bacterial lipopolysaccharide (IBS5), which is not lethal to mice not conditioned in advance, becomes lethal. The factor named

IEM-y-inducing factor (ISIR), and later called interleukin-18 (1-18), isolated in pure form from 1200 g of liver of mice treated with R. aspez. Degenerate oligonucleotides obtained from the amino acid sequences of purified 1-18 were used to clone the cDNA of mouse II-18 (Momisk et al., 1992). 11-18 is a protein of 18-19 kDa with 157 amino acids, which has no obvious similarity with any peptide in the database. Matrix RNA 1I-18 and interleukin-12 (1-12) are well detected in Kupffer cells and activated macrophages. Recombinant 1І/-18 induces IERM-gamma more strongly than 1ІІ/-12, apparently due to a separate cascade (Momisk ei ai., 1992). Like endotoxin-induced serum activity, I/-18 itself does not induce IEM, but acts primarily as a co-stimulator with mitogens or 1I-2. II/-18 enhances cell proliferation, apparently through

I/-2-dependent cascade, and enhances the production of cytokines TI and migo and reveals synergism when combined with 1-2 and 9) in the sense of increased production of IRM-u (MaiighemuzKivia!., 1990).

It has been shown that neutralizing antibodies to murine III-18 prevent the lethality of a low dose of I! P5 for mice previously conditioned with R. aspev. There are other reports on the importance of IEM as a mediator (se) of ARDS lethality in preconditioned mice. For example, neutralizing anti-IEM-y antibodies protect mice from Schwartzman-like shock (Rapitsg?i et al., 1998), and galactosamine-treated mice deficient in IEM-y receptors were resistant to ARDS death, which is called (Wut, 1990). Therefore, it did not come as a surprise that neutralizing antibodies to murine II-18 protect mice, pre-conditioned by R. "o aspez, from lethal I P5 (Momisk, ei., 1992). Treatment with anti-mouse 1-18 also protected surviving mice from severe hepatic cytotoxicity. in.

After the mouse form was cloned, in 1996 the cDNA sequence for

I--18 people (OKatiga ei a/i., 1995). Recombinant human II-18 exhibits natural II-18 activity (OKatiga ei aYi., 1995). Recombinant human 1-18 does not show direct IEM-y-inducing activity in relation to human T-cells, but acts as a co-stimulator of the production of IEM-y and other cytokines of T-helper cells-1 (TR1) (Okatiga 70 eka, 1995) . To date, 1-18 is considered primarily as a co-stimulator of the production of cytokines TA1 not) with (IEM-u, 1--2 and the colony-stimulating factor of granulocytes-macrophages) (Igaki, 1978), as well as a co-stimulator "with EAz-ligand-mediated cytotoxicity mouse clones of natural killer cells (Momisk, 1989).

By cloning II-18 from affected tissues and studying the expression of the II-18 gene, a close connection of this cytokine with autoimmune disease was revealed. A mouse with non-obese diabetes (MY) spontaneously develops autoimmune insulin and diabetes that can be accelerated and synchronized by a single injection of cyclophosphamide. In the early stages of insulitis in the pancreas of MOO mice by reverse PCR

Ge) transcription showed the presence of mRNA 1-18. Levels of mRNA 1-18 rapidly increase after treatment with cyclophosphamide and precede the increase in the level of IEM mRNA and then the development of diabetes. It is interesting that such kinetics resemble the kinetics of mRNA 1-12-p40, and a close correlation of individual mRNA levels is obtained. - 70 Cloning of cDNA 1II-18 from pancreatic RNA followed by sequencing shows identity with

Ф sequence 1I/-18, cloned from Kupffer cells and pre-activated IP mimo macrophages. Also, macrophages of MOJ mice responded to cyclophosphamide by expression of the II-18 gene, while macrophages of parallel treated Vair/s mice did not respond. Therefore, the expression of II-18 in the autoimmune MOO-mouse is regulated abnormally and is closely related to the development of diabetes (Momisk ei ai!., 1992). 59 I--18 may play a role in immunoregulation or inflammation by increasing functional activity

GF) of Eaz-ligand on TA1 cells (Sophii ei ai., 1997). I/-18 is also expressed in the cortical substance of the adrenal 7 gland and, therefore, may represent a secreted neuromodulator, playing an important role in the organization of the immune system after severe stress (Spagheg, 1986).

Ip mimo I/-18 is formed by cleavage of pro-II-18, and it turns out that its endogenous activity is responsible for the production of IEM with lethality mediated by R. aspez and I RB5. Mature 1-18 is produced from its precursor with the help of II-1p-converting enzyme (II-beta-converting enzyme, ISE, caspase-1).

Receptor II -18 consists of at least two components acting together during ligand binding. High-65 and low-affinity IIII-18 binding sites were found in T-cells stimulated with murine 1-12 (Movzpitayu et al., 1998), which suggests a multi-chain receptor complex. So far, two receptor subunits have been identified, and both belong to the 1ІІ/-1 family of receptors (Ragpei et al., 1996). Signal transduction

I--18 is involved in the activation of ME-KV (bibopaiyoegzai, 1997).

Some 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 formed either by alternative splicing of pre-mRNA, common for a cell surface receptor, or by proteolytic cleavage of a cell surface receptor. Such soluble receptors have been described, including, among others, soluble receptors 1-6 and IRM-u (Makatiga et al., 1989), TME (Ogo et al., 1996; Epdietapp et al., 1989), 1-1 1-4 (Doyp, 1986), IRM-o/r (Migizpita apa Madaia, 1990) and others. It turns out that one 70 cytokine-binding protein called osteoprotegerin (OROS, also known as osteoclast inhibitory factor -

OSIR), a member of the TMEK/Raz family, is the first example of a soluble receptor that exists only as a secreted protein (Apadegsop, 1997; VoiIop, 1980).

Recently, a soluble protein with a high affinity for II-18 was isolated from human urine, and human and mouse cDNAs were described (Momisk ei a!., 1999; UMO 99/09063). The protein is named II-18-binding protein (II-188P).

I-18BP is not an extracellular domain of one of the known III/-18 receptors, but a secreted naturally circulating protein. It belongs to a new family of secreted proteins. The family also includes some proteins encoded by poxviruses with high homology to II/-18BP (Momisk et al., 1999). And -18BP is constitutively expressed in the spleen, belongs to the superfamily of immunoglobulins. It has limited homology with the receptor

I--1 type II. Its gene is localized on human chromosome 1413, and the exon encoding the transmembrane domain is 8.3 kb in the genomic sequence. not detected (Momisk ei ai., 1999).

Four human II -18BP isoforms and two mouse isoforms resulting from mRNA splicing were identified in different cDNA libraries and expressed, purified and tested for binding and neutralization of biological activity

I/-18 (Kigp eyi ai., 2000). Isoform 1ІІ/-18В8Р. human (II -188Ra) showed the highest affinity to 1-18 with a fast "on" (harium op-gaie) and a slow "off" (viom/oi-gaie) and a dissociation constant (K(a)) of 399 pMm. Ha 1 -18VRse shares the common Id-domain with II-18VRa, except for 29 C-terminal amino acids; K(a) I-- 18V8Ps is 10 times less (2.94NM). However, II-18ВРа and I/-18В8Рс neutralize I/-18 29595 at a molar excess of one or the other. I/-188Рб and /-188Ра isoforms lose the complete Id-domain and lose the ability to bind or neutralize I/-18. Mouse isoforms 1І/-18ВРс and ІІ-188Ра, possessing identity with the Id-domain, also neutralize 295956 mouse І/-18 at a molar excess of both. However, murine II-188vRa, which Ge) shares a common C-terminal motif with human I/-188Ra, also neutralizes human I/-18. With the help of molecular modeling, a large excess of electrostatic and hydrophobic binding sites in the Id-domain of II-18BP was identified, which may be responsible for its high-affinity binding to the ligand (Kigp "I ei a!., 2000).

It has recently been suggested that interleukin III-18 is involved in the development of pathogenicity in chronic and inflammatory diseases, including endotoxin shock, hepatitis and autoimmune diabetes (Kapizhatiga apa h-

Okkatiga, 1998). Another indication of the positive role of I/-18 in the development of liver damage comes from experiments, the results of which were published by Tzytsi ei ai. (Tevytsi et al.,, 1999), where an elevated level of 1-18 was shown in a mouse model in lipopolysaccharide-induced acute liver damage. However, the mechanism of action of multifunctional factor II-18 in the development of liver damage has not yet been clarified.

Liver damage can have various causes. This can happen, for example, due to viral or bacterial infections, alcohol abuse, immune disorders or cancer. Viral hepatitises, caused, for example, by hepatitis B virus or hepatitis C virus, are poorly managed "» diseases that affect a large number of people around the world. The number of known hepatitis viruses is constantly growing. In addition to hepatitis B and C viruses, at least four others have been identified so far the viruses that cause viral hepatitis are called hepatitis A, O, E, and O. -I Alcoholic liver disease is another common disease associated with chronic alcohol use Immune hepatitis is a rare autoimmune disease that is poorly managed treated Damage

F of the liver also includes damage to the bile ducts. Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the destruction of intrahepatic bile ducts. tsu In some studies, liver damage in diseases such as alcoholic hepatitis, liver cirrhosis, viral hepatitis, and primary biliary cirrhosis has been shown to be associated with T helper cell-1 (TA1) responses. In one of the works, a new model of liver damage in mice was created by targeted delivery of ovalbumin-containing liposomes to the liver followed by adoptive transfer of ovalbumin-specific TA1 cells.

The combined treatment of mice with ovalbumin-containing liposomes and the carrier of T1 cells caused an increase in serum transaminase activity in parallel with an increase in IPM-y levels in the serum. In sharp contrast, transfer of ovalbumin-specific Tp2 cells resulted in increased serum I/-4 levels but did not cause liver damage. Liver damage was blocked by antibodies against IEM-y and by antibodies against TME-y tumor necrosis factor. Such results show that TI1 cells are the main effector cells in acute liver damage (Mizpitiga apa OpNia, 1999). In a number of other works 60, it is shown that spontaneous hepatitis is detected in mice with overexpression without any pathogen or any other stimulant (OKatoyo ei ai., 1998).

Another study related to TA1 reactions in primary biliary cirrhosis (PBC). PBC is an autoimmune disease of the liver characterized by destruction of the intrahepatic bile ducts. As a rule, it is believed that such damage to the bile ducts is caused by cellular immune mechanisms, in particular, with the participation of 65 T cells. Recently, it has been suggested that the relative strength of T1 and T2 reactions is an important factor in the pathophysiology of various autoimmune diseases. In this study, the balance of subsets in PBC was assessed by detecting cytokines specific for two subsets of T cells, i.e., IEM-y for ТН1 cells and ЙЙ-4 for cells

And. Cells positive for IEM-u-matrix RNA (mRNA) and 1--4-mRNA were counted in the liver sections of 18 patients with PBC and 35 control patients who had chronic active hepatitis C, extrahepatic biliary obstruction and a healthy liver, with using non-isotopic hybridization of IP 8 and immunohistochemistry.

Mononuclear cells that express IRM-»y and 1-4 mRNA in PBC were collected in the inflamed inlet tracts of the liver, but were rarely present in liver sections with extrahepatic biliary obstruction, alcoholic fibrosis, or healthy liver. The number of cells positive for IRM-y and I/-4 mRNA in the PBC of the liver was significantly higher than in the control liver (P "0.01). In addition, in PBC of the liver, the expression of 70. IEM-y mRNA was detected more often than the expression of 1-4, and the expression levels of IEM-y mRNA are more correlated with the degree of inflammatory activity at the entrances. Cells positive for IRM mRNA were detected mainly near the damaged bile ducts, which were surrounded by lymphoid aggregates. The results show that TNA1 cells are a more prominent subpopulation of T cells in lymphoid infiltrates in PBC (Nagaaa ei!ai!., 1997).

The pattern of cytokines upon recognition of viral antigens is also believed to have a profound effect on 72 the resolution of viral infections and viral clearance. In one of the works, it was investigated whether the cytokine imbalance, focused on the response of type TA2 cells, plays any role in chronic hepatitis B. Cytokine profiles of peripheral blood mononuclear cells associated with chronic hepatitis B were analyzed by RT-PCR.

After stimulation of the surface antigen of hepatitis B (NozAd), the expression of IBM-u, 1-2, 1-4 and 1-10 was detected

A190, 890, 4190 and 5090 patients, respectively. Among these cytokines, the expression of the cytokine TII in IEM was associated with high serum levels of AZT/AGI T (aspartate aminotransferase/alanine aminotransferase), which are typical markers of liver damage. It has not been shown that Tp2-type cytokines have a protective effect on hepatocytes. As a conclusion, the production of cytokine TI7 by IRM-y, HBvAd-reactive cells is associated with damage to hepatocytes in chronic hepatitis B (ee ei aiYi.,, 1999). There are reports of high levels

EAb-ligand and its receptor (СО95) in the liver of patients with hepatitis B (I tso ei aii., 1997). It is believed that c 29 EAz-ligand is one of the main cytotoxic factors leading to apoptosis of hepatocytes. Gee)

Another study identified factors associated with the development of liver damage in 30 hepatitis C/RNA (HCV/CMA)-positive chronic hepatitis patients.

Necroinflammatory and architectural damage was assessed using the Izpak scale. Activated stellate cells of the liver (HC) were visualized by immunohistochemistry for o-smooth muscle actin (azmMA) and quantified by the method of morphometry. NSM/KMA in plasma was assessed using the OT-PCR method. In order to investigate the type of immune response involved in the development of liver damage, IEM-y-positive cells (as the expression "

TA1-like reaction) was assessed by immunohistochemistry and quantified by morphometry. It was found that most of all NS were determined near areas of lobular necroinflammation or fibrous septa (from the litter. Parenchyma, "ZMA- and Sirius red-positive, significantly correlates with evaluations of necroinflammation and M architecture. IRM-y-positive cells were found around the entrance areas, associated with inflammatory infiltrates, which correlates well with architectural damage. Therefore, it was concluded that activation

NZO Its development of liver damage is associated with a Tp1-like reaction (Vagopi ei ai!., 1999). Similarly, in the case of hepatitis C, RAZ-ligand and its receptor were detected in the liver and serum of patients with hepatitis C (Nigataivy, 1994; OKa?gaki, 1996; Pio, 1998). It was found that cytokines YOU! and other markers YOU! associated with alcoholic hepatitis and liver cirrhosis.

Inflammatory stimuli and lipid peroxidation activate nuclear factor kV (ME-kV) and activate;" proinflammatory cytokines and chemokines. In one of the works, the relationship between pathological liver damage, endotoxicosis, lipid peroxidation and activation of ME-kV and imbalance between pro- and anti-inflammatory cytokines was evaluated. Rats (5 individuals in the group) were given ethanol and -I food containing saturated fat, palm oil, corn oil or fish oil by intragastric infusion. In control rats, ethanol was replaced with an isocaloric amount of dextrose. Pathology was analyzed and the levels of endotoxin, lipid peroxidation, ME-B and matrix RNA (mRNA) of pro-inflammatory cytokines (TME, g» P/-1-beta, IRMUu and 1-12), C-chemokines (regulated in activation expressed and secreted by normal shu 20 T-cells (KAMTE5), monocyte chemotactic protein |MSRI|-1, macrophage inflammatory protein |MIR'|-1a),

C-X-C-chemokines (cytokine-induced neutrophil chemoattractant IIMSII, MiIR-2, IR-10 and epithelial 4) neutrophil-activating protein (EMA/)-78) and anti-inflammatory cytokines (1-10, II -4 and 1Й/-13 ). Rats exhibiting necroinflammatory damage (fish oil and ethanol and corn oil and ethanol) show activation of ME-kV and increased expression of pro-inflammatory cytokines C-C and chemokines C-X-C. These groups also had the highest levels of endotoxin and lipid peroxidation. Levels of mRNA 1-10 and 1/-4 were lower in the group indicating inflammatory liver damage. Thus, the activation of ME-KV occurs in the presence of pro-inflammatory stimuli and leads to increased expression of pro-inflammatory cytokines Tp1 and chemokines (Mayii, 1999). In alcoholic liver diseases, the amount of RAZ-ligand and its receptor also increased, which once again suggests that TAI1 cytokines are involved in autoimmune processes induced in alcoholic hepatitis (Szaye, 1995; Taier, 1995). ., 1998; Rioge et al., 1999).

TM is also found to be a common metabolic pathway in the pathogenesis of alcohol-related liver necrosis. Elevated levels of TME in the liver and serum have been confirmed in animal models of alcoholic liver disease and in human alcoholic liver disease. Such deregulated TME metabolism has been postulated to play a role in many metabolic complications and liver damage in alcoholic liver disease (Sgome bo ei a|.,, 1997; MeoSiap apa Sopep, 1989). For example, the study found that patients with alcoholic hepatitis have higher levels of TMEo (on average 26.Zng/l; 9595 CI - 21.7-30.9) than healthy people

(b, 4ng/l; CI - 5.4-7.4). In patients who died later, the level of TMEo is higher (34.7 ng/l; CI - 27.8-41.6) than in those who remained alive (16b,blgUl; CI - 14.0-19.2 ). In patients with alcoholic hepatitis, TMP-o levels are positively correlated with serum bilirubin (g-0.74; P-0.0009) and serum creatinine (g-0.81; P-0.0003). In patients with alcoholic hepatitis, TME levels are higher than in patients with inactive alcoholic cirrhosis (11/Tng/l; CI - 8.9-13.3) and heavy alcoholics without liver disease (b.4ng/l; CI - 5.0-7.8). In patients with abnormal kidney function, TME levels are lower (by 14.Ing/l; Ci - 5.4-22.8) than in patients with alcoholic hepatitis. Therefore, it was concluded that an increase in the level of TME-o in alcoholic hepatitis is most noticeable in severe cases, which suggests that TME-o plays a role in pathogenesis (Viga et al., 1990).

