Pharmaceutical composition for treating non-alcoholic fatty liver or non-alcoholic steatohepatitis, comprising Anti-ICAM-1 antibody as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for treating non-alcoholic fatty liver or non-alcoholic steatohepatitis, the pharmaceutical composition comprising an anti-ICAM-1 antibody as an active ingredient. The present invention takes into account that non-alcoholic steatohepatitis can be treated by regulating inflammatory responses through ICAM-1 targeting in liver tissue, and is based on the discovery that using the anti-ICAM-1 antibody in immunosuppressive therapy following a liver transplant effectively suppresses inflammatory response in hepatocytes and stably maintains liver function, and the expression of genes associated with the mechanism of non-alcoholic steatohepatitis is effectively regulated in inflamed liver tissue through administration of the anti-ICAM-1 antibody. Through this, the anti-ICAM-1 antibody according to the present invention is expected to be effectively used for treating non-alcoholic fatty liver or non-alcoholic steatohepatitis.

Description

Pharmaceutical composition for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis comprising anti-ICAM-1 antibody as an active ingredient

[0001] The present invention relates to a novel pharmaceutical composition of an anti-ICAM-1 antibody, and more particularly, to a pharmaceutical composition of an anti-ICAM-1 antibody for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis.

[0002] This invention claims priority to Korean Patent Application No. 10-2024-0081818, filed June 24, 2024, the entire contents of which are incorporated herein by reference.

[0003] Non-alcoholic steatohepatitis (NASH) is a condition in which fat accumulates, potentially causing liver damage or cirrhosis. It has characteristic pathogenesis depending on the progression stage. Specifically, it starts with insulin resistance and dysregulation of glucose / lipid regulation, and progressively progresses from non-alcoholic fatty liver disease (NAFLD) to NASH (divided into stages F0, F1, F2, F3, F4) and further progresses to cirrhosis (compensated → decompensated). Recently, due to concerns about the mixing of pathogenesis and the allowance of free alcohol consumption, it is also called metabolic dysfunction-associated steatohepatitis (MASH).

[0004] Non-alcoholic fatty liver disease (hereinafter referred to as 'NAFLD') is a liver disease that is rapidly increasing along with metabolic syndrome such as obesity, diabetes, and high blood pressure. It can progress to cirrhosis or liver cancer. Recently, the incidence of NASH, a severe inflammatory form of NAFLD, and NASH-related cirrhosis have been rapidly increasing, and many studies are being conducted to develop treatments for it.

[0005] Specifically, global big pharma such as AstraZeneca and Gilead Science have failed in their attempts to enter the NASH treatment market one after another, and Intercept Pharmaceuticals' Ocaliva (active ingredient: obeticholic acid) failed to obtain FDA approval for NASH treatment again in 2023, following its successor in 2020. Currently, there are five types of candidate drugs for NASH treatment: farnesoid X receptor (FXR) agonists, thyroid hormone receptor (THR)-β agonists, glucagon-like peptide (GLP)-1 agonists, and peroxisome proliferator-activated receptor (PPAR) and fibroblast growth factor (FGF) 21 agonists. However, there is still no breakthrough treatment that can lead the market. Considering the burden of the disease, it is a disease with high unmet needs where patients' demands for treatment options are not being met, and the development of treatments for it is urgently needed.

[0006] Meanwhile, ICAM-1 (Intercellular Adhesion Molecule 1) is a 90 kDa type I cell surface glycoprotein consisting of five extracellular immunoglobulin superfamily domains, numbered from the N-terminus to the C-terminus, designated as domains 1 to 5, a transmembrane domain, and an intracellular domain, which mediates leukocyte / leukocyte interactions, such as interactions between T cells and antigen-presenting cells, and leukocyte extravasation into tissues during inflammatory processes. Studies on MD-3, a monoclonal antibody that binds to human ICAM-1 protein, have confirmed its effectiveness in treating autoimmune diseases, such as autoimmune encephalitis and rheumatoid arthritis, in non-human primate models, and the interaction between ICAM-1 on endothelial cells and LFA-1 (leukocyte function-associated molecule-1) on T cells is known to regulate T cell adhesion and migration, and can induce allograft rejection. Furthermore, the interaction between ICAM-1 on dendritic cells and LFA-1 on T cells induces T cell activation, which can induce immune responses such as inflammation. Therefore, the inhibitory effect of anti-ICAM-1 antibodies on transplant rejection has been confirmed. However, it is not yet known whether anti-ICAM-1 antibodies can be applied to the treatment of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

[0007] The present invention aims to address various issues, including those described above, and to provide a novel pharmaceutical composition capable of more effectively treating NASH. However, the scope of protection of the present invention is not limited to the aforementioned objective.