TME mediates many biological actions of endotoxin. Recent studies have shown that administration of TME can cause liver damage and that TME can mediate the lethality of the hepatotoxin galactosamine. One of the strongest inducers of TME is endotoxin. Because patients with alcoholic liver disease often have endotoxemia and because many of the clinical manifestations of alcoholic hepatitis are known biological effects of TME, its activity in patients with alcoholic hepatitis was evaluated. Basal and lipopolysaccharide-stimulated TME release from peripheral blood monocytes - the main source of TME production - was determined in 16 patients with alcoholic hepatitis and 16 healthy volunteers. Eight of 16 patients with alcoholic hepatitis and only two of 16 healthy volunteers had detectable spontaneous TME activity (p less than 0.05). After stimulation with lipopolysaccharides, the average level of TME release from monocytes in patients with alcoholic hepatitis significantly - more than twice - exceeded the level of TME release in healthy people (25,313.7 vs. 10.9-42.4 units per ml, p less than 0.005) . Therefore, it was concluded that monocytes of patients with alcoholic hepatitis have a significantly increased spontaneous and lipopolysaccharide-stimulated release of TME compared to healthy volunteers (MesSiaip apa Sopep, 1989).

Lipopolysaccharide (I P5)-binding protein (BP) and CO 14 play key mediating roles in the activation of cells by endotoxin. It has been postulated that ARZ formed in the intestine is involved in the promotion of pathological EM 29 liver damage in alcoholic liver disease. It was shown that the levels of CO 14 and BP in Kupffer cells and hepatocytes, respectively, increased in rats given intragastric ethanol in oil for 4 weeks. CO 14 mRNA expression can also increase in non-myeloid cells.

Enhanced expression of BP and CO 14 contributes to a rapid increase in the I Ro-induced expression of various proinflammatory cytokines and correlates with the presence of pathological liver damage in alcoholic liver damage (5y ei ix, a1., 1998; I ikKaii ei ai., 1999). "-

Arthritis is a disease that consists in inflammation of the joints. Joints swell, lose mobility, become painful, red or burn. Symptoms may include weight loss, fever, or weakness. When these symptoms persist for more than two weeks, it may mean inflammatory arthritis, such as rheumatoid arthritis. The most common type of arthritis is degenerative joint disease

Zo (osteoarthritis). -

Medicines that are usually prescribed for arthritis are non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs include aspirin and aspirin-like medicinal products. They reduce inflammation, which is the cause of joint pain, immobility and joint swelling. However, NSAIDs are non-specific medicinal products with a number of side effects, including gastric bleeding

Also Opimegeyu oi MuUavpipdyup opo Agiygiyz, Egedegisk Mayivep (Spaipttap), lmmly.ogpor.mavpipdiop.edi.). In addition to C with M5ZAIIUO, to weaken the signs and symptoms of osteoarthritis and rheumatoid arthritis in adults, c" celebrextm - cyclooxygenase inhibitor (COX-2) is used. It is also indicated for the treatment of patients with familial adenoma polyposis.

UMO 01/00229 describes the combination of tumor necrosis factor (TNF) antagonists and COX-2 inhibitors for the treatment of inflammation. and TME antagonists are also used to treat arthritis. Antagonists of TME are described, for example, in UMO

Gee! 9103553.

Recent studies have shown that interleukin II/-18 plays the role of a pro-inflammatory factor in metabolism in the joints. Oyeee ey a). (1999) showed that II-18 is produced by articular chondrocytes and induces pro-inflammatory - 20 and catabolic reactions. In chondrocytes, mRNA 1-18 is induced by 11-13. Chondrocytes produce precursor 1I--18 and in response to 1-1 stimulation secrete mature form 1-18. The study of the effect of I/-1 on chondrocytes also showed that it inhibits TOB-p-induced proliferation and enhances the production of nitric oxide. II-18 stimulates the expression of several genes in articular chondrocytes of healthy people, including inducible nitric oxide synthase, inducible cyclooxygenase, II/-6, and stromelysin. Gene expression is associated with the synthesis of the corresponding proteins.

Treatment of the articular cartilage of a healthy person with I/-18 increased the release of glycosaminoglycans. Indicated

GF) results identify II/-18 as a cytokine that regulates chondrocyte reactions and contributes to the degradation of 7 cartilages.

Localization of interleukin-1 D-converting enzyme (ICE)/caspase-1 in osteoarthritic human tissues and its role in the maturation of interleukin-i-beta and interleukin-18 are shown by Zapa sei aI. (1999). Zara is here. studied the expression and production of caspase-1 in the cartilage and synovium of a healthy person and in the case of osteoarthritis (OA), quantified the level of ISE in OA chondrocytes and checked the relationship between the topographical distribution of ISE, interleukin-1p (1-18) and I1--18 , as well as apoptosis of chondrocytes. The experiments conducted in this study showed that ISE was expressed and synthesized both in the synovial membrane and in human b5 cartilage, and a significantly greater number of cells were stained in OA-positive tissue than in healthy tissue.

ISE production is localized mainly in the outer and upper intermediate layers of articular cartilage.

The production of mature II-1-beta in OA cartilage explants and chondrocytes was completely blocked by treatment with a specific ISE inhibitor, which also markedly reduces the number of II/-18-positive cells.

The correlation between active HER -1-beta and ISE suggests that ISE may promote the development of OA by activating this proinflammatory cytokine and that II-18 may play some role in cartilage pathology.

I will remember her. (1999) predicted for 1-18 the role of a pro-inflammatory factor in rheumatoid arthritis. I will remember her. found significantly more mRNA 1-18 and protein in synovial tissues in rheumatoid arthritis than in osteoarthritis (control). It is also shown that the combination of 1-12 or 1-15 with II-18 causes the production of IEM in the synovial tissues of the hip. In addition, administration of I/-18 to mice immunized with collagen/incomplete Freund's 7/0 adjuvant alleviates the development of erosive inflammatory arthritis, suggesting that I-18 may be a proinflammatory factor in mimo.

However, to date, it has been shown that, in addition to chemical compounds, only the blockade of TME0 and I/-1Dd by using receptors or mononuclear antibodies weakens collagen-induced arthritis in mice (SIA, a mouse model of rheumatoid arthritis) (UMiShatve et al., 1994), and is therefore suggested as a treatment for 75 rheumatoid arthritis.

The term "chronic or idiopathic inflammatory bowel disease" encompasses at least two conditions: the disease

Crohn's disease and non-specific ulcerative colitis. Both diseases are diseases of the intestinal tract, and the disease

Crohn's usually affects the small intestine. When the colon is also involved in the pathological process, the differential diagnosis that distinguishes this disease from non-specific ulcerative colitis can be problematic.

Chronic inflammation and ulceration in Crohn's disease usually begins either with obstruction of the small intestine or with pain in the abdominal cavity resembling acute appendicitis; other descriptions may refer to Her complications. The course of the disease is chronic, and there may be exacerbations and remissions, despite treatment. The onset, as a rule, takes place in early youth, and approximately half of all cases refer to the age of 20-30 years and 90965 - to 10-40 years. Slightly more men than women are affected.

Microscopy reflects massive phenomena. The inflammation is intermittent: it is focal or spot-like. and)

Accumulations of lymphocytes and plasma cells are found mainly in the mucous membrane and the layer of tissue under the mucous membrane, but, as a rule, all layers are affected (transmural inflammation). A classic microscopic feature of Crohn's disease is the presence of mast cells surrounded by a cluster of lymphocytes. Gee)

The occurrence of idiopathic inflammatory bowel diseases has significant geographical differences. These diseases occur more often in Northern Europe and the United States than in the countries of Southern Europe, Africa, South --

America and Asia, although increasing urbanization and economic prosperity leads to more frequent cases in "I regions of Southern Europe and Japan (Sepega! apa Zuzietaiis Raiyoidou, Spigspi! I imipdviope, Z eiyyiop 2000, p

ALL of Opadegmos, Ea.).

In Crohn's disease, there are clinically two main groups, and the first includes patients whose disease is in long-term remission for three years from the onset of the disease, and the second includes patients who have been sick for longer than three years.

Whatever the etiology, there is evidence of persistence and inconsistency of activation of T cells and macrophages in "Crohn's disease with increased production of pro-inflammatory cytokines, in particular, interleukins (I) 1, 2, 6 and 8, interferon (IEM)-y, and necrosis factor tumors (TME)-o. Crohn's disease is characterized by long-term (chronic) inflammation accompanied by fibrosis. The process of fibroblast proliferation and collagen deposition may be mediated by the transfer of growth factor Dr, which has some anti-inflammatory effect, namely, fibroblast recruitment, matrix synthesis, and deactivation of cells in the inflammatory zone, but it is also likely that many other mediators may be involved.

Nonspecific ulcerative colitis is a nonspecific inflammatory disorder in the colon that begins in the colon and rectum and immediately spreads to varying degrees. Unlike Crohn's disease, nonspecific ulcerative

FO colitis is limited to the colon.

There is increasing evidence that nonspecific ulcerative colitis results from altered autoimmune reactivity, but mucosal damage may also result from inappropriate --o/ 20 T-cell activation and indirect damage caused by cytokines, proteases, and metabolites and reactive oxygen of macrophages and neutrophils. The last mechanism of damage to the epithelium of the colon is called ii; damage with a "harmless witness". Proof of support for autoimmunity is the presence of autoreactives

T-lymphocytes and autoantibodies against colon epithelial cells and endothelial cells, and cytoplasmic autoantibodies against neutrophils (AMSA). However, nonspecific ulcerative colitis should not be considered an autoimmune disease in which mucosal damage is a direct consequence.

GF! immune reaction to autoantigens (Sepega! apa Zuvietaiiis Raiioiodu, cited above).

When it comes to treating Crohn's disease, most people are first treated with medications that contain mesalamine, a substance that helps fight inflammation. Patients who are not helped by it or who cannot tolerate it can receive other mesalamine-containing medicines, usually known as 5-ABA. The 60 possible side effects of mesalamine drugs include nausea, vomiting, heartburn, diarrhea, and headache.

Some patients take corticosteroid drugs to fight inflammation. These drugs are most effective in active Crohn's disease, but they can cause severe side effects, including increased susceptibility to infection.

Drugs that suppress the immune system are also used to treat Crohn's disease. Most often, b-mercaptopurine and the related drug azathioprine are prescribed. Immunosuppressants work by blocking the immune response that contributes to inflammation. Such medicines can cause side effects such as nausea, vomiting and diarrhea, and can reduce a person's resistance to infection. When patients are treated with a combination of corticosteroids and immunosuppressive drugs, the dose of corticosteroids may eventually be reduced. Some researchers believe that immunosuppressive drugs can increase the effectiveness of corticosteroids.

The US Food and Drug Administration has approved the drug infliximab for the treatment of moderate to severe Crohn's disease unresponsive to standard therapy (mesalamines, corticosteroids, immunosuppressants) and for the treatment of open draining fistulas. Infliximab, the first drug approved specifically for Crohn's disease, is a monoclonal antibody against tumor necrosis factor 7/0 (TME). Anti-TMEs remove TME from the bloodstream before it reaches the intestines, thereby preventing inflammation.

Antibiotics are used to treat overgrowth of microflora in the small intestine caused by stenosis, fistulas, or previous surgery. For this common problem, your doctor may prescribe one or more of the following antibiotics: ampicillin, sulfonamide, cephalosporin, tetracycline, or metronidazole.

Diarrhea and cramping abdominal pain often improve when the inflammation subsides, but additional drug therapy may also be needed. Some antidiarrheal agents can be used, including diphenoxylate, loperamide, and codeine. Patients who are dehydrated due to diarrhea are usually treated with fluids and electrolytes.

There remains a need for effective therapies for the treatment and/or prevention of inflammatory bowel diseases, particularly Crohn's disease and non-specific ulcerative colitis, with reduced or, ideally, no side effects.

Both histological and immunological observations show that cell-mediated immunity and

T-cell activation is a key feature of CO. Studies on humans and experimental models of cancer suggest that with CO, the local immune response tends to develop mainly according to the Tp1 type (Oeszgestvaikh ei a!., 1997), and that locally released cytokines, such as IEM-u, I--1r and TME-o, contribute to (8) the promotion and spread of the inflammatory reaction (Keiitipoa ei ai., 1996).

Cytokine I/-18 plays an important role in the Tp1-mediated immune reaction together with cytokine 1-12 by stimulating the secretion of IPM-y, increasing the cytotoxicity of natural killer cells and stimulating the differentiation of T1 cells (Ozsnpiyu ei ai., 1996).

I--18 acts together with 1I-12, II/-2, antigens, mitogens and also, possibly, other factors, to induce -- production of IEM-»u. I--18 also enhances the production of ZM-SZE and 1-2, enhances the proliferation of T cells, which is induced by anti-CO-3, and enhances Raz-mediated killing of natural killer cells. Mature 11-18 is produced from its precursor 1I--1 by rD-converting enzyme (ISE, caspase-1). Receptor 1-18 consists of at least two components acting together during ligand binding. High- and low-affinity binding sites -

I--18 was detected in T-cells stimulated by murine 1-12 (OKatoyo ei ai., 1998), which suggests a multi-chain receptor complex. So far, two receptor subunits have been identified, and both belong to the family of I/-1 receptors (OKatoyo et al., 1999). Signal transduction of I/-18 is involved in "activation of ME-KV" (Maivytoyo ei a!., 1997).

Recently, it has been suggested that IL/-18 takes some part in inflammatory bowel diseases (Rigato et al., 1999; Mopieieope et al., 1999). h Rigato ei ai. (1999) characterized the expression and localization of I/-18 in colonic samples and identified mucosal cell populations in patients with Crohn's disease. Using the scheme of semiquantitative RT-PCR, it was found that the number of mRNA 1/-18 transcripts in freshly isolated intestinal epithelial cells and mononuclear cells of the lamina propria of patients with CO increases compared to patients with non-specific ulcerative colitis and controls without inflammation. There were more transcripts of mRNA 1/-18 in intestinal b epithelial cells compared to mononuclear cells of the lamina propria. Immunohistochemical analysis of the cut tissues of the colon revealed the localization of II-18 both in the mononuclear cells of the own plate of the i- (in particular, macrophages and dendritic cells), and in the intestinal epithelial cells. Western blotting of sh--uU 20 showed that the band of 18.3 kDa, corresponding to both recombinant and mature protein 1-18, in the biopsy of the intestinal mucosa is detected mainly in CO, in comparison with OS; another band of 24 kDa, corresponding

I) the inactive precursor of II-18, is determined by biopsy of non-inflamed areas both in CO and in OS, and is the only form detected in controls without inflammation.

MopieYeope ei ai. (1999) confirmed these results. Intact tissue of the intestinal mucosa and mononuclear cells of the lamina propria of 12 patients with Crohn's disease, 9 with nonspecific ulcerative colitis and

Gee! 15 men (control) without inflammatory bowel disease were tested on II -18 by the method of semiquantitative

RT-PCR and western blotting. Transcripts 1-18 were detected in the tested samples. However, increased accumulation of de mRNA 1-18 was detected in samples of both the mucous membrane and mononuclear cells of the lamina propria during the disease

Crohn's disease, when compared with non-specific ulcerative colitis and control. In Crohn's disease, more than 60 1-18 transcripts accumulate in mucosal samples taken from affected areas. The 18 kDa band corresponds to mature

I/-18 was mainly detected in samples of the mucous membrane in Crohn's disease. In mucosal samples of controls without IBO, 1/-18 was present as a 24kDa polypeptide. Accordingly, the active subunit (p20)

I/-1-beta-converting enzyme (CE) was expressed in samples with either CO or OS, while in the colonic mucosa of controls without IBO, ISE was synthesized only in the form of a precursor (p45). 65 Oaueg (1999) considered different and partly contradictory functions of I/-18. As a result, I/-18 is a pleiotropic interleukin that has the functions of both increasing and decreasing inflammation. On the one hand, it increases the production of pro-inflammatory cytokines, such as TME-o, thus promoting inflammation. On the other hand, it induces the production of MO, an inhibitor of caspase-1, thus blocking the maturation of 1-1p and 1-18 and possibly attenuating inflammation. Such an uncertain role of I/-18 casts doubt on the effectiveness of I/-18 inhibitors in inflammatory diseases. In addition, due to the interaction of a large number of different cytokines and chemokines in the regulation of inflammation, the beneficial effect in the treatment or prevention of inflammatory diseases by blocking only one of the participants is unpredictable.

This invention is based on the conclusion that II-18 inhibitors are effective for the treatment and/or prevention of various diseases and disorders.

The first object of this invention is a new agent for the treatment and/or prevention of liver damage. Therefore, the invention relates to the use of the I/-18 inhibitor for the production of a medicinal product for the treatment and/or prevention of liver damage in the acute or chronic stage. More specifically, the invention relates to the treatment and/or prevention of alcoholic hepatitis, immune hepatitis, fulminant hepatitis, liver cirrhosis and primary biliary cirrhosis.

The second object of this invention is a new means for the treatment and/or prevention of arthritis. Therefore, the invention also relates to the use of I/-18 inhibitors for the preparation of a medicinal product for the treatment and/or prevention of arthritis. The beneficial effect of I-18 inhibitors includes reducing the severity of the disease, as well as preventing the spread of the disease. These results are surprising because the prior art described above does not suggest that blockade of one specific factor involved in arthritis, namely interleukin I/-18, can lead to relief of arthritis or even to cure the affected joint arthritis.

In a mouse model, it was also found that the introduction of the II/-18 inhibitor significantly reduces cartilage erosion. Thus, the present invention also relates to the use of inhibitor 1-18 in the manufacture of a medicinal product for the treatment and/or prevention of cartilage destruction. Ha

The third purpose of this invention is a new means for the treatment and/or prevention of inflammatory bowel disease (IBD), in particular, Crohn's disease and non-specific ulcerative colitis. Therefore, the invention also relates i) to the use of inhibitor 1-18 for the manufacture of a medicinal product for the treatment and/or prevention of IVI.

According to the present invention, it has now been found that in the biopsies of patients with Crohn's disease, the concentrations of 1I/-18BP mRNA and protein increase in areas of inflammation of the mucous membrane. In addition, two different II-18 inhibitors have been shown to protect animals from the disease in a mouse model of inflammatory bowel disease.

The use of combinations of I/-18 inhibitor and/or interferon and/or TME antagonist and/or COX-2 inhibitor are also considered according to the invention. In order to apply gene therapeutic approaches to the delivery of II inhibitor II/-18 to diseased tissues or cells, other aspects of the invention relate to the use of expression vectors containing the coding sequence of inhibitor II/-18 for the treatment and/or prevention of disease i-th

35 states. The invention also relates to the use of endogenous gene activation of I/-18 inhibitors and to the use of cells created by genetic engineering methods to express I/-18 inhibitors for the prevention and/or treatment of liver damage, arthritis and IVF.

Fig. 1 shows a histogram showing the level of IEM in serum (pg/ml) after the injection of different amounts of recombinant PLT8BP (0; 3.04; 0.4; 4 mg/kg) into mice 1 hour before ection of the IRO. Blood samples are taken after 5 hours. after the injection of ARZ and by the EGISA method, the presence of circulating IEM in them is analyzed. - c Fig. 2 presents a histogram showing the level of alanine aminotransferase (AG T) in serum. Mice are injected with increasing doses of human recombinant I18BP (0; 0.04; 0.4; 4 mg/kg) before injection of IR to mice sensitized by R. aspevz. Blood samples are taken after 5 hours. after the injection of I! RB5, and measure blood pressure in the serum. ZE - Frenkel sigma: 1 unit of 5E A5T/AI T will form 4.82x10"" micromoles of glutamine per minute at pH 7.5 at 25960. Fig. 3 shows the survival time of mice after injection of I/P5. Mice are injected with different doses

He" of human recombinant I18BP (0; 0.04; 0.4; 4 mg/kg) in 20 minutes. before injection | P5 mice sensitized by R. aspez. Triangles - 4mg/kg; small diamonds - 0.4; large diamonds - 0.04; mugs - II 18VE missing (only I RB). Fig. 4 presents a histogram showing the level of IEM in serum, measured after 5 hours. after -k 70 injections of various amounts of I 18BP (0; 0.4; 4 mg/kg, which is administered 20 minutes before the injection of IR5 to mice sensitized by R. aspev. that Fig. 5 shows the survival of mice, etc. injected with either a polyclonal antiserum against 1I/-18 or normal rabbit serum (MOH - control) according to ZOhv. before injection of 40 mg/ml (lethal dose) and P5 obtained from E. soya (Fig. 5A) or 5. (purpitigist (Fig. 58). Triangles: mice are injected with antiserum against II-18; 29 circles: mice are injected with MOH. The X-axis marks the days after infection with II R. a histogram showing the mean s-SEM (mean squared error) for a group of five mice treated as follows.Mice are injected intraperitoneally (i.p.) with antiserum against oI/-16, soluble TME-o receptors ( TMEzKr5b5) or the carrier (physiological solution) immediately after intravenous (i.m.) administration of concanavalin A (Sop A; Fig. bA) or REA (Rzeidotopaz aegidiposa, Fig. 6B). 60 jahr«0.01; and re0, 001 against one tel ki SopA or REA; Ж p«0.01 against or TMEzKr5b5 or anti-!-18, factorial AMOMA.

Figure 7 shows the action of I/-188P. on clinical indicators in a mouse model of arthritis. Fig. 7A is a diagram showing the clinical parameters measured after daily i.r. (intraperitoneal) administration to mice of various amounts of either II-18Vr, or IRM-D, or the carrier (MasSi). Designation: shaded triangles - for 10000 ME IRM-D; unshaded triangles - 1Omg/kg I.-18VR; inverted triangles - Zmg/kg I--18ВР; diamonds -

1mg/kg 1 -18BP; circles - 0О.5mg/kg I/-188Р; unshaded squares - 0.25mg/kg I/-18BP; and shaded squares - Masi. Treatment days are plotted on the X-axis, clinical indicators (average values) are plotted on the x-axis

U. Statistical indicators are calculated using the Mann-Whitney test. Fig. 7 shows a histogram showing the LIS (area under the curve) obtained from the graph in Fig. 7A. n - the number of animals.

Figure 8 shows the effect of II -18VR on swelling of the paws. Fig. 8A presents a diagram showing the results obtained when measuring the thickness of diseased hind paws (swelling) in individual animals treated with different amounts of II -18VR. The change in the thickness of the paws in mm from the beginning of processing is plotted along the X axis. The markings are the same as on

Fig. 7. Fig.88 is a histogram showing AC obtained from Fig.8A. n - the number of animals. 70 Fig. 9 shows the analysis of the number of diseased hind paws later in acute arthritis, that is, the spread of the disease to other joints. Designation: shaded squares - Mass (control); triangles - 1TOmg/kg

I/-18В8Р; inverted triangles - Zmg/kg I/-18ВР; diamonds - mg/kg I/-18BP; mugs - 0О, Bmg/kg II/-188ВР; and unshaded squares - 0.25mg/kg II -188В R.

Fig. 10 shows a histogram showing indicators of cartilage erosion of diseased joints.

Fig. 11 shows the histopathology of mouse joints. At the end of the experiment, the paw where the arthritis developed first is cut off, fixed and processed as described below in example 10. Fig. 11A: a healthy joint of a mouse;

Fig. 118: a joint of a mouse with arthritis; Fig. 11C: a joint of a mouse treated with pi-188BP.

Figure 12 presents a histogram showing the levels of antibodies against type I collagen! isotype Id (unshaded columns) or IdsSga (shaded columns) in mice treated with Zmg/kg I/-188P or saline (vehicle), respectively. Measurements were performed on the 4th day (04) or on the 8th day (08) of the disease.

Fig. 13 presents a histogram showing the levels of I/-b, in pg/ml, in animals treated with 1, 3 or 10 ug/kg I--188P, 10,000 IU interferon Dr (IEM-p), normal mouse serum (MM5 ) or physiological solution (Masi)), respectively. with

Fig.14 shows the expression of PI/-18BP and mRNA transcripts of 1I/-18 in intestinal biopsies of patients suffering from active Crohn's disease, nonspecific ulcerative colitis or healthy individuals. The characteristic RT-PCR products 1I-188P, 1-18 and the required gene (p-actin) are visible (Fig. 14A). Relative quantification of ethidium bromide-stained bands is performed using Kodak Reed image analysis software! and given as the ratio of the target gene to p-actin. The target genome is II-18 in Fig. 14B8 and co 1--188P in Fig. 14C.

Fig. 15 shows the expression of mRNA transcripts of PIG/-18BP and NOMES (endothelial cells of the human umbilical vein) and protein expression of TNRI1 (human monocyte cell line). RNA is isolated from untreated ChI endothelial cells (medium) and endothelial cells stimulated with IL-1f, TMERo, IEMu. Positive control: colon of a patient with Crohn's disease; negative control: no cDNA. The expression of II-188P and 11-18 is analyzed by the method of semi-quantitative RT-PCR (Fig. 15A). The culture supernatant of untreated (medium) their" cells and cells after 24-hour activation of I.-1D, TMERo, IEMu or NOMES (Fig. 158) or TNRI (Fig. 15C) are analyzed for the production of protein II-188BP and I-18 by the method EP IZA.

Fig. 16 shows the change in body weight between 1 and 10 days in a mouse model of IBO after intraperitoneal (IR) administration of either saline (weight) or II-18BP (8mg/kg). Weight change is expressed as a percentage of body weight change compared to day 1. Mean values and ZEM for two groups of 8 mice per group are given. Fig. 17 shows the results of analyzes of colons, caudal lymph nodes, and spleens obtained from mice with IVO treated with II-18BP and untreated. Fig. 17A shows the mass, in mg, of the last bcm of the colon. Figure 178 shows the total number of cells present in the caudal lymph node. Fig. 17C shows the percentage of cells in the spleen that stain positively for СХО4"/СО69". The data represent the average values of the values and ZEM. The sign 7" shows a significant difference. Fig. 18 shows the amount of IRMUu (Fig. 18, A and B) and TMERo (Fig. 18; C and 0), produced after 48 hours by the cells of the caudal lymph node (Fig. 18, A and C), and spleen cells (Fig. 18, B and 0) after stimulation of cSOrUsSor28, present in the supernatant. Average values and 5EM are given. Fig. 19 shows the content of TMEo, (Fig. 19A) and IEMu (Fig. 198) in colon homogenates. Data corrected for -70 colon mass. Mean values and ZEM are given. " sign indicates significant difference.

This invention is based on the discovery of the beneficial effect of II-18 inhibitor in various diseases and disorders. c The term "inhibitor" in the context of this invention refers to any molecule that modulates the production and/or action of II-18 in such a way that the production and/or action of II-18 is attenuated, reduced, or partially, substantially, or completely prevented or blocked . The term "inhibitor" is intended to denote inhibitors of the production of 25 I/-18, as well as inhibitors of the action of II-18.

GF) The production inhibitor can be any molecule that negatively affects the synthesis, processing or maturation of II-18. Inhibitors considered according to the invention can be, for example, suppressors of interleukin II/-18 gene expression, antisense mRNAs that reduce or prevent the transcription of mRNA 1-18 or that lead to mRNA degradation, proteins that disrupt the correct spatial 60 packaging, or partially or significantly prevent II-18 secretion, proteases that destroy 1-18 immediately after synthesis, inhibitors - proteases that cleave pro-I/-18 to generate mature I/-18, such as caspase inhibitors i1, etc.

An inhibitor of I/-18 action can be, for example, an antagonist of II-18. Antagonists can either bind to the I1I/-18 molecule or isolate the molecule itself with sufficient affinity and specificity to partially or completely neutralize I/-18 or the 1II/-18 binding site(s) responsible for binding 1// -18 with its ligand (for example, like its receptors). An antagonist can also inhibit the signaling cascade

II-18, which is activated in cells after the binding of II-18 and the receptor.

The inhibitor of I/-18 action can also be soluble I/-18 receptors or molecules resembling

Deceptors, or agents that block I/-18 receptors, or antibodies to 1-18, such as polyclonal or monoclonal antibodies, or any other agent or molecule that prevents binding of II-18 to its targets, thereby reducing or the activation of mechanisms of intracellular or extracellular reactions mediated by II -18 is prevented.

According to the first aspect of the present invention, II/-18 inhibitors are used for the production of a medicinal product for the treatment and/or prevention of liver damage. Preferably, the invention relates to the use of the II-18 inhibitor for the manufacture of a medicinal product for the 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" as used herein includes a variety of pathological conditions. Some of these states, which are considered in this invention, are explained in detail above in the section "Prerequisites of the creation of the invention". Other liver diseases that can be treated and/or prevented according to the invention include, for example, pyrogenic liver abscess. It is also called a bacterial liver and is a cavity in the liver that produces pus. There are many causes of liver abscess. It can develop from an abdominal infection, such as appendicitis, diverticulitis, or a perforated bowel; infections in the bloodstream; infections from the biliary (hepatic secretion) tract or trauma where a clogged liver becomes infected.

The most common microorganisms that cause liver abscess are Ezpegispia soii, Rgoieizv ucidagi and

Epiegorasieg aegodepevz. The frequency of the disease is 1 case per 10,000 people.

Alcoholic liver diseases can be treated and/or prevented using inhibitors 1-18 according to the invention. These include acute and chronic inflammation of the liver caused by alcohol abuse. with

Alcoholic hepatitis usually occurs after several years of heavy drinking. The longer the duration of alcohol use and the greater the consumption of alcohol, the greater the probability of developing i) liver disease. Malnutrition develops as a result of low calorie alcohol, reduced appetite and malabsorption (inadequate absorption of nutrients from the intestinal tract). Malnutrition contributes to liver disease. Ethanol toxicity to the liver, individual susceptibility to alcohol-induced liver disease, and genetic factors also contribute to the development of alcoholic liver disease.

According to the present invention, it is possible to treat and/or prevent liver cirrhosis with the use of I/-18 inhibitors. Cirrhosis is a chronic liver disease that causes damage to liver tissue, scarring of the liver "E (fibrosis; nodular regeneration), progressive decline in liver function, formation of excess fluid in the abdominal cavity (ascites), bleeding (coagulopathy), increased pressure in blood vessels (portal hypertension) and ISE)

With disorders of brain functions (hepatic encephalopathy). A 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 the spleen and liver. Excessive alcohol consumption is the leading cause of cirrhosis. Other causes include infectious diseases (such as hepatitis), diseases and defects of the biliary drainage system (such as biliary stenosis or obstruction), cystic fibrosis, and increased absorption of iron and copper. The type of cirrhosis depends on the cause of the disease. Severe complications can occur with cirrhosis. Cirrhosis is the 9th leading cause of death in the United States. Neurological problems (such as hepatic encephalopathy) may occur. ;" Increased accumulation of fluid in the abdominal cavity (ascites) is caused by low protein content in the body, increased sodium content, and increased pressure in the blood vessels of the liver (portal hypertension). Portal hypertension can cause increased pressure, enlargement and thickening of blood vessels in the esophagus (esophageal varices). Bleeding and blood coagulation problems may appear. Increased pressure in the blood vessels and blood coagulation problems can increase the possibility of severe and dangerous

Me. for life hemorrhage. Another disorder covered by the term "liver damage" 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. Different types can be found in the blood of patients with chronic active hepatitis

F circulating antibodies. Other autoimmune diseases may be associated with chronic active hepatitis or may occur in relatives of patients with chronic active hepatitis. Such diseases are thyroiditis, diabetes mellitus, non-specific ulcerative colitis, Sootrzg-positive hemolytic anemia, proliferative glomerulonephritis and Sjögren's syndrome. Risk factors may include the specified diseases, or risk factors associated with chronic active hepatitis. The frequency of the disease is 4 cases per 10,000 people.

F) Biliary atresia is another disorder in the field of the term "liver damage". It is an obstruction of the bile ducts, caused by their insufficient development, as a rule, before birth (in the womb). Biliary atresia is caused by abnormal and inadequate development of bile ducts inside and outside the liver. The purpose of the biliary system is to remove waste products from the liver and remove bile salts, which are necessary for the digestion of fats in the small intestine. In this condition, the release of bile from the liver into the gallbladder is blocked. This can lead to liver damage and cirrhosis of the liver, which, if left untreated, is ultimately fatal.

According to the invention, inhibitors 1-18 are also used for the manufacture of a medicinal product for the treatment 65 and/or prevention of chronic active hepatitis, also called chronic aggressive hepatitis. It is a long-term inflammation of the liver that damages liver cells. Causes of chronic active hepatitis include viral infection, drug reaction/absorption, metabolic disorders, or autoimmune diseases. The reason may also be implicit. The disease is characterized by necrosis or death of liver cells, active inflammation and fibrosis, which can lead to liver failure, cirrhosis and death. The frequency of the disease is 1 case per 10,000 people, risk factors are autoimmune diseases, the previous - more than 6 months - infection with hepatitis C or a positive reaction to hepatitis A or hepatitis B antigen.

Chronic persistent hepatitis is a moderate non-progressive form of inflammation of the liver, and is also a disease covered by the term "liver damage" according to the present invention.

According to the invention, inhibitors 1-18 are also used for the manufacture of a medicinal product for the treatment 70 and/or prevention of primary biliary cirrhosis (PBC). PBC is an inflammatory condition that results from obstruction of bile secretion in the liver, causing damage to liver cells. The bile ducts in the liver become inflamed for an unknown reason. The disease most often affects middle-aged women. Symptoms appear gradually, and the first symptom is itching of the skin. There is inflammation of the bile ducts in the liver. Eventually, liver cirrhosis develops. The disease can be associated with autoimmune disorders. The frequency of the disease is 8 cases / 5 per 100,000 people. Inhibitors 1-18 discussed in this description can also be used to treat acute liver poisoning, for example, caused by a large amount of paracetamol. Such acute liver poisoning can occur due to an overdose of paracetamol, either accidental or intentional.