[0008] The present invention provides a pharmaceutical composition for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, comprising an anti-ICAM-1 antibody as an active ingredient.

[0009] The present invention also provides a method for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ICAM-1 antibody.

[0010] The present invention also provides a method of treating a subject suffering from nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, comprising administering to the subject a therapeutically effective amount of an anti-ICAM-1 antibody.

[0011] The present invention also provides a use of a composition comprising an anti-ICAM-1 antibody as an active ingredient for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis.

[0012] In addition, the present invention provides a use for preparing a non-alcoholic fatty liver disease or non-alcoholic steatohepatitis treatment preparation comprising an anti-ICAM-1 antibody as an active ingredient.

[0013] In one embodiment of the present invention, the anti-ICAM-1 antibody may include, but is not limited to, a CDR derived from a heavy chain variable region and a CDR derived from a light chain variable region of an antibody produced from a hybridoma deposited as KCLRF-BP-00264.

[0014] In another embodiment of the present invention, the anti-ICAM-1 antibody may be, but is not limited to, an animal antibody, a chimeric antibody, or a humanized antibody.

[0015] In another embodiment of the present invention, the anti-ICAM-1 antibody may be, but is not limited to, a full-length antibody, or a scFv, (scFv)2, Fab, Fab', or F(ab')2 of the anti-ICAM-1 antibody.

[0016] The present invention relates to a pharmaceutical composition for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis. In the present invention, it was confirmed that an anti-ICAM-1 antibody effectively suppresses inflammatory responses in hepatocytes and stably maintains liver function when applied as an immunosuppressive therapy after liver transplantation, and also effectively regulates the expression of genes related to the mechanism of nonalcoholic steatohepatitis in liver tissue where inflammation is induced through administration of the anti-ICAM-1 antibody. Since it is expected that nonalcoholic steatohepatitis can be treated by regulating inflammatory responses through targeting ICAM-1 in liver tissue, the anti-ICMA-1 antibody according to the present invention is expected to be effectively utilized as a therapeutic agent for nonalcoholic steatohepatitis.

[0017] Figure 1 illustrates the group-specific immunosuppressive therapy performed in an embodiment of the present invention.

[0018] Figure 2 shows the survival rate by group according to immunosuppressive therapy.

[0019] Figure 3 shows the results of confirming the expression levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IFN-γ) and chemokines (MCP-1, RANTES, and IP-10) in liver allografts of the No-IS, Con-IS, and MD-3 groups.

[0020] Figure 4: Liver FoxP3 in Con-IS and MD-3 groups + CD4 + This is the result of confirming changes in the proportion of Treg cells.

[0021] Figure 5a shows the results of histological analysis of liver tissue isolated from animal models. H&E staining (top) shows: (#2, No-Is, POD6) lymphocyte-dominant inflammation in the portal tract with endothelial inflammation (asterisk) and bile duct damage (black arrow). (#3, Con-IS, POD52) mononuclear cell-infiltrated portal tract with endothelial inflammation (asterisk) and bile duct damage (black arrow). (#4, Con-IS, POD66) mild portal mononuclear infiltration and portal fibrosis, with obliteration of interlobular bile ducts (white arrow, area of ​​bile duct obliteration). (#11, MD-3, POD216) focal accumulation of foamy macrophages in the interlobular bile and sinusoids (black arrow) without the portal tract. (#10, MD-3, POD730) Moderate mixed inflammatory cell infiltrate including eosinophils in the hepatic fibrillary sinus with neutrophils and eosinophils in the microvasculature. CD4, CD8, CD20, CD68, and IgG are shown in the respective tissues (200X). Immunohistochemical staining for CD4, CD8, CD20, CD68, and IgG is shown.

[0022] Figure 5b shows the results of H&E staining and immunohistochemical staining for CD4, CD8, CD20, CD68, C4d, and IgG in liver tissue isolated from each animal in the humanized antibody-treated MD-3 group (200X).

[0023] Figures 6 to 8 show the AST and ALT levels (Figure 6), ALP and GGT levels (Figure 7), and T-bil and CRP levels (Figure 8) by group according to immunosuppressive therapy.

[0024] Figures 9a and 9b show the results of proteomics analysis performed in the MD-3 treatment group.