As shown below in the examples, the authors of this invention unexpectedly found that inhibitors 1-18 are particularly effective in the prevention and treatment of fulminant hepatitis (acute hepatitis). Therefore, the invention is preferred

Refers to the prevention and/or treatment of fulminant hepatitis.

According to the second aspect of the present invention, II/-18 inhibitors are used for the manufacture of a therapeutic agent for the treatment and/or prevention of arthritis.

The term "arthritis" as used herein includes all the different types of arthritis and arthritic conditions, both acute and chronic arthritis, as indicated, for example, in Noterade oi She Oeragtepi oi sch Opporaedisev oi (She Opimegeiu oi UMazpipdyup op Agyigov (mlylu.oppormazpipdiyup.edi) Examples of arthritic conditions are ankylosing spondylitis, back pain, carpal tunnel syndrome, Ehlers-Danlos syndrome, (8) gout, juvenile arthritis, lupus erythematosus, myositis, osteogenesis imperfecta, osteoporosis, polyarthritis , polymyositis, psoriatic arthritis, Reiter's syndrome, scleroderma, arthritis with intestinal disease, disease

Behcet's, childhood arthritis, degenerative joint disease, fibromyalgia, infectious arthritis, Dyme's disease, Marfan syndrome, osteoarthritis, osteonecrosis, Paget's disease, polymyalgia rheumatoid, pseudogout, reflex sympathetic dystrophy, rheumatoid arthritis, rheumatism, Sjogren's syndrome, familial adenoma - - polyposis, etc.. "g

Preferably, according to the invention, I/-18 inhibitors are offered for the treatment and/or prevention of inflammatory arthritis. Inflammatory arthritis is classified as chronic arthritis, according to the persistent, long-lasting or reversible course of the disease. Cha

In a preferred embodiment of the invention, inflammatory arthritis is rheumatoid arthritis (RA). CA causes inflammation in the lining of joints (synovial membrane, epithelium with a single layer of cells) and/or internal organs. The disease tends to persist for many years, usually affects many different joints in the body, and can eventually cause damage to cartilage, bone, tendons, and ligaments. Joints that can be affected by CA are joints located, for example, in the neck, shoulders, elbows, hips, wrists, hands, hands, knees, and feet. In many cases, the joints are inflamed symmetrically with KA.

KA is common in approximately 195 populations in the United States, and is distributed across all ;" ethnic groups and age. It occurs worldwide, and the ratio of women to men with CA is 3 to 1.

As shown below in the examples, the II-18 inhibitor has been shown to have a very effective beneficial effect on cartilage erosion. Therefore, the invention also relates to the use of the I/-18 inhibitor in the manufacture of a medicinal product -I for the treatment and/or prevention of cartilage destruction, i.e. to the use of the I/-18 inhibitor as a chondroprotective agent. Inhibitor I/-18 can be used for any condition in which it occurs

Me, destruction or erosion of cartilage. Cartilage destruction is a progressive deterioration of the structural integrity of the articular cartilage. It occurs, for example, in conditions that affect articular cartilage, such as rheumatoid arthritis, juvenile arthritis or osteoarthritis, but also, for example, in infectious synovitis. - The third aspect of this invention relates to the use of inhibitor 1-18 for the manufacture of a medicinal product

F means for the treatment and/or prevention of inflammatory bowel disease. This application is based on the finding that the I/-18 inhibitor is activated in the inflamed mucosa of patients with CO. It is also based on the conclusion that administration of various II-18 inhibitors has a protective effect in a murine model of colitis.

Activation of I/-18 in the diseased mucous membrane in patients with CO has already been described in the technique | Mopiveieope ei ai., 1999; Riggago et al., 1999). (F) After the presence of large amounts of II-188R is shown. in the inflamed areas of the mucous membrane of the intestine, according to the invention, it became even more surprising to find a clear beneficial effect of II -188BP, introduced systemically, on the development and symptoms of colitis in an animal model. Although II -18VR. already activated endogenously in the intestine of patients with CO, as shown below in the examples, the amount of I/-18BP in the body capable of production, as it turns out, is insufficient to fight the disease.

According to the invention, the inflammatory bowel disease is preferably Crohn's disease or non-specific ulcerative colitis.

In a preferred embodiment of the present invention, the I/-18 inhibitor is selected from caspase-1 65 (ICE) inhibitors, antibodies directed against 1-18, antibodies directed against any of the subunits of the I receptor! -18, inhibitors of signaling cascade 1-18, antagonists of II/-18, competing with I/-18 and blocking the receptor 11-18,

of P/-18-binding proteins, isoforms, muteins, fusion proteins, functional derivatives, active fractions or their derivatives with a cyclic rearrangement, possessing the same activity.

The term "1/-18-binding proteins" is used in this description as a synonym for "1-18BP". It covers

I/-18-binding proteins according to the definition given in MUO 99/09063 or in Momisk ei ai., 1999, including splice variants and/or isoforms of II-18-binding proteins according to the definition given in Kigp ei AI., 2000. In particular, according to the present invention, useful isoforms a and c I/-18B8P. a person Proteins useful according to the present invention can be glycosylated or non-glycosylated, they can come from natural sources, such as urine, or, preferably, they can be obtained by recombinant methods. Expression of recombinants can be carried out in prokaryotic expressing systems, similar to E. soy, or in eukaryotic, and preferably in mammalian expressing systems.

The term "muteins" used in this description refers to analogs of I/-18BP or analogs of viral II-18BP, in which one or more amino acid residues of natural II-18BP or viral I/-188P. replaced by different amino acid residues or deleted, or one or more amino acid residues added to the natural sequence of I/-18BP or viral II/-18BP, without a significant change in the activity of the obtained products compared to wild-type II-18BP or viral II-188BP. Such muteins are obtained by known methods of synthesis and/or methods of site-directed mutagenesis or by any known method suitable for this purpose.

Any such mutein has an amino acid sequence essentially duplicating the sequence of II-18BP or substantially duplicating the sequence of viral II/-18BP so that the activity is substantially similar to that of II-18BP. One of the activities of I/-18BP is its ability to bind II/-18. As long as the mutein has substantial binding activity to II/-18, it can be used in the purification of II/-18 by methods such as affinity chromatography, and thus can be considered to have activity substantially similar to that of II - 18 BP. Thus, it is possible to determine whether any given mutein has activity substantially similar to that of II-18BP by conventional experiments involving determination of whether such mutein binds or does not bind to an appropriately labeled 1-18, for example, by simple (8) competitive sandwich analysis, for example, radioimmunoassay or EHIZA analysis.

Polypeptide I/-18BP muteins or viral I/-18BP muteins that can be used in accordance with the present invention, or the nucleic acid encoding them, include a limited number of essentially corresponding Hezo sequences in the form of substituted peptides or polynucleotides that can be obtained by conventional methods of an ordinary specialist in this field of technology without unnecessary experimentation, based on the instructions and guidance presented in this description. "Mr

Preferred changes for muteins according to the present invention are changes known as "conservative" substitutions.

Conservative substitutions of amino acids of IIII/-18BP polypeptides or viral I-188P proteins may include i) identical amino acids within a group that has essentially similar physico-chemical properties, and with substitutions between i-members of the group, the biological function of the molecule will be preserved ( Sgapipat, 1974). It is clear that insertions or deletions can also be made in the above-mentioned sequences without changing their function, in particular, if the insertions or deletions involve only a few amino acids, for example up to thirty, preferably up to ten, and amino acids critical for functional conformation, for example, "Cysteine residues. Proteins and muteins obtained with the help of such deletions and/or insertions are included in the scope of the present invention.

Preferably, when the identical amino acids are the amino acids listed in the table. I. It is preferable if the amino acids listed in the table are identical to the amino acids. II; and most preferably, when identical amino acids are the amino acids indicated in the table. PI. U.S

F g o - s z o te 000 mertustne uviv yu v b5 ssh Avr, Guv, Avp, sip, Niv, Aga, SI o 6 emertteuvytya 2 sch o (Se) - ch i-o zvi- ch i Z s . 6 rvmaiaya 000

I" 1 -

F z z u

F

Examples of amino acid substitutions in proteins that can be used to produce muteins of polypeptides o or I-18BP proteins or viral I/-188P muteins for use in the present invention include any known methods, for example, disclosed in US Patent No. 33653 , 4959314, 4588585 and 4737462, Magk eyi ko aI;; 5116943, Koiyz ei aYi., 4965195, Matep ei aiI.; 4879111, Spopd eyi aiYi.; and 5017691, ee ei ai.; and proteins with lysine substitution, presented in US patent No. 4904584 (Zpamu ei ai.). for The term "fusion protein" refers to a polypeptide containing I/-188P or viral II/-188P, or its mutein or fragment, fused to another protein, which, for example, has a long residence time in body fluids. I/-18BP or viral II/-18BP, thus, can be hybridized with another protein, polypeptide or similar element, for example, an immunoglobulin or a fragment thereof.

The term "functional derivatives" used in this description includes derivatives of 11-18B8P or viral 65 1I-188P and their muteins and fusion proteins, which can be obtained by functional groups present in the side chains of residues of the M- or C-terminal groups , by methods known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, that is, they do not destroy the activity of the protein, which is basically similar to the activity of II-18BP or viral II-188P, and do not add toxic properties to the compositions containing them.

Such derivatives can include, for example, side chains of polyethylene glycol, which can mask antigenic sites and prolong the residence time of II/-188P. or viral I/-18BP in body fluids. Other derivatives include aliphatic ethers formed with carboxyl groups, amides formed from carboxyl groups by reaction with ammonia or primary or secondary amines, M-acyl derivatives of free amino groups of amino acid residues formed with acyl groups (eg, alkanoyl or carbocyclic aroyl groups), or O-acyl derivatives of free hydroxyl groups (for example, groups of 7/o beryl and threonyl residues) formed with adyl groups.

As "active fractions" of I/-18BP or viral IP/-18BP, their muteins and fusion proteins, the present invention encompasses any fragment or precursor of the polypeptide chain of the protein molecule as such or together with associated molecules or residues attached thereto, for example residues of sugars or phosphates, or protein molecule aggregates or sugar residues as such, provided that the specified fraction has an activity basically similar to the activity of II -188V R.

In another preferred embodiment of the invention, the I/-18 inhibitor is antibodies to 1-18. Antibodies against

I/-18 can be polyclonal or monoclonal, chimeric, humanized or even fully human. Recombinant antibodies and their fragments were characterized by high-affinity binding to II-18 IP and low toxicity. The antibodies that can be used in the invention are distinguished by their ability to treat 20 patients for a period sufficient to obtain excellent regression or relief of the pathogenic condition or any symptom or group of symptoms associated with the pathogenic condition and low toxicity.

Neutralizing antibodies are easily obtained from animals such as rabbits, goats or mice by immunization

I/-18. Immunized mice are particularly useful for providing a source of B cells for the production of hybridomas, which in turn are cultured and receive monoclonal antibodies against II-18 in large quantities. high school

Chimeric antibodies are immunoglobulin molecules characterized by two or more segments or parts obtained from animals of different species. Typically, the variable region of the chimeric antibody i) is derived from a non-human mammalian antibody, such as a murine monoclonal antibody, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. Preferably, both areas and the combination have low immunogenicity, which is determined by conventional methods (BeiYoi ai ai., 1994). Humanized Gezo antibodies are immunoglobulin molecules created by genetic engineering methods, in which the mouse constant regions are replaced by the corresponding parts of human immunoglobulin, while preserving the mouse antigen-binding regions. The obtained murine-human antibodies mainly have reduced immunogenicity and improved pharmacokinetics in the human body (Kpidni ei a/!., 1993).

Thus, in another preferred embodiment, antibodies to ІІ/-18 are humanized antibodies to ІІ/-18. Preferred examples of humanized antibodies to 1I/-18 are described, for example, in the European patent application EP 0974600.

In another preferred embodiment, antibodies to 1-18 are fully human. The technology for obtaining human antibodies is described in detail, for example, in MO 00/76310, UMO 99/53049, 5 6162963 or ASH5336100. Fully human antibodies are mainly recombinant antibodies obtained from transgenic animals, for example, "Xenomice", containing all or part of the functional human Id loci. z c In a very preferred embodiment of this invention, the 1-18 inhibitor is I/-18BP. or its isoform, . mutein, fusion protein, functional derivative, active fraction, or cyclized derivative. Indicated and? its isoforms, muteins, fusion proteins or functional derivatives retain the biological activity of II -188BP, in particular, binding to II -18, and, preferably, have essentially at least activity similar to that of II -18BP. Ideally, such proteins have a biological activity that is even higher compared to the activity of unmodified -I I-18BP. Preferred active fractions have activity superior to that of II-18BP, or have other advantages such as increased stability or less toxicity or immunogenicity, or are easier to obtain in large

Me. quantities, or easier to clean. their Sequences of II -18ВР and its splice variants/isoforms can be taken from MUO 99/09063 or from Momisk eii ai., 1999, 5o atakoz in Kit ega!., 2000. - Functional derivatives of I -18В8Р. can be conjugated to polymers to improve protein properties such as

Ф stability, half-life time, bioavailability, tolerance by the human body or immunogenicity. To achieve this goal )/-188P. can be combined, for example, with polyethylene glycol (PEG). PEG processing can be carried out by known methods, described, for example, in UUO 92/13095.

So, in the preferred embodiment of the present invention II -18B8R is treated with PEG.

In another preferred embodiment of the invention, the I/-18 inhibitor is a fusion protein containing the whole

F) I/-18-binding protein or its part, fused with all immunoglobulin or its part. It will be clear to a person skilled in the art that the resulting fusion protein retains the biological activity of I/-18BP, in particular, binding to II/-18. Hybridization can be direct or through a short peptide linker, which can be short, for example, 1-3 amino acid residues long, or longer, for example, 13 amino acid residues long. This linker can be a tripeptide, for example, with the sequence E-B-M (Si0-Ppe-Med), or a linker sequence of 13 amino acids containing

SiISH-RINe-c1u-AIa-C1u-I ei-Ma!-G etzsh-(SiIu-(Iu-Sip), embedded between the sequence -18VR ii / the sequence of immunoglobulin. The resulting fusion protein has improved properties, such as increased residence time in 65 body fluids (half-life), increased specific activity, increased expression level, or the fusion protein is more easily purified.

In a preferred variant, I/-18BP is hybridized with the constant region of the Id molecule. Preferably, it is hybridized with regions of the heavy chain, similar, for example, to the SNI and SNZ Id domains. a person Preparation of specific fusion proteins containing II-18VR and part of immunoglobulin is described, for example, in example 11

MURO9/09063. Other isoforms of Id molecules are also suitable for obtaining fusion proteins according to the present invention, such as isoforms of Ids o or Ids, or other classes of Ids, similar, for example, (YAM or IdA. Fusion proteins can be monomeric or polymeric, hetero or homopolymeric.

Interferons are known mainly for their inhibitory effect on virus replication and cell proliferation.

Interferon-y, for example, plays an important role in the promotion of immune and inflammatory reactions. It is reported that 70 interferon p (IEM-p, type I interferon) plays the role of an anti-inflammatory factor. Data from published studies

TpapiarnuPoroshiiv her a. (1999), show that IRM-D has a beneficial effect in the treatment of rheumatoid arthritis, as shown in a mouse model of the disease - a model of collagen-induced arthritis (CIA). Such a beneficial effect of IEM-D is confirmed below in the examples.

The invention also relates to the use of a combination of inhibitor I/-18 and interferon in the manufacture of 75 medicinal products for the treatment of arthritis, in particular, rheumatoid arthritis.

Interferons can also be conjugated to polymers to increase protein stability. The conjugate of interferon Dr and polyol polyethylene glycol (PEG) is described, for example, in UUO 99/55377.

In another preferred embodiment of the invention, interferon is interferon B (IPM-B), and more preferably - IEM-D and.

The inhibitor of production and/or action of II-18 is preferably used simultaneously with interferon or separately from it.

In another embodiment of the invention, the II-18 inhibitor is used in combination with a TME antagonist.

TME antagonists show their activity in several ways. First, antagonists can bind to the TME molecule itself or isolate it with sufficient affinity and specificity and partially or substantially neutralize the TME epitope or epitopes responsible for binding to the TME receptor (hereinafter referred to as "sequestering antagonists"). A sequestering antagonist can be, for example, antibodies directed against TME.

On the other hand, TME antagonists can inhibit the TME signaling cascade activated by a cell surface receptor after TME binding (hereinafter referred to as "signaling antagonists"). Both groups of X antagonists are useful, either alone or together, in combination with an II-18 inhibitor in the treatment of arthritis, in particular, rheumatoid arthritis.

Antagonists of TME are easily identified and evaluated by conventional screening of candidates for their effect on the activity of native TME and migo on sensitized cell lines, for example, on human B-cells, in which TME causes proliferation and immunoglobulin secretion. The analysis includes the analysis of TME compositions at

From different dilutions of the antagonist-candidate, for example, from 0.1 to 100-fold dilution of the molar amount -

TME used in the assay and controls without TME or with antagonist alone (Tissi et al., 1992).

Sequestering antagonists are preferred TME antagonists for use in the present invention.

Among the sequestering antagonists, polypeptides that bind to TME with high affinity and "possess low immunogenicity" are preferred. Soluble molecules of the TME receptor and neutralizing antibodies to TME are especially preferred. For example, soluble TME-BKI and TME-KII are useful in the present invention. Truncated forms of the indicated C c receptors, which contain the extracellular domains of the receptors or their functional parts, are even more preferred "with antagonists according to the present invention. Truncated soluble TME receptors of type | and type IIII are described, " for example, in EP 914431.

The truncated forms of TME receptors are soluble and are found in urine and serum as inhibitory binding of TME proteins in ZOkDa and 40kDa, named TVRI and TVRII, respectively (EpdeItapp et al., 1990). According to the invention, simultaneous, sequential or separate use of II-18 inhibitor with TME antagonists and/or interferon is preferred.

Ф According to the invention, TVRI and TVRI are preferred antagonists of TME for use in combination with inhibitor 1-18. Derivatives, fragments, regions and biologically active parts of receptor molecules are functionally similar to receptor molecules and can also be used in this invention. Such biologically active equivalents or derivatives of the receptor molecule include a portion of the polypeptide or sequence encoding the receptor molecule that is of sufficient size and capable of binding to TME with such affinity that the interaction of TME with the membrane-bound receptor is inhibited or blocked. .

In another preferred embodiment, the TME antagonist for use according to the invention is soluble human TME-KI (TVRI). Natural and recombinant soluble TME receptor molecules and methods of their preparation are described in 22 European patents EP 308378, EP 398327 and EP 433900.

GF) Inhibitor, II/-18 can be used simultaneously, sequentially or separately with TME inhibitor. It is beneficial to use a combination of an antibody or antiserum against II/-18 and a soluble TME receptor that has TME inhibitory activity.

In another preferred embodiment of the invention, the drug also includes a COX inhibitor, preferably a COX-2 inhibitor. COX inhibitors are known in the art. Specific COX-2 inhibitors are described, for example, in UMO 01/00229.

The invention also relates to the use of a combination of I/-18 inhibitors and/or interferons and/or TME antagonists and/or COX-2 inhibitors. Such a combination is suitable for the treatment and/or prevention of arthritis, in particular rheumatoid arthritis, and for the treatment and/or prevention of liver damage and for the treatment and/or prevention of inflammatory bowel disease, in particular Crohn's disease and non-specific ulcerative colitis. Active components can be applied simultaneously, sequentially or separately.

In a preferred embodiment of the present invention, the I/-18 inhibitor is used in an amount of approximately 0.0001-10 mg per kg of body weight, or approximately 0.01-5 mg per kg of body weight, or approximately 0.1 mg per kg of body weight, or approximately 1-2 mg per kg of body weight. In another preferred embodiment, the III-18 inhibitor is used in an amount of approximately 0.1-100 Ωkg per kg of body weight, or approximately 1-100 Ωkg per kg of body weight, or approximately 10-5O0μg per kg of body weight.

The invention also relates to the use of an expression vector containing the coding sequence of an inhibitor 1-18 in the preparation of a medicinal product for the prevention and/or treatment of arthritic conditions, 7/0 In particular, rheumatoid arthritis, for the treatment of liver damage and for the treatment of inflammatory bowel disease. Thus, a genotherapeutic approach is used to treat and/or prevent the disease.

Then it is beneficial to express the inhibitor I/-18 ip zysh, and in this way I/-18 is effectively blocked directly in the tissue(s) or cells affected by the disease.

In order to treat and/or prevent arthritis, a gene therapy vector containing a sequence inhibitor of the production and/or action of I/-18 can be injected, for example, directly into the diseased joint, thus avoiding the problems associated with systemic introduction of gene therapeutic vectors, similar to dilution of vectors, achievement and targeting of target cells or tissues and side effects.

The use of a vector to induce and/or enhance the endogenous production of II-18 in a cell that is normally silent regarding the expression of the inhibitor 1/-18 or expresses insufficient amounts of the inhibitor is also contemplated according to the invention. The vector may contain regulatory sequences functional in cells desired to express the I/-18 inhibitor. Such regulatory sequences can be, for example, promoters or enhancers. The regulatory sequence can then be inserted into the right locus of the genome by the method of homologous recombination, thus operably linking the regulatory sequence to the gene whose expression is to be induced or enhanced. This technology is usually called "endogenous gene activation of sch (EOA)", and it is described, for example, in UUO 91/09955.

A person skilled in the art will understand that using such a method it is also possible to stop i) the expression of 1/-18, i.e., to introduce a negative regulatory element into the II/-18 gene locus, similar, for example, to a silent element, and in this way expression of II-18 is deactivated or prevented. One skilled in the art will understand that such deactivation or "silencing" of I/-18 expression has the same effect as using an II-18 inhibitor to prevent and/or treat disease.

The invention also relates to the use of a cell genetically modified to produce an inhibitor -

I/-18, for the manufacture of a medicinal product for the treatment and/or prevention of liver damage, arthritis or inflammatory bowel disease.

The invention also relates to pharmaceutical compositions, in particular, useful for the prevention and/or re)treatment of inflammatory arthritis, liver damage or inflammatory bowel disease, containing a therapeutically effective amount of an I/-18 inhibitor and a therapeutically effective amount of interferon. As an I/-18 inhibitor, the pharmaceutical composition may contain caspase-1 inhibitors, antibodies against 1-18, antibodies against II-18 receptor subunits, inhibitors of the 1-18 signal transmission cascade, II-18 antagonists competing with

Y-18 and blocking the /1/-18 receptor, and proteins binding 1/-18, their isoforms, muteins, fusion proteins, " functional derivatives, active fractions or derivatives with cyclic rearrangements, possessing the same 4 4 //-ptsh) with activity. . I/-18BP and its isoforms, muteins, fusion proteins, functional derivatives, active fractions or derivatives with a cyclic rearrangements, described above, are preferred active ingredients of pharmaceutical compositions.

The interferon contained in the pharmaceutical composition is mainly IEM-D. In another preferred embodiment, the pharmaceutical composition contains therapeutically effective amounts of inhibitor I--18, optionally interferon and TME antagonist. Antagonists of TME can be antibodies,

Fos neutralizing the activity of TME, or soluble truncated fragments of the TME receptor, are also called TVRIs and TVRIs. The pharmaceutical composition according to the invention may also contain one or more COX inhibitors, preferably COX-2 inhibitors. - The definition of "pharmaceutically acceptable" is intended to mean any carrier that does not affect

Ф efficiency of the biological activity of the active ingredient and non-toxic to the host to which it is administered.

For example, for parenteral administration, the active protein(s) can be incorporated into a standard dosage form for injection in a vehicle such as saline, dextrose solution, serum albumin, and Ringer's solution.

The active ingredients of the pharmaceutical composition according to the invention can be administered to an individual in various iF) ways. Methods of administration include intradermal, transdermal (eg, in slow-release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural, topical, and intranasal routes. Any other effective route of administration can be used, such as absorption through epithelial or endothelial tissue or gene therapy, where a DNA molecule encoding an active agent is administered to a patient (eg, by means of a vector) that causes expression and secretion of the active agent ip mimo . In addition, according to the invention, the protein(s) can be administered together with other components of biologically active agents, such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and fillers. 65 For parenteral (e.g., intravenous, subcutaneous, intramuscular) administration, the active protein(s) may be administered as a solution, suspension, emulsion, or lyophilized powder, in combination with a pharmaceutically acceptable parenteral vehicle (e.g., water, physiological solution, dextrose solution) and additives that maintain isotonicity (for example, mannitol) or chemical resistance (for example, preservatives and buffer substances). The composition is sterilized by methods that

They are used naturally.

The bioavailability of the active protein(s) according to the invention can also be improved by using conjugation procedures that increase the half-life of the molecule in the human body, for example, by attaching the molecule to polyethylene glycol, as described in the international patent application PCT UUO 92/ 13095.

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 1-18, the residual cytotoxic activity of any kind detected by the antagonists, the route of administration, the clinical condition of the patient (including including the desirability of maintaining a non-toxic level of endogenous II activity -18). "Therapeutically effective amount" is that amount of inhibitor 1-18, which, when administered, leads to inhibition of the biological activity of II/-18. The dosage administered to an individual in the form of a single or multiple doses will depend on various factors, including the pharmacokinetic properties of the inhibitor

I/-18, the method of administration, the patient's condition and its characteristics (gender, age, body weight, state of health, volume), severity of symptoms, concomitant treatment, frequency of treatment and desired effect. Regulation and manipulation in the established dosing intervals are within the capabilities of specialists in this field of technology, as well as methods of determining the ip migo and ip mimo inhibition of II -18 in an individual.

According to the invention, the I/-18 inhibitor is used in an amount of approximately 0.0001-10 mg per kg of body weight, or approximately 0.01-5 mg per kg of body weight, or approximately 0.1-Zmg per kg of body weight, or approximately 1 -2 mg per kg of body weight. Other preferred amounts of I/-18 inhibitor are amounts of about 0.1-100 Ωkg per kg of body weight, or about 1-10 Ωkg per kg of body weight, or about 10-5 Ωkg per kg of body weight.

A preferred method of administration according to the invention is subcutaneous administration. According to the invention, intramuscular administration is also preferred.

In other preferred embodiments, the II-18 inhibitor is administered daily or every other day. and)

Daily doses are usually given in divided doses or in a delayed-release form that is effective in obtaining the desired results. The second or subsequent administration can be carried out in a dosage that is the same as the initial or previous dose administered to the individual, or less or more than it. The second or further introduction of Hezo can be carried out during or before the onset of the disease.

According to the invention, the I/-18 inhibitor can be administered to an individual in a therapeutically effective amount (87 prophylactically or therapeutically before, simultaneously with, or after other types of treatment or drugs "g (eg, regimens with several drugs), in particular, with interferon and/ or a TME antagonist and/or COX inhibitor Active agents administered simultaneously with other therapeutic agents may be administered in the same regimen or in other compositions. uh-

The invention also relates to a method of obtaining a pharmaceutical composition, which includes mixing an effective amount of inhibitor 1/-18 and/or interferon and/or TME antagonist and/or COX inhibitor with a pharmaceutically acceptable carrier.

Having a description of the invention, it will be easier to understand it by referring to the following examples, which are given with "

For the purpose of illustration and are not intended to limit the present invention. with with Examples. Part I. Examples 1-8 related to the use of II-18 inhibitors in liver damage and? Example 1. Obtaining II -188P with the label Niv LI -18V Р-Н from i89)

Purified recombinant human I/-18B8P containing the Piv label ((I-piY-II-18BP-Niz) was obtained in

CHO cells. The production of recombinant proteins in eukaryotic cells is known to specialists in this field - I technique. Well-known methods are available for the construction of suitable vectors containing the coding DNA

I/-188P, and suitable for transfection of eukaryotic cells in order to obtain recombinant II-18BP. For me) expression in cells of DNA encoding II/-188P (see, for example, Momisk ei ai!., 1999), were cut out and inserted into them expressing vectors suitable for transfection of cells. On the other hand, such DNA can be obtained by the PCR method with the appropriate sense and antisense primers. The resulting DNA constructs were then inserted into appropriately engineered eukaryotic expression vectors by methods well known in the art

F (Mapaiiv, 1982). The recombinant protein was purified to a purity of more than 952 and was found to be biologically active ip micgo and ip mimo and has a high affinity for its ligands.

Example 2. Protective effect of II-18VR against endotoxin-induced death in a mouse model

A mouse model was used to test whether an II-18 inhibitor would I/-18BP. to protect mice from a high dose of lipopolysaccharides (II RB). | PO causes acute liver damage and, as a result, rapid death of mice. iF) C57VI/6 mice were injected intraperitoneally (i.r.) with 4 mg/kg of recombinant II-188R. human (g 18vRNIZz), which contains a piz label (obtained during recombinant protein production). After 1 hour, bOmg/kg was injected

And RB (lethal doses). The survival of mice in this group was compared with the survival in the group of animals that received only ARZ (without II--188).

Five mice out of 7, injected with gpII-18VR-pis, survived after the injection of I! P5, in contrast to the control group, where all animals died within 3 days.

Blood samples were taken through Bhod. after injection of ARZ in the absence or in the presence of increasing doses of gpI--18vR-piz and analyzed by the EGISA method for the presence of circulating IRM (Fig. 1). Doses of g-188BP-pis at 0.4 b5 and 4 mg/kg caused a 2-fold decrease in the content of IEM-y in the serum. This inhibitory effect was lost at lower doses of p-18BP (0.004 and 0.04 mg/kg).

Example 3. II-18VR has a protective effect against liver damage in a mouse model of the disease

A mouse model of fulminant hepatitis was used to test the action of II-18BP. Mice developed acute liver damage when they were additionally administered Rgoriopirasiegitst aspez (R. aspev) and lipopolysaccharide (I RB).

Mice were injected with increasing doses of gpII/-18BP-half (4; 0.4; 0.04; Ωg/kg) in different. moments of time (1 hour, 20 minutes, simultaneously) before injection of I P5 to C57VI/6 mice sensitized by R. aspez. When pII-18VR-pis was injected with hydr. at the same time as I P5, there were no surviving mice, and the levels of circulating IEM-u and TME-o remained unchanged. Unexpectedly, gpI/-18BP (4 and O4 mg/kg) caused a decrease in circulating alanine aminotransferase (a marker of liver damage) in 70956 mice, as shown in Fig. 2. 70 In addition to the above, the survival of mice was checked (Fig. 3). When -18BP was administered ip 20 min earlier

IRB5, the two highest doses of I/-18BP (4 and O4 mg/kg) slowed down the death of mice by 1 hour. in comparison with the control group, where instead of II -188P. mice received Masi.

The results of measuring the content of IEM in serum are shown in Fig. 4. gpI/-18BP (4 mg/kg) inhibited levels of circulating IEM by 9095 and circulating alanine aminotransferase by 80905 (not shown).

When gpII--188R was administered i.p. 1 hour earlier I P5, survival curves and levels of circulating IEM were similar to the indicators observed in the case when gPPI/-188P was administered i.r. for 20 minutes earlier than I P5, but circulating alanine aminotransferase levels remained unchanged (not shown).

In addition to the above, mouse liver tissue was analyzed by staining with hematoxylin and eosin, as well as tunnel microscopy. The livers of mice previously induced with severe hepatitis show severe necrosis when compared with healthy liver tissues. In contrast, the liver tissue of treated mice

I/-18BP, reveals significantly fewer necrotic foci than untreated mice.

Example 4. Antibodies against II-18 protect against lethal endotoxemia

In order to assess whether blockade of II-18 with anti-II-18 antibodies will protect mice from the effects of lethal doses of bacterial lipopolysaccharides, C57VI/6) mice were first injected with neutralizing rabbit anti-mouse antibodies "M to 1-18 (polyclonal) or serum healthy rabbits (MOB) as a control. Through ЗОхв. after treatment (5) with antibodies, lethal doses of I P5, obtained either from E. soy (Fig. 5A) or from 5. Shurpitigisht (Fig. 5B), were injected.

Experiments included 10-12 mice per group and were performed twice on two different occasions.

As can be seen in Fig. 5A, treatment of mice with antiserum against I/-18 prevents mortality caused by 4 Ωg/kg I RB from E. soyii. 10,095 mice survive versus 1,095 surviving mice treated with the serum of healthy rabbits (p<0.005).

Figure 5B shows that antibody-treated mice are also protected from the lethal action of 5. (purpitigiitis (5095 survivors vs. 096 survivors; p<0.05).

Example 5. II-18 and TME-y blockade protects mice from hepatotoxicity caused by SopA and REA co

Two experimental models of hepatotoxicity were used to evaluate the role of II-18 and TME-o in injury

From the liver. Both injections of concanavalin A (SopA) and Rzendotopaz aegidipose (REA) caused damage in mice. liver and were models of hepatitis mediated by T cells.

Mice C57VI/62) were treated with antiserum against II/-18 or soluble receptor TME-o TMEzkrB55.

Serum levels of alanine aminotransferase (A!T) were measured as indicators of liver damage (Fig.b). "

As can be seen in Fig.bA, both the antiserum against II/-18 and the soluble TME receptors significantly reduced the levels of dItT induced by SopA in the serum in comparison with the control injection of the same carrier (apyrogenic C with saline). Co-administration of soluble TME receptor and antiserum against 1-18 results in "complete inhibition of SopA-induced liver damage."

I--18 leads to 93595 and 8395 inhibition of AI T serum levels, respectively. A combined blockade of both leads to 999p5 protection. and Example 6. Levels of II -18-binding protein in plasma are increased in patients with chronic liver disease

Ge") Levels of I -18BP in plasma were measured in 133 patients with chronic liver disease (CI 0) of various etiologies and 31 healthy people (control) by the method of specific EGI5A using monoclonal antibodies to II -18. shk Levels of I/-18BP in plasma were significantly higher in patients with SI O (12.1-0.89 ng/ml; mean Я-ZEM) than in healthy people - 70 people (4.96-0.4 ng/ml, p "e0.001). Cirrhotic patients had significantly higher levels than CI patients without

F of cirrhosis (19.2341.28ng/ml, p-b7/, against 6.49-40.51, p-6b, p«0.001). Patients in stage B by classification

Spiid-Rodpy have higher levels of IIII/-18BP than patients in stage A (22.48-42.44ng/ml vs. 9.57--1.25ng/ml, p-0.001). However, there is no significant difference between Spa stages in C (22.48-244ng/ml vs. 20.62-4.75ng/ml, 5B p-0.7). Levels of I-18BP in plasma were positively correlated with OT, bilirubin and erythrocyte sedimentation rate.