[0025] The present invention relates to a pharmaceutical composition for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, comprising an anti-ICAM-1 antibody as an active ingredient. The inventors of the present invention devised the present invention by confirming that nonalcoholic steatohepatitis can be treated if the inflammatory response is controlled by targeting ICAM-1 in liver tissue based on the mechanism in which LSEC (Liver Sinuisoidal Endothelial Cells) increase ICAM-1 (Intracellular adhesion molecule-1) by Kupper cells activated in the liver when nonalcoholic steatohepatitis occurs, and that an anti-ICAM-1 antibody used in immunosuppressive therapy after liver transplantation not only effectively suppresses the inflammatory response in hepatocytes and stably maintains liver function, but also effectively regulates the expression of genes related to the nonalcoholic steatohepatitis mechanism in liver tissue where inflammation is induced by administration of the anti-ICAM-1 antibody.

[0026] Accordingly, the present invention provides a pharmaceutical composition for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, comprising an anti-ICAM-1 antibody as an active ingredient.

[0027] In the present invention, "anti-ICAM-1 antibody" refers to an antibody that specifically binds to intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is a cell surface glycoprotein expressed on the surface of endothelial cells or immune cells, and is also referred to as CD54 (Cluster of differentiation 54). The ICAM-1 is composed of five extracellular immunoglobulin (Ig) superfamily regions, a transmembrane region, and an intracellular region. The ICAM-1 according to the present invention may be a mammalian ICAM-1, and may be, for example, human ICAM-1 (e.g., NCBI accession numbers NP_000192.2, etc.), monkey ICAM-1 (e.g., NCBI accession numbers NP_001266532, etc.), but is not limited thereto. In one embodiment of the present invention, the antibody may exhibit cross-reactivity to human ICAM-1 and monkey ICAM-1. In another embodiment of the present invention, the antibody may bind to region 2 of ICAM-1.

[0028] In one embodiment of the present invention, the anti-ICAM-1 antibody is characterized by specifically recognizing the second domain (domain 2) from the N-terminal region among the five extracellular immunoglobulin domains of ICAM-1. Preferably, the antibody according to the present invention can modulate an immune response by inhibiting the differentiation, maturation, or function of dendritic cells. Preferably, the anti-ICAM-1 antibody according to the present invention acts as an antagonist of ICAM-1, thereby inhibiting the maturation of dendritic cells by ICAM-1 without interfering with the binding of ICAM-1 and LFA-1.

[0029] The anti-ICAM-1 antibody according to the present invention is sufficient if it can specifically bind to ICAM-1 and inhibit its activity or function, and is not limited to a specific type, but may be an antibody or fragment thereof comprising a complementarity determining region derived from a heavy chain variable region and / or a complementarity determining region derived from a light chain variable region of a monoclonal antibody produced from a hybridoma deposited as KCLRF-BP-00264 (Korea Cell Line Research Foundation, May 4, 2011). Specific information on the anti-ICAM-1 antibody can be found in Republic of Korea Patent No. 10-1434029. Throughout this specification, the anti-ICAM-1 antibody may also be referred to as MD-3. The antibody may be present in a whole cell including a hybridoma, or a lysate or medium thereof, and may be purified and isolated therefrom in a partially or substantially pure form. Purification is to remove other cell byproducts other than antibodies, such as cell components, nucleic acids, proteins, etc., and can be performed using known methods such as alkaline / SDS treatment, CsCl separation, column chromatography, and agarose electrophoresis. The complementarity determining region (CDR) is a region among the variable regions of an antibody that specifically binds to an antigen and is a region that determines the specificity of the antibody. The positions of CDR1, CDR2, and CDR3 of the MD-3 antibody, respectively, according to IMGT analysis (http: / www.imgt.org / ) are between FR (frame region) 1-FR2, FR2-FR3, and FR3-JR (joining region), and the base sequences of the FR1, FR2, FR3, and J regions of the mouse IgG1 heavy chain and kappa light chain are known.

[0030] In the present invention, "antibody" means an immunoglobulin molecule that immunologically reactive with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, and functional fragments thereof. In addition, the antibody in the present invention may include a form produced by genetic engineering, such as an animal antibody (e.g., a mouse antibody or a monkey antibody), a chimeric antibody (e.g., a humanized murine antibody, a human-monkey antibody), and a heterologous antibody (e.g., a bispecific antibody). The antibody comprises a variable region of a heavy chain and / or a light chain (VH, heavy chain variable region; VL, light chain variable region). The variable region includes a portion that forms an antigen-binding site of an antibody molecule as a primary structure, and the antibody of the present invention may be composed of a complete antibody including two full-length heavy chains and two full-length light chains, as well as some fragments including the variable region. As described above, in one embodiment of the present invention, the anti-ICAM-1 antibody may be an antibody produced from a hybridoma deposited under the name KCLRF-BP-00264 (Korea Cell Line Research Foundation, May 4, 2011), in which the antibody of the present invention is a complete antibody including two light chains and heavy chains.