A negative correlation with prothrombin time was revealed. (F) As a conclusion, the results showed that the levels of I-18BP in the plasma during SI O increase and correlate with the severity of the disease, regardless of the etiology of the disease. Despite the endogenous antagonist of pro-inflammatory II-18, it turns out that elevated levels of II-18VR. is not sufficient to counteract the countless pro-inflammatory mediators in UTI.

Example 7. Suppression of alcoholic hepatitis with II -188R

Four groups of rats (5 per group) were given ethanol and food containing corn oil by intragastric injection for 4 weeks. In the control animal, ethanol was replaced with dextrose in an isocaloric manner.

Rats were injected daily with mouse II-18VR (mg/kg) or saline. The analysis of pathology was carried out on 65 liver sections, and the serum content of liver enzymes, TME-o, Raz-ligand and IPM-u was measured. In rats that were given ethanol and injected with physiological solution, necro-inflammatory damage and expression of liver enzymes, TME-o, Ravz-ligand and IREIM-u were observed.

Rats injected with II-18VR are protected from necroinflammatory damage, and levels of liver enzymes, TME-o,

Rav-ligand and IRM are significantly reduced in them (29090).

Example 8. Suppression of hepatitis induced by Sop A using II -188P.

VaiBr/s mice were injected with 12 mg/kg concanavalin A (Sop A) together with an injection of murine II-18BP (Img/kg) 2 hours before or without Sop A administration and daily thereafter. Liver damage was assessed by determining serum levels of liver enzymes, TME-o, Rav-ligand and IEM-u. The histopathology of the liver of such mice and mice treated only with concanavalin A was compared.

Preliminary processing of I/-188P. significantly reduces the serum levels of liver enzymes and TMEs with no evidence of inflammation in histopathological examination compared to control animals not treated with Sop

AND.

Part II. Examples 9 and 10 relating to the use of II-18 inhibitors in arthritis

Example 9. Obtaining II -188Р with a Niz label (TI -18В R-Niz (ad)

For the experiment described in detail below in example 10, recombinant

I-188R. human, containing a label with b residues of Piv (I-I -18VR-Niv (ad), and was purified as described in Kigpei aI.,, 2000. The recombinant protein was purified to a degree of more than 9595 and its biological activity was detected by ip migo and ip mimo with high affinity for its ligands.

Production of recombinant proteins in other eukaryotic systems without tags or with tags that facilitate the purification of recombinant proteins is known to specialists in this field of technology. Well-known methods are available for the construction of suitable vectors containing DNA encoding I/-18B8P and suitable for transfection of eukaryotic cells in order to obtain recombinant II-188P. For expression in cells of the coding DNA

I/-18BP. (see, for example, Momisk et al., 1999), excised and inserted into expression vectors suitable for transfection of cells. On the other hand, such DNA can be obtained by PCR with appropriate sense and CM antisense primers. The resulting DNA constructs were then inserted into appropriately constructed eukaryotic expression vectors by methods well known in the art (Mapiaiiv, 1982).

Example 10. Blockade of endogenous II-18 in a mouse model of arthritis

Methods

Induction of collagen-induced arthritis (CA)

SIA was induced in male OVA/1 mice (age 8-12 weeks) by immunization with native bovine collagen type "-

And (SI), as described earlier (Ria(eg-2uregk ei a!., 1995). From the 25th day after immunization, mice were checked daily for the onset of the disease.

Processing pi -188R-bpiv p

Treatment of OVA/ mice immunized with SP was started at the first appearance of clinical signs of the disease. Recombinant 1/-18BP. human, containing a tag of 6 histidines (gpI/-18ВР - bpiv), was used to neutralize endogenous and -

I/18 in mice treated with collagen. gpII-18VR-6bpiz was injected intraperitoneally (i.r.-) daily for 7 (seven) days at 5 different concentrations - 10, 3, 1, 0.5 and 0.25 mg/kg/injection. Control mice received only vehicle (0.995% solution by weight) as a placebo. "

Evaluation of the development of the disease

Clinical evaluation (clinical indicators) t s At the first appearance of clinical symptoms, the mice were checked every day by a staff member associated with the treatment. h Each limb was evaluated for the severity of the disease (scale 0-3.5, maximum number of evaluations - 14/mouse). The development of edema (inflammation) was measured on the paws on which the signs of the disease appeared first using precise compasses (Rgosievi 2T, Kgoeriip I apdepitezwiesypik).

The development of the disease was evaluated daily for 8 days from the beginning, after which the mice were killed, and the paws were collected for histopathological examination. b Histological assessment of cartilage erosions and assessment of microinflammation

At the end of the experiments, that is, on the 8th day after the onset of the disease, the mice were killed, and the paws on which the signs of the disease appeared first were cut off. The joints were fixed, decalcified and filled with paraffin. 20 standard sections were made and stained with hematoxylin/eosin/safranin 0. Each joint was evaluated by 2 researchers who were not familiar with the treatment protocol (absence of destruction of cartilage or bone - 0; localized erosions of cartilage -

I) 1-2; more numerous erosions - 3; general destruction of cartilage and the presence of bone erosion - 4). The final scores for each mouse were the average of the scores for all joints evaluated. Evaluation of microinflammation under a microscope or synovitis was carried out on a scale from 0 to 4 as follows: absence of inflammation - 0; slight thickening of the litter layer and/or few infiltrating cells in the layer under the litter - 1-2; thickening of the litter layer and/or

Gee! a more pronounced influx of cells of the layer under the litter - C; the presence of cells in the synovial space and synovium strongly penetrated by many infiltrating cells - 4. ko Determination of anti-collagen antibodies

Antibodies against bovine type I collagen were tested using a solid-phase enzyme-linked immunosorbent assay (ELISA). Ids1 and Idsz2a titers were measured. Briefly, tablets were sensitized with 1 µg of bovine collagen, and then the nonspecific binding site was blocked with 0.1 M ethanolamine solution (Zidta). Serial dilutions (1:2) of sera were added and then incubated with isotype-specific goat anti-mouse peroxidase (Zocipegp Vioiespoiodu Avzovzia(ez, Vippipuoapat, AIII., OBA)) and substrate (5-aminosalicylic acid, Zidta).

Tablets were read at 492 nm. Titers were expressed as the mean of the 50th dilution, which gives a value half the maximum.

Analyzes 1-6

Levels I--6 were determined by commercial EGISA (KU systems, Minneapolis, Minnesota, USA). Biological activity

I/-6 was determined by proliferation analysis using VU cells. Briefly, in the round-bottom wells of a titration microplate, 5x103 cells of VO were placed in 200 μl per well of ERMI 1640 medium with 595 ECZ and incubated for 3 days using recombinant human I/-b6 as a standard (systems K 80,

Minneapolis, Minnesota, USA). At the end of the incubation, 0.5 μCi of ZINTIGIMIDIN (MEM-YUBiropy, Boston,

Minnesota, USA) per hole. After three hours, the cells were harvested, and thymidine incorporation was determined. The limit of detection for the bioassay for II -6 was pg/ml.

Statistical analysis 70 The significance of differences was assessed using the Mann-Whitney test using the Zidtaviai statistical analysis program and the StarpRaai Rgizt program.

The results

A mouse experimental model of SIA (collagen-induced arthritis) was used to evaluate the effectiveness

I/-18BP. as a remedy for arthritis. Administration of collagen and incomplete Freund's adjuvant to OVAL 75 mice causes the development of erosive inflammatory arthritis and represents an ideal opportunity to investigate the therapeutic potential of II/-18VR. For this purpose, endogenous 1-18 is neutralized using II-188P, and the effect on various pathogenic parameters is evaluated.

Studies are conducted depending on the dose. Three groups of OVA/1 mice with collagen-induced arthritis are treated therapeutically (ie, after the onset of the disease) with 5 doses of 1ІІ/-18ВР i.p. (intraperitoneal). 11-18 VR. in concentrations of 10, 3, 1, 0.5 or 0.25 mg/kg are administered at the first clinical signs of the disease. An injection of physiological solution (sodium chloride, Masi) is used as a control. In addition, inject i.r. 10,000 IU of interferon d (IEM-r) to evaluate the effect of IEM on this experimental model of arthritis. The effect on disease severity is monitored daily by visually evaluating each individual paw as described above. Mice are killed on the 8th day after the onset of the disease. with

The following indicators are evaluated: o - visual clinical indicators (each paw, scale 0-3.5) (Fig. 7, A and B); - swelling/swelling of the joints (in mm, measured with a compass) of the first sick paw, provided that it is the hind paw (Fig. 8); - the number of paws affected by the disease (Fig. 9); - indicators of erosion of the first diseased paw (destruction of cartilage - 0-4, Fig. 10); (Se) - in the histological analysis of the paw that became the first to develop arthritis (Fig. 11); "- - levels of antibodies against type II collagen (Fig. 12); - levels 1-6 (Fig. 13). "

Clinical severity of the disease

As can be seen in Fig. 7, A and B, the clinical severity of the disease is significantly reduced in the groups treated with 1mg/ml (P«e0.01) and 0.bmg/ml (0.01«P«0.05) and -18BP . Mice receiving a low dose of gpII/-188P (0.25 mg/ml) or a high dose of 1 ug/ml had a clinical score similar to that of the placebo group. Dose 1 mg/ml I. -188R. effective approximately as much as interferon p (marked in Fig. 7A as IEMB).

Inflammation and swelling (swelling) of the joints "

Macroinflammation (swelling) was studied by measuring paw swelling from day 1 after the onset of the disease to day 8 after the end of the experiment. The results are shown in Fig. 8, A and B. The effective doses of II/-18BP were 1, 3 and 10 mg/kg. Administration of lower doses does not have a beneficial effect on swelling of the paws. As can be seen in Fig. 8, A and B, "" interferon p (IRMB) at a concentration of L0000OME shows a beneficial effect on the swelling of the paws." Microsynovitis is checked after the end of the experiment on histopathological sections and expressed as indicators on a scale ("synovitis index"). The results of inflammation (swelling) and indicators of synovitis are summarized in the table. 1.

Although treatment with hppII-18BP at a dosage of 1 and Zmg/kg leads to a tendency to reduce swelling, treatment with any dose of II-18BP has only a limited effect on inflammatory synovitis (Table 1). (22) with with"

Phew

F)

GF Fig. 9 shows that the number of paws affected by the disease decreases after the introduction of II-18BP. In particular, therapeutic injections of I-18BP at doses of 1 and 00.5 mg/kg reduced the number of paws involved in the disease, which shows that the blockade of I-18 ip mimo delays the spread of arthritis to other joints. Treatment 1 and 0.5mg/kg I. -188Р. can even cause the normalization of arthritic joints.

Protection against destruction of joints

Treatment of gpII/-188R mice. leads to protection of joints from destruction (Fig. 10). The system of semi-quantitative assessment shows that a dose-dependent protective effect is visible on bone erosion, significant at 10 and Zmg/kg (P «0.05,

Fig. 10). Mice receiving mg/kg gpI/-18BP showed less erosion than mice receiving vehicle alone. because the protective effect was not detected at doses of 0.5 mg/kg and 0.25 mg/kg. Of interest was the fact that the protective effect on the joints at doses of 3 and 1Omg/kg gpI/-18BP. comparable and even more pronounced than the favorable effect of L0000ME interferon Dd (EM-r).

Fig. 11 shows the histology of a healthy (A) and diseased (B) joint in comparison with the joint of an animal treated with II -188P (C). Sections were made at the end of the experiment from the paws of the first ones that developed arthritis.

The joint of an arthritic mouse shows severe destructive arthritis with cartilage loss and erosions and numerous infiltrating cells in the inflamed synovium. In the joint of a mouse treated with gpI/-18BP, the cartilage looks almost healthy, despite the presence of cells of the inflammatory zone in the synovial space. Cartilage tissue is not only presented in greater quantity, but also looks smoother. 70 Anti-1I-18 treatment modulates the levels of anti-collagen type II antibodies

STA mice have increased levels of antibodies (DS1 and IDS02a against collagen type II) in the bloodstream. Antibodies of the isotype

Ido1 was associated with diseases mediated by TNaI, while antibodies of the isotype dose and isotype

Id02B5 are associated with diseases mediated by TNI1.

The isotypes of antibodies IdC1 and Idbg2a against collagen type II (SI) were determined in the serum of animals treated with 75 1-188P. (Fig. 12). The content of dS1 and Idb2a anti-SI isotypes does not change significantly on the 4th or 8th day (04, 08) of the clinical disease. However, after 8 days of processing gpII-18VR. a 2.6- and 3.4-fold decrease in the IdSiILdsga ratio is observed at doses of 1 and Zmg/kg, respectively. Figure 12 shows an experiment in which it is used

Zmg/kg. Essentially the same results are obtained using mg/kg of PP -18V8P. A reduced ratio of IdsS1Ldog2a antibodies against SII indicates a reduced concentration of antibodies against type II collagen of the IdS2a isotype and an increased

The concentration of antibodies against type II collagen of the Y9DO1 isotype, which suggests that in this model of arthritis there is a shift towards the disease mediated by TN2.

Reduction of II levels -6 after neutralization 1-18

In order to get an idea of the effect of II/-18 blockade, the content of I/-b6 in the serum of animals treated with II-18BP was measured. Fig. 13 shows that the content of biologically active 1-6 significantly decreases in animals that receive 1I-188BP, at all measured doses, i.e., 1, 3 and 1Omg/kg, just as in the case of interferon Dd (IEMB ).

Immunoactive levels of II/-6, measured in the serum of animals treated with Zmg/kg gpII-18VR, are significantly lower (P «0.0023), i) than in animals treated with physiological solution. Serum levels of IL-6 in diseased animals treated with 1, 3, or 10 mg "/-188P or 10,000 IU IEM-r are similar to levels in normal mouse serum (MM5) obtained from healthy animals, i.e., from animals without inflammatory disease. (Te)

The obtained results show that 1II/-18 regulates the levels of I/-6b at the onset of the disease. Since 1I/-6 was a marker of inflammation, these results show that treatment of diseased mice with II -18BP reduces inflammation in the animal. --

The following conclusions can be drawn from the experiments described above: "I - introduction of II -18BP reduces the clinical severity of arthritis; - I-18BP inhibits the further development or spread of the disease; and - input II -18V8R. reduces swelling; h- - input II -18BP. reduces the destruction of cartilage; - after treatment II -18В8Р. serum levels of II--6 decreased, and the IDS1/Ldog ratio decreased.

The data presented above show that neutralization of the biological activity of I/-18 after the onset of the disease is a disease-modifying antirheumatic therapy. The results clearly show that II-18 blockade reduces the clinical development of arthritis and, more importantly, delays the development of cartilage and bone destruction. Therefore, blockade of 1-18 with II-188P, anti-II/-18 antibodies, or any other 1-18 blocking agent represents a new disease-modifying antirheumatic therapy. "» The above description of specific embodiments of the invention shows the general nature of the invention, so that others skilled in the art, applying the existing knowledge, can easily modify and/or adapt such specific embodiments for different applications without departing from the basic concept, therefore, such adaptation and modifications should be -And included, and intended to be included, within the scope and limits of equivalents of the variants described. It should be understood that the phraseology or terminology used in this description is intended to be descriptive and not restrictive. b Part II Examples 11 and 12, related to inflammatory bowel disease Example 11. Expression of II-18BP by endothelial cells and macrophages during active Crohn's disease Collection of samples

Intestinal mucosal tissue samples are extracted when samples taken from patients with CO or OS are exhausted. 4) Fourteen patients with COPD (three men and eleven women) were included, the average age was 37.8 years (range 20-78 years), the duration of the disease was 8.3 years (range 1-21 years). In eight patients, the disease is localized in the ileum and in six - in the colon. Twelve patients are taking immunosuppressive drugs. Diagnosis

CO is assigned with the help of histopathological examination and is based on the following criteria: the presence of ulcers, an increased number of inflammatory infiltrate cells, and transmural inflammation. Seven patients with active COPD and seven patients with inactive disease are identified. There were no significant differences in age, localization of the disease, gender, drug therapy, and duration of the disease between patients with active and inactive CO.

The average age of 5 patients with OS (three men and two women) was 37.6 years (range 30-44 years). In all patients, the disease is localized in the colon, and all were treated with immunosuppressive drugs. The average duration of the disease is 4 years (range 1-9 years). Control samples are taken from 5 patients undergoing resection for disorders unrelated to IVO (three men and two women). The average age of this group is 55.2 years (range 24-76 years). In all patients, the disease is localized in the colon.

Semi-quantitative RT-PCR for human 1-18 and II -18yr 65 Total RNA is extracted from frozen samples of intestinal tissue of patients with CO, with OS or controls.

RNA extraction is carried out using Trizol (Birso) according to the manufacturer's recommendations. The obtained samples are evaluated quantitatively by measuring the absorbance at 2 bOhm. RNA integrity is analyzed by electrophoresis on 195 agarose gels. cDNA is synthesized from 1 μg of total RNA using the reverse transcription system

Rgoteda according to the manufacturer's scheme. PCR reactions are carried out in a total volume of bOmcl in the presence of 1 E

AtriaTad DNA polymerase (RegKip EIteg, Kospe, USA), 2.5 mM amtTR (Ategzpat, USA) and 5 Opmol of forward and reverse PCR primers. The reaction mixtures are incubated in a thermocell RTO-200 ReSheg EPesi (Mu Kezeagsp, USA) under the following conditions: denaturation - іхв. at 942C, renaturation - iv. at 552C, and stretching - 1 min. at 72 96.

Determine the optimal number of cycles for I.-18BP, II-18, and D-actin until saturation of the bands (respectively, Z1, 28, and 25). Create the following PCR primers based on the published sequences (АЕ110799, 049950, ХООЗ51): 70. 1-18, reverse - 5-ВСОТСАСАСТСАОСТАА-3; direct - 5-ВССТАСАОСТАОOSTОТАА-З"; И -188Р, direct - 5-АССТОТСТАСТОСАСТОАА-У; reverse - 5-ССАСОААСАТАССААСТСТО-3"; rD-actin, reverse - B-VSAOOAOASAATOATSTTOATSTTO-3; direct -5-OSTSASSATOSATOATOATATOSOS-3U. In order to exclude the amplification of genomic DNA contaminating the samples, PCR reactions are carried out in the absence of cDNA template.

PCR products (1μl) are analyzed on 195 agarose gels during electrophoresis in 1xTAE buffer. The size of the 75 PCR products is confirmed by comparison with a 1 tbp chain. ((irso) after staining the gels.

Determine the relative number of bands stained with ethidium bromide in UV light using analytical software Kodak Ridna! Zsiepsevs, and the results are given in the form of the ratio of the target gene (PI--188P, MI--18) and mandatory genes (pr-actin).

Obtaining monoclonal antibodies directed against FDI -188R

VAH V/s mice were injected subcutaneously in four limbs, as well as in the nape of the neck, with 5 μg of the isoform gpII-18VR-bniz (isolated in pure form from the cells of the ovary of the Chinese hamster, Ipiegrpagt Iaroghaiyogiev, Mev 7iopa,

Israel) in RV5 with an adjuvant (MRI-TOM emulsion, KIVI Ittipospet Kezeagsy, Ips.) on days 0, 7 and 28. 4 days after the 3rd immunization, lymph nodes are obtained and hydrolyzed with 2.4 μg/ml of collagenase (collagenase IM , UMohipipdiyup,

Viospetisa! Sogr.) and 0.195 DNase (Zidta). Then the selected cells are merged with myeloid cells Zr2/O with the use of PEG-1000 (RHOKA). Cells are resuspended in OMEM-E12, 1095 EC (birso) in the presence of NAT (5) (hypoxanthine, aminopterin, thymidine) and distributed in 96-well plates at a concentration of 5 x 10" cells/ml. Samples of supernatants of hybridoma cultures are screened for the presence of reactive antibodies in the analysis by direct screening. For this, tablets for EGISA are sensitized with goat anti-mouse antibodies (ab) (Chaskwop

Ittipo Kezeagsp, Mijap Apaiuisa, Switzerland) added to the supernatants of gshybridoma cultures and then is) biotinylated gpI/-18VR-bpase (isolated in pure form from CO5 cells as described in Momisk et al., "-- 1999), with or without the addition of hPPI/-18 (isolated in pure form from recombinant E. soy, Zegopo

Rpagtaseciisa!| Kezeagsi! Ypsilhe, Geneva), and finally detected by streptavidin and horseradish peroxidase (HRP) " (Chaskwop Ittipo Kezeagsi, Miiap apaimisa, Switzerland) using o-phenylenediamine (ORO) (Zidta). "at

Non-neutralizing antibodies are selected and subcloned. Mouse monoclonal antibodies IdsI 95-H20 were used in this study. in.

Immunohistochemical studies of the localization of cells positive in relation to II -18 years

Tissue samples are quickly frozen and stored at -80C. Serial cryostat sections (1μm) were obtained, placed in the environment on glass plates coated with poly-I-lysine Zirepgov/Riiz (Roihuabo, Riap-Iev-Otsaiev, "

Switzerland) and fixed in ice-cooled acetone. Localization of the II-18B8R protein. of a person is analyzed by immunohistochemistry using Mar 95-H20. After a brief rehydration in PB5, the sections pre-C with are incubated for 30 minutes in PB5 with the addition of 295 fatal calf serum (EC) (Satega, Ontario, "from Canada), 195 human serum (AB serum, Tgappeysiop Sepieg, Annemass, France) and 0.595 bovine serum albumin (BSA) (Zidta, St. Louis, MO, USA). Endogenous peroxidase activity is blocked by placing the plates in a solution of PB5 with 295 ESS, 195 human serum, 0.595 B5A and 195 hydrogen peroxide (H2O»z) (Bika, Switzerland) for 71 hours. After washing with PBO5, the sections are incubated overnight with undiluted culture supernatant Mab 95-H20. After another wash with PB5, the sections are incubated for 1 hour with

Ge» with biotinylated goat anti-mouse antibodies (daskwap Ittipo Kezeagsi, Miiap Appaiuiisa, Switzerland) (μg/ml) in РВ5 containing 0.595 VBA. Staining sensitivity was increased by incubating with the avidinON/biotinylated NCR complex (Mesiavsiaip Eilishe AVS Kiy, Mesiog Irogagiez, California, USA) for 20 h. Then the plates are washed with RVZ and detected using a solution of 3095. H 205, 3,3-amino-9-ethylcarbazole (AES) (Zidta) and M,M-dimethylformamide (MegSK) in acetate buffer, pH 5. After m. counterstaining with hematoxylin (Zidta) sections are covered with glycerin, and coverslips are used. Mouse antibodies are still used as an isotype control (system K and 0).

In order to identify the cellular localization of human I-18BP, two immunohistochemical 252 studies with staining on sections of the intestinal mucosa are performed. After re-hydration in RV5 during

GF) 10 min. sections are pre-incubated in RVZ with the addition of 295 EC5, 195 human serum and 0.595 B5A. For co-localization with endothelial cells, sections were incubated overnight with biotinylated Mar 95-H20 de (20 μg/ml) mixed with mouse anti-human POP1 conjugated with RITS (1:50) (Reagtipdep, CA,

USA), in RVB/0.595 VZA. For co-localization with macrophages, sections are incubated overnight with biotinylated Mar 60 95-H20 (20 μg/ml) mixed with anti-human COb68 conjugated with RITS (1:25) (Oako, Denmark). After washing in РВ5 for 1 hour, streptavidin Texas-K (Zociegp Vioiespoiodu Avzosia (ez, AI, USA) is added).

The plates are washed again, and the sections are covered with film, and coverslips are used. As an isotype control, biotinylated mouse antibodies Ide1 (Rpagtipdep) are used, followed by Texas-Kea streptavidin.

Cell Culture Human Umbilical Vein Endothelial Cells (NOMES) were cultured (Ciopeia Sogr., San Diego, CA)

using a medium for growing endothelial cells (ESM) with the addition of recombinant human epidermal growth factor (PES) (TOng/ml), hydrocortisone (μg/ml), gentamicin and amphotericin B (5μg/ml), bovine brain extract (BVE) (Zmg/ml) and 295 fatal calf serum (EU) (Siope-ysv Sogr., San Diego, CA), according to the manufacturer's recommendations. A layer of human fibronectin (1 Ωkg/cm7) (Voepjipdeg, Mannheim) was previously applied to the dishes with tissue cultures. Cells were incubated in a humid chamber with 595 CO", and experiments were carried out using NOMES cells in the 3rd passage. Cells

NOMES treated 1I-1rd human (1Ong/ml), TMERo, (TOng/ml) and IEMUu (2Ong/ml) (systems K and 0, Germany) for 24 hours. At the end of the cultivation period, the cells were collected, DNA was isolated and subjected to RT-PCR for the analysis of 70 transcripts of 1I/-18BP and 1-18 mRNA. Supernatants were collected, and protein expression of II-188BP and 11-18 was analyzed in them by the EGISA method.

The human monocyte line TNR-1 was maintained in suspension culture in KRMI medium with the addition of 1090 heat-inactivated ES, I-glutamine (2mM), penicillin-streptomycin (1U/ml) (Sirso VK, and Me

Thesppoiodiyev) and d-mercaptoethanol (50 μM) (Rioka). Cells were incubated in a humid chamber at 596 °C and maintained at 1:10 every 5 days. Three days before the experiment, human monocytes were differentiated at a density of 0.4X10 cells/ml with vitamin OZ (8OHM) (Viotoi Rezeagsi I Arogaogieev, USA), and they were left for attachment.

After differentiation, I RBZ (10 ng/ml) (SaIriosnet), 1-1 human dD (1 TOng/ml) was added to the cell culture,

TMEo, (TONg/ml) and IEMUu (2Ong/ml). After 48 hours, the supernatants were collected, and the expression of II-188P and

I--18 by the EGISA method.

Product measurement! -188P and 1-18 people

Presence of I/-188R. were evaluated by the EGISA method in NOMES cell-free supernatants, unstimulated and stimulated for 24 hours. with a mixture of cytokines (1-1 p, TMEo, IRMu) as well as the TNR-1 cell line, unstimulated and stimulated for 24 hours. a mixture of cytokines (GR, 1-1 p, TMRo, IEMu). To do this, immobilized Mar (clone 657.27 in the amount of 0.5 μg per 10 μl per well) was applied overnight to two tablets,

IpFegrpapt I arogagiez, Mez yiopa, Israel) directed against gpI/-18VR (isoform a). Then (o) soluble gpI./-188P was determined using rabbit polyclonal antibodies (dilution 1/10000) directed against gpI/-188P-bpiz (isolated in pure form from Chinese hamster ovary cells, Ipiegrpagt

I arogyaugies, Mez opa, Israel), followed by incubation with affinity purified goat anti-rabbit to, with peroxidase conjugated (diluted 1/20000) (daskwop Ittipo Kezeagsp, Miiiap appaiuiis, Switzerland).

Immobilized Mar, as well as rabbit polyclonal antibodies, were tested by the Western blotting method to confirm the specificity to III-18BP. Recombinant gpIII-18VR-bpiz was used as a standard. Sensitivity "

EPIZA 100 Opg/ml. In parallel, the amount of II-18 was determined using a set for EGI5A for human II-18 (MVI, Ittipoyesi). The sensitivity of EGISA was 12.5 pg/ml. (Se)

Expression of gpII-18VRa-Nivb in CHO cells of M

Recombinant II/-188R. human (gpII/-18VRa, label Nizb) was isolated in pure form from the cells of the ovary of the Chinese hamster (Ipiegrpagt I arogayugiez, Mez 7iopa, Israel).

The results

Expression of tRNA-18BP transcripts in intestinal biopsies « 20 Analysis of 1/-18BP mRNA expression. was carried out by the OT-PCR method in tissue homogenates obtained during the operation of colon samples of patients with active CO or inactive CO, or with OS and without inflammation of intestinal tissues (Fig. 14). Transcripts of I/-18BP and actin were detected in all tested intestinal homogenates. In a similar way, transcripts 1-18 were detected in all tissue homogenates in the case of CO, OS or control without IBO (Fig. 14A).

The ratio of II -18BP or II -18 to control actin mRNA levels shows a significant increase (as described below) of 415 transcripts in both II/-188BP and I/-18 biopsies obtained from patients with active CO compared with -1 biopsies of patients with inactive SO, OS or controls without IVO (Fig. 14, B and C). Such data showed that

I-18VR. is activated in mucosal tissue during active CO, and were the first evidence that the level (o) expression of II -18BP clearly distinguishes active CO from inactive CO, OS, and controls without IBO. Statistical analysis of II-18BP mRNA transcript expression in intestinal biopsies

Analysis of variance was performed by pooling all available data. The feedback clearly shows a statistical outlier, which refers to the results for one of the patients from the group with active CO (a very high ratio of OO for

Ф I--18, namely, 16252, and a low ratio of OO for II -18ВР, namely, 1058). The only and very atypical pair of measurements does not give the possibility of not recognizing the sample as unbiased according to the AMOMA model (the value of p according to the criterion

Shapiro-Wilks test for normality of residuals "0.0001). Therefore, it was decided to ignore the indicated pair of measurements in the VAT statistical analysis.

The AMOMA model used takes into account the group factor (control, active CO, inactive CO and OS), (F) protein (II -18 or II -18ВР) and the number of patients (23 patients). There is a significant difference in the group (value p«0.0001). It is also interesting to note that the difference in OO relations between I/-18 and I/-188P is not significant (value p-0.369). In addition, the correlation between II/-18 and II-18BP expression was also performed. The correlation coefficient between 1-18 and I -18BP is equal to 0.67, which suggests the presence of a strict relationship between the OO ratio measured for II -18 and Iii -18BP. Next, for the results related to the group effect, the ZspepPe method was used to compare different groups. It can be concluded that the ratio of ОЙ for active CO is significantly higher than for control (43280), OS (12590) and especially inactive CO (4580) both for the expression of II -18 and for the expression of II -188ВР.

Immunohistochemical localization of I-18BP in intestinal tissues In order to evaluate the cellular expression of I/-18BP in IP, using specific monoclonal antibodies against PII/-188P, immunohistochemical studies were performed on cryostat sections obtained from intestinal tissues of patients with active CO and controls without IVO. II-18BP-positive cells were found in the lamina propria, submucosal tissue layer, and in the muscular lamina of the mucosa (not shown). Positively stained cells, present in the lamina propria and the tissue layer under the mucous membrane, possessed abundant cytoplasm, vesicular reticular nuclei and morphologically resembled tissue macrophages. In the muscular plate of the mucous membrane, positively stained cells had numerous cytoplasm with a lumen open for some time in the middle, which implies positive staining of capillaries, and morphologically resembled endothelial cells.

Large vessels were also specifically stained with monoclonal antibodies against PI/-18BP. A significant increase in the number of positively stained cells, observed in samples obtained from patients with active 7/0 CO, compared to samples obtained from controls without IVO, correlates with increased expression of II/-18ВР, observed in RT-PCR analysis . Adjacent sections were incubated with the appropriate mouse isotype control.

Identification of cells that produce II-18VR in biopsies of the mucous membrane

Cells positive for I./-18BP, present in inflamed intestinal tissues, were identified with 7/5 using specific markers of macrophages (anti-COb8) and endothelial cells (anti-SBZ1) (not shown).

СОрб8-positive cells (green color), and I. -18ВР - positive cells (red color) are found in the lamina propria and the tissue layer under the mucous membrane of intestinal tissues with active CO (not shown). СОЗ31-positive cells (green color), and I/-18ВР - positive cells (red color) are found in the tissue layer under the mucous membrane. In order to confirm that macrophages and endothelial cells are also 1I/-188P positive, both colors - green from anti-СОб68 or anti-СОЗ31 and red from anti-II -18ВР - were analyzed together, while showing that all cells binding antibodies against II-18BP were either СОб8-positive or

СОр31-positive (orange-yellow color). Double immunostaining showed that macrophages and endothelial cells were the main source of I/-188P staining in inflamed tissues obtained from patients with CO. high school

Expression of mRNA-18Br and protein by endothelial cells

In order to investigate the ability of human endothelial cells to produce II-18BP, as well as to confirm (8) the result obtained by immunostaining and RT-PCR on general biopsies, further experiments were carried out by the RT-PCR method on human umbilical vein endothelial cells (NIOMES) ( Fig. 15A).

Endothelial cells were treated with a mixture of cytokines (MII-1 dD, TMEo, IEMUu) and collected after 24 hours for He3o RNA extraction and PCR analysis. The ratio of the content of I/-188P to the levels of control mRNA of actin showed an increase after 24 hours. the number of I/-18BP transcripts in treated cells compared to unstimulated cells. In addition, it turned out that 1I-188P mRNA is apparently significantly expressed in endothelial cells. "AND

The mRNA level of II -188P was also analyzed, and its slight increase was found in the treated cells. However, MENK

I--18BP was not expressed in unstimulated endothelial cells. at

The study by the EGISA method of culture supernatants of unstimulated cells (medium) and NOMES, treated with it for 24 hours, showed that the protein I/-188P. present both in the medium and in stimulated cells, increasing 30 times after 24-hour stimulation (Fig. 158).

Expression of protein II -18rBr by monocyte cell line (TNR-1)

Expression of 1-18 and I/-188P. were analyzed in the culture supernatants of unstimulated and stimulated "differentiated TNR-1 cells by the EGISA method (Fig. 15C). In this experiment, an increase in the expression of I/-18BP was detected after 48-hour stimulation of GR, MI-1 dD, TMRo, IRMu. At the same time, the secretion of 1-18 increases after stimulation of ts (Fig. 15C). "» Summary

This study characterized the expression and localization of I-18BP in the mucosal tissue of patients with Crohn's disease and nonspecific ulcerative colitis. Using the scheme of semiquantitative RT-PCR, it was found that the number of 1I-188R mRNA transcripts in biopsies of the mucous membrane of patients with active disease

Crohn's is higher compared to biopsies from patients with non-specific ulcerative colitis and controls without inflammation.