[0031] In the present invention, a humanized antibody refers to an antibody in which the antibody framework is a human antibody, but a portion of the CDR region is modified to include only the portions essential for specific antigen binding among the CDRs of the species from which the existing antibody molecule was derived. For example, the remaining CDR regions and the light and heavy chain frameworks of a monkey or mouse-derived antibody, excluding the portions essential for specific antigen binding, may be replaced with human antibodies.

[0032] In the present invention, a "chimeric antibody" refers to an antibody in which at least a portion of the variable region and constant region are derived from different species. For example, the variable region may be mouse-derived and the constant region may include human-derived. A chimeric antibody also refers to a class-switched antibody, for example, an antibody that has been converted from an IgG type to an IgE type. A chimeric antibody can typically be produced through recombinant DNA technology. In one embodiment of the present invention, the anti-ICAM-1 antibody is a chimeric antibody, and may be one in which the variable regions of the light and heavy chains of an antibody produced from the hybridoma are grafted onto a human antibody, such as a lambda light chain and an IgG4 heavy chain constant region.

[0033] In the present invention, the term "fragments" of an antibody refers to (functional) fragments that possess the antigen-binding function of the antibody, and is used to mean scFv, (scFv)2, Fab, Fab', and F(ab')2, as well as nanobody fragments, etc. The definitions of the above fragments are well known in the art. Preferably, the fragment of the antibody that binds to a cancer cell-specific antigen according to the present invention is scFv.

[0034] The present invention provides a method for treating nonalcoholic steatohepatitis in a subject, comprising administering to the subject a therapeutically effective amount of an anti-ICAM-1 antibody.

[0035] The present invention also provides a method for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ICAM-1 antibody.

[0036] The term "therapeutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the disease, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrent medications, and other factors well known in the medical field.

[0037] The therapeutically effective amount of the composition of the present invention may be, but is not limited to, a concentration of 1 to 50 mg / kg. That is, the pharmaceutically effective amount (i.e., dosage) of the anti-ICAM-1 antibody in the composition according to the present invention is not limited and may vary depending on the condition of the patient and the severity of the disease, but preferably, a single dosage of the anti-ICAM-1 antibody may be 1 to 50 mg / kg, 1 to 40 mg / kg, 1 to 30 mg / kg, 1 to 20 mg / kg, 1 to 15 mg / kg, 1 to 10 mg / kg, or 1 to 5 mg / kg.

[0038] The content of the anti-ICAM-1 antibody in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9 wt% or 0.001 to 50 wt% based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0039] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0040] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0041] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0042] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0043] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0044] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0045] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0046] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0047] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0048] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0049] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0050] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0051] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0052] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0053] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0054] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease. Specifically, the effective dose of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0055] In the present invention, the term "subject" means a subject requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0056] In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method.

[0057] In the present invention, “treatment” means any action in which a target disease and its metabolic abnormality symptoms are improved or beneficially changed by administration of a pharmaceutical composition according to the present invention, and “improvement” means any action in which a parameter related to a target disease, for example, the severity of symptoms, is reduced by administration of a composition according to the present invention.

[0058] The present invention also provides a use of a composition comprising an anti-ICAM-1 antibody as an active ingredient for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis.

[0059] In addition, the present invention provides a use for preparing a non-alcoholic fatty liver disease or non-alcoholic steatohepatitis treatment preparation comprising an anti-ICAM-1 antibody as an active ingredient.

[0060] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0061]

[0062] [Experimental Materials and Methods]

[0063] 1. Laboratory animals

[0064] Male rhesus macaques (Macaca mulatta) weighing 4–7 kg (Mokeyking Biotechnology Co. Ltd., Guangzhou, China) were used for ABO-compatible, cross-match-negative, and MHC-mismatched orthotopic liver transplantation (orthotopic LT). Before liver transplantation, serological tests were performed to confirm the absence of herpes virus B, Schmidt-lupin virus, simian T-cell lymphotropic virus, and simian immunodeficiency virus. All animals received humane care according to the standards outlined in the “Guide for the Care and Use of Laboratory Animals.” The experimental protocol was approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital (IACUC number: 18-0179).

[0065]

[0066] 2. ABO blood type and MHC type

[0067] ABO blood group identification was performed using specific primers (Sci Rep. 2018 Jan 31;8(1):1957). MHC typing for Manu-A (class I) and DRB (class II) was also performed using sequence-specific primers (PCR-SSP) (Immunol. 2000;61(10):1013-1020;Tissue Antigens. 1999;54(3):254-263.).

[0068]

[0069] 3. Blood, coagulation, and biochemical monitoring

[0070] Blood monitoring was performed, including white blood cell counts, hemoglobin levels, and platelet counts. Blood coagulation tests included prothrombin time (PT) and activated partial thromboplastin time (aPTT). For biochemical analysis, blood levels of total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total bilirubin, and C-reactive protein (CRP) were measured.

[0071]

[0072] 4. Immune cell subtype analysis

[0073] Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll separation (Ficoll-Paque PLUS; GE Health Sciences, Uppsala, Sweden). Liver allografts were dissected and digested with 200 U / mL collagenase (Sigma-Aldrich, St. Louis, MO) and 30 U / mL DNase (Roche, Basel, Switzerland) at 37°C for 1.5 h with continuous shaking. Single-cell suspensions were then stained with various antibodies to analyze immune cell types. Flow cytometry was performed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA), and the results were analyzed using FlowJo (Tree Star, Ashland, OR).

[0074]

[0075] 5. Enzyme-Linked ImmunoSPOT (ELISPOT) analysis and DSA and CMV monitoring

[0076] IFN-γ-secreting T cells were assessed using an ELISPOT assay (MabTech, Nacka Strand, Sweden). To monitor donor-specific antibodies (DSA), recipient serum was incubated with donor plasma membrane cancer cells, and anti-monkey IgG or IgM was added. DSA levels were then measured by flow cytometry. Rhesus cytomegalovirus (RhCMV) DNA in plasma was quantified by real-time PCR (Bioneer, Daejeon, Korea).

[0077]

[0078] 6. Real-time PCR

[0079] Total RNA was isolated from PBMCs or liver tissues using TRIzol (Invitrogen, Carlsbad, CA). RNA was reverse transcribed into cDNA using oligo (dT) 12-18 and SuperScript II reverse transcriptase (Invitrogen). Oligonucleotide primers and TaqMan probes for measuring TNF-α, IL-1β, IL-6, IL-10, TGF-β, IFN-γ, MCP-1, RANTES, IP-10, PD-1, CTLA-4, and GAPDH were designed using Primer Express (Applied Biosystems, Foster City, CA). Real-time PCR was performed using an ABI PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems). mRNA expression levels were normalized to GAPDH levels.

[0080]

[0081] 7. Histological analysis

[0082] Liver grafts were obtained after death or euthanasia of the mouse models. For hematoxylin and eosin (H&E) and Masson's trichrome staining, liver grafts were sectioned into 5-mm sections from paraffin-embedded tissue. For immunohistochemistry, slides were incubated for 30 minutes with the following primary antibodies: anti-CD4 (H-370, Santa Cruz Biotechnology, Santa Cruz, CA), anti-CD8 (polyclonal, Abcam, Cambridge, UK), anti-CD20 (L26), anti-CD68 (KP1), anti-IgG (polyclonal, Dako, Glostrup, Denmark), and anti-C4d (polyclonal, Roche). After washing and peroxidase blocking, tissues were incubated with Envision+System horseradish peroxidase-labeled polymeric anti-rabbit antibody (Dako). Two investigators independently counted the number of periportal immune cells and calculated the mean. Liver allograft pathology was assessed by a liver pathologist according to the Banff criteria.

[0083]

[0084] 8. Statistical Analysis

[0085] Data are presented as mean ± standard error. Survival rates were assessed by Kaplan-Meier analysis and compared using the log-rank test. A P value less than 0.05 was considered statistically significant. Statistical analyses were performed using Prism 6 (GraphPad, San Diego, CA).

[0086]

[0087] 9. Proteomics analysis

[0088] Protein concentrations in liver tissue were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Samples were then subjected to the in-situ digestion method, and each sample was mixed with 8 M urea in 100 mM ammonium bicarbonate lysis buffer and homogenized for 30 s at 4°C using a hand-held homogenizer. Subsequently, each sample was reduced with 10 mM dithiothreitol (DTT) at 37°C for 30 min, alkylated with iodoacetic acid (IAA) for 1 h at 25°C in the dark, and sequentially washed with lysis buffer and 50 mM ammonium bicarbonate (ABC). The proteins were digested with trypsin (enzyme-to-protein ratio 1:25, w / w) at 37°C for 16 h. The resulting peptide mixture was transferred to a new tube, and trypsin activity was stopped by acidification with 1% trifluoroacetic acid (TFA). The digested peptides were purified using a C18 cartridge (SOLA HRP 96-well plate C18 cartridge), and eluted with 80% acetonitrile dissolved in 0.1% formic acid in water. The peptides were dissolved