F Immunohistochemical analysis of biopsies of the mucous membrane showed that the I/-18BP protein is localized in endothelial cells and macrophages infiltrating the mucous membrane during Crohn's disease. Expression of shu 20 I--18В8Р. by endothelial cells and activated macrophages is confirmed in primary human umbilical vein endothelial cells (NOMES) and the TNRI monocyte cell line stimulated by IP migo. After 4) stimulation, the specified cells secrete biologically active II -188V R.

Example 12. Treatment with II-18 inhibitors improves experimental colitis

Materials and methods

Mice and induction of colitis

All experiments were approved by the Animal Research Ethics Committee of the University of Amsterdam. o Mice VAY V/s received in Nagiap Zrgadoe Yuamieu Ips. (Horst, the Netherlands). Mice were kept under standard conditions and provided with drinking water and food (AM-II 1Omm Nore Ragtz, The Netherlands).

Experiments were performed on female mice of VAI V/s at the age of 8 and 10 weeks. Colitis was induced by rectal 6o administration of two 2-mg doses (with a 7-day interval) of 2,4,6-trinitrobenzenesulfonic acid (TMV5) (Zidta

Spetis! So., St. Louis, Missouri, USA) in 4095 ethanol (MegsK, Darmstadt, Germany) using a vinyl catheter, which was inserted into the anus at Ccm (10 mice per group). At the time of instillation, mice were anesthetized with isoflurane (1-chloro-2,2,2-trifluoroethylisoflurane difluoromethyl ether, AbBoi I arogaigiez tsa. Oceeprogotzdi, Kent, OK) and were held upright for 60 seconds after instillation. Control 65 mice were subjected to an identical procedure, but saline solution was installed. All mice were killed on day 9 after the first administration of TMV5 (ie, 48 hours after the second administration of TMV5).

Mice were treated intraperitoneally with II -188R. of a person in 50 μl of 0.995 physiological solutions.

NI--18BP is a 76-fold histidine-tagged recombinant human protein produced in an expression system

SNO NII-18BP is biologically active, as it inhibits the production of IEM in the KO-1 cell line and reduces the production of IRM by mouse spleen cells (Kigp eg a!., 2000).

Assessment of inflammation

Body weight was recorded daily. After killing, spleens, caudal lymph nodes and colons were collected.

The colon was removed through a moderate incision and opened longitudinally. After the removal of fecal material, the mass in the wet state of the distal part in the BSM was recorded and used as an indicator of 70 disease-related thickening of the intestinal wall. Then, the colon was divided longitudinally into two parts, one of which was used for histological evaluation.

Histological analysis

Colons, divided longitudinally, were rolled, fixed in 495 formalin, and embedded in paraffin for routine histology. Two researchers who treated mice in a blinded manner evaluated the following parameters: 1) percentage of affected area, 2) hyperplasia of follicular aggregates, 3) edema, 4) erosion/ulceration, 5) crypt loss, and b) infiltration of mono- and polymorphonuclear cells. The percentage of the affected area and the loss of the crypt were evaluated on a scale ranging from 0 to 4 as follows: 0 - normal state; 1 - less than 1095; 2 -1095; Z - 10-5095; 4 - more than 5095. Erosion was defined as 0, if the epithelium is unchanged, as 1 - in the case of damage to the lamina propria, as 2 - in the case of damage to the layer of tissue under the mucous membrane, and as З - in the case that the cover by ulcers is transmural. The degree of severity of other indicators was assessed on a scale from 0 to 3 as follows: 0 - absence; 1 - weak manifestation; 2 - moderate; and Z is heavy. Such a scale contains from 0 to 26 points maximum.

Colon homogenates

Homogenates were obtained from the colon using a tissue homogenizer in 9 volumes of Greenburger's buffer for lysis (ZOOMM Masi, 15 mM Tris, 2 mM MosIi), 2 mM triton (X-100), pepstatin A, leupeptin, aprotinin (all 2 ng/ml ), pH 7.4). Tissues were lysed for 30 minutes on ice followed by two-fold centrifugation (1 rpm, 14,000 rpm). Before use, homogenates were stored at -2026.

Cell culture and EGISA for the determination of cytokines

To obtain a suspension of cells of the spleen and caudal lymph nodes, mesh filters with (Se) filter cells (Wesiop/Oiskipwop I arzhage, New Jersey, USA) were used. Cells were suspended in medium

KRMI 1640 (Wiomupichakeg-Woephipdeg, Verviers, Belgium) containing 1095 EC and ciproxin (1 Ωkg/ml) (Zidta --

Spetis! So., St. Louis, Missouri, USA). Spleen cells were centrifuged with sterile Ficol (Rpagtasia, "Zh

Uppsala, Sweden), mononuclear cells were transferred to KRMI, and cell counts were performed in the cell suspension. co

Cells with a total number of 1x10? per mouse was incubated three times in wells of 200 μl. ERMI (Wiomuakeg Eigore, A Satrgeh Sotrapu, Verviers, Belgium) containing 1095 fetal calf serum. -

Cells were stimulated by pre-coating a layer of antibodies against POPs (concentration 1:30; clone 145.2C11) and soluble antibodies against CO28 (concentration 1:1000; RPagtipdep). After 48 hours, the supernatants were removed, and the concentrations of IEM-u (Rpagtipdep) and TME-o (KO vzuvietv, "

Abingdon, United Kingdom).

Flow Cytometry With s Isolated spleen cells were washed with GRasz buffer (PB5 containing 0.595 B5A, 0.0 mmol/l EDTC, and 0.0195 "» sodium azide) and kept on ice for the remainder of the procedure. Cells (2.10 9 cells per well, " 9b-well microplate with an M-shaped well, Sgepeg V. M. Iarog (esppiek, AIrep aap de Ki)p, the Netherlands) was incubated with the following antibodies (ptAbBvg): rat anti-mouse CO4 conjugated with Su-chrome (clone vMa4-5), rat anti-mouse COb9 conjugated to Ris, and rat anti-mouse CO25 conjugated to it. ES (Rpagtipdep, San Diego, CA). Lymphocytes were directed using the front and side scatterers

Ge") using the RGASZsap flow cytometer, in combination with the Raszsap software (Vesiop

Oisciphop, Moshpiaip Miem, USA), and 5000 cells were counted. The results were expressed as percentages of missed cells, t-positive in relation to the tAbBz used. -about 70 Statistical analysis

Values are given as mean and SD for the treated group. Differences between groups are analyzed using the nonparametric Mann-Whitney O-test. Mass changes are subsequently analyzed by one-way analysis of variance. P "0.05 is considered significant. ZRO statistical software (ZR Ips., Chicago, USA) is used for all analyses. 52 Results

F! I/-18BP protects against weight loss in a murine model of colitis

In order to investigate the role of 1/-18 in experimental colitis, and in particular, the protective effect of 1/-18-binding protein (I/-188P), TMB5-colitis was induced in ВАЙВ/с mice. This model includes the local influence of trinitrobenzenesulfonic acid (TMB5) in 4095 ethanol. It causes a delayed-type hypersensitive 60 reaction to a hapten(trinitrophenyl)-modified autoantigen, and the reaction is a T-type reaction with increased production of pro-inflammatory cytokines.

Mice were treated daily intraperitoneally (ir) with human I-18B8R or control.

Daily intraperitoneal doses in the range from 12.5 μg to 5 μg pPI/-188P did not affect the severity of the disease (data not presented). However, the use of a dose of 2000 µg of PI./-18B8P, administered intraperitoneally daily, was effective in reducing the weight loss associated with the induction of the disease.

As expected, intrarectal instillation of TMV5 leads to diarrhea and exhaustion. As can be seen in Fig. 16, animals in both treated groups lost 1595 of their initial weight on day 3. However, unlike control mice, beginning on day 4 after instillation of TMB5, animals treated with PII -18VR quickly recovered the initial weight loss, returning to baseline body weight on day 8. In control mice, a second administration of TMV5 on day 8 again leads to significant weight loss, which is essentially prevented by administration of MI-18VR. Administration of PII-18VR to saline-treated mice has no effect (data not presented). Therefore, the introduction of PII-188BP significantly attenuated the weight loss associated with colitis induced by TMV5 (p<0.05).

Effect on inflammation parameters 70 On the 10th day, the mice were killed, and the mass of the last bsm of the colon was determined (Fig. 17A). In TMB5-colitis, the mass of the colon increased in comparison with the mass of the colon in mice treated with saline. Such weight gain is significantly less in mice treated with PII-18VR 2 (181.bmg -11.4 compared to 268mg -27.3 in mice treated with physiological solution (p«0.05). It was previously reported that TMV5-colitis is associated with increased migration of cells into the caudal lymph nodes (Satodaiyo et al., 2000).1II/-188P 75 treatment reduced the number of cells infiltrating the caudal lymph node compared with the number of cells in the caudal lymph node of TMB5-mice (TMB5-colitis mice), treated with physiological solution (Fig. 17B).

Changes in the expression of CO69, which is an early marker of T-lymphocyte activation, were determined by Raszsap analysis (Fig. 17 C).

The percentage of splenic СО4" cells expressing СОб6О in TMB5-treated mice was 11.495, but in

The percentage of СО4"/СО69" in TMB5 mice treated with PII .18-BP was 7.395 (Р«0.05).

Production of cytokines by the spleen, caudal lymph nodes and colon homogenates

In order to investigate the effect of PII -18VR on the ability of T-lymphocytes to synthesize pro-inflammatory cytokines after activation of the T-cell receptor, cells were isolated from the caudal lymph node and spleen and stimulated for 48 hours with antibodies against СО3З/СО28. The production of IEM in and TMEo was measured in the supernatants. (Figure 18). No significant differences were observed between the production of cytokines in mice treated with PII -18BP, with | control mice. Therefore, the neutralization of I/-18 MI/-18VR does not cause a general decrease in the ability of

T-lymphocytes respond to the activation of the T-cell receptor.

Homogenates of the colon were analyzed for the content of cytokines in them, determining the local production of cytokines (Fig. 19). No differences were found in the levels of IEM in the colon homogenates of TMV5 mice and TMV5 mice treated with PII -18BP (134 pg/ml.-7.8 and 139 pg/ml.-23, respectively). However, TMEou levels in colon homogenates of mice treated with PII/-18B8R significantly decreased - from 11Opg/ml - 3 in TMB5-mice to 5Opg/ml2.1 in "k-

TMB5-mice treated with PI! -188R.

Histological results of

In order to investigate whether the reduction of inflammatory parameters mediated by PII -188R also affected (se) histological indicators, histopathological analysis was performed on paraffin sections. The total score of 30 inflammation in histology is significantly reduced for mice treated with PII -18BP, in comparison with the assessment of control mice (15.9--1.1 in untreated mice to 9.8-1.3 in mice treated with PII -18VR), mainly due to a decrease in the number of leukocytes infiltrating the mucous membrane (p « 0.05). A noticeable result is the complete absence of mucosal ulceration in mice treated with PI/-188P (p "0.05). The results are summarized in Table 2 below. An almost complete prevention of ulceration in mice is noticeable , from processed FDI -188R.

I" 00010 we, treated with fisol, romin TMvv vysh, treated with myslvVe, etc.

F ich » z

F

5 o ko Polyclonal antibodies against those! -18 protected against the disease in a murine model of colitis induced by dextran sulfate sodium in In this model, dextran sulfate sodium (055) was given in drinking water, starting from day 0 until the day the animals were killed. Polyclonal antibodies against I/-188P were injected i.r. on days 0, 4 and 8. The doses were 200 and 400 μl of rabbit serum. The highest dose (400 μl) produced a significant reduction in weight loss, clinical score, rectal bleeding, and colonic shortening (not shown). Treatment with rabbit anti-ties! -18-antibodies slowed down the initiation of the disease and prevented its development (not shown). 65 Summary

Example 12, presented above, demonstrates that neutralization of II-18 by administration of either PII-188P or a polyclonal antiserum against II-18 effectively reduces the severity of experimentally induced colitis in mice.

Mice treated daily with PII-18VR, intraperitoneally, quickly recovered after the primary

Weight loss compared to control mice. Other parameters of colon inflammation, measured by colon mass and cell influx into the caudal lymph node, were reduced in PII-18VR-treated mice.

The histopathology of the treated mice was characterized by a decrease in the severity of tissue destruction (coverage by ulcers), and the number of infiltrating cells significantly decreased. Action of pPI/-188R. was also systemic, as demonstrated by reduced expression of СОб9 by spleen cells. 70 Local production of TME o, measured in homogenates of the colon, significantly decreased in TMB5 mice treated with PII-18B8R. This circumstance shows that TME o; plays an important role in the development of the disease. Levels of IEM U

TMB5-mice and TMB5Z-mice treated with PPI/-18BP are compared, which can be explained by redundancy

IMERU-inducing stimuli.

As a result, the data presented above demonstrate the beneficial effect of I/-18 inhibitors in the treatment of 75 inflammatory bowel diseases.

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Claims (24)

2 Formula of the invention 1. Use of an I/-18 inhibitor for the manufacture of a medicinal product for the treatment and/or prevention of cartilage destruction, where the I/-18 inhibitor is selected from antibodies against 1I/-18, antibodies against each of the subunits of the I/-18 receptor, 1 /-18-binding proteins, their isoforms, muteins, fusion proteins, functional derivatives, active fractions or derivatives with a cyclic rearrangement, having essentially the same activity as the I/-18-binding protein. 2. Use according to claim 1, where the cartilage destruction is associated with rheumatoid arthritis, juvenile rheumatoid arthritis or infectious synovitis. 3. Use of the II/-18 inhibitor for the manufacture of a medicinal product for the treatment and/or prevention of psoriatic arthritis, where the I/-18 inhibitor is selected from antibodies against 1II/-18, antibodies against each of the subunits of the receptor 1/-18, 1I/- 18-binding proteins, their isoforms, muteins, fusion proteins, functional derivatives, active fractions or derivatives with a cyclic rearrangement, having essentially the same activity as the I/-18-binding protein. 4. Use according to any of the preceding clauses, where the II-18 inhibitor is an antibody against II-18. 5. Application according to claim 4, where the antibody against II-18 is a humanized antibody against II-18. 6. Application according to any of claims 1-4, where the antibody against II-18 is an antibody against human II-18. 7. Use according to any of the preceding clauses, where the II-18-binding protein is treated with polyethylene glycol. 8. Use according to any of the preceding clauses, where the P/-18 inhibitor is a fusion protein that contains all or part of the II-18-binding protein fused to all or part of the immunoglobulin, and where the fusion protein connects with II -18. (at) 9. Application according to claim 8, where the fusion protein contains the entire constant region of the immunoglobulin or its part. 10. Application according to claim 9, where the immunoglobulin is an immunoglobulin of the IdDs1 or Ids2 isotype. 11. Use according to any of the previous items, where the medicinal product additionally contains interferon 12. Application according to claim 11, where the I/-18 inhibitor is used simultaneously, sequentially or separately from interferon-B. - 13. Application according to any of the previous items, where the drug additionally contains an antagonist of tumor necrosis factor I (TME). 14. Application according to claim 13, where the TME antagonist is a soluble TME receptor. i-th 15. Application according to claims 13 or 14, where the I/-18 inhibitor and/or interferon is used simultaneously, consecutively or separately from the TME antagonist. 16. Use according to claims 13 or 14, where the I/-18 inhibitor is used in an amount from about mg per kg of body weight to about Zmg per kg of body weight. " 17. Use according to any of the preceding clauses, where the II-18 inhibitor is administered subcutaneously. 18. Use according to any of the preceding clauses, where the II-18 inhibitor is administered intramuscularly. - with 19. Use according to any of the preceding clauses, wherein the II-18 inhibitor is administered daily. c 20. Use according to any of the preceding clauses, wherein the II-18 inhibitor is administered every other day. "» 21. Use of an expression vector containing the coding sequence of the 1/-18 inhibitor in the manufacture of a medicinal product for the treatment and/or prevention of cartilage destruction, where the 1-18 inhibitor is selected from II/-18-binding proteins, their isoforms, muteins , fusion proteins, functional derivatives, active -I fractions or derivatives with a cyclic rearrangement, which have essentially the same activity as II -18-binding protein. 22. Application according to claim 21 for gene therapy. F 23. Use of a vector for the induction and/or enhancement of endogenous production of the I/-18 inhibitor in a human cell in the manufacture of a medicinal product for the treatment and/or prevention of cartilage destruction, where the 1-18 shu 20 inhibitor is selected from the II/-18-binding proteins, their isoforms, muteins, fusion proteins, functional derivatives, active fractions or derivatives with a cyclic rearrangement, which have essentially the same activity as II-18-binding protein. 4) 24. Use of a cell genetically modified to produce an I/-18 inhibitor in the manufacture of a medicinal product for the treatment and/or prevention of cartilage destruction, where the I/-18 inhibitor is selected from I/-18-binding proteins, their isoforms, muteins , fusion proteins, functional derivatives, active fractions or derivatives with a cyclic rearrangement, having essentially the same activity as II-18-binding protein. F) name) 60 b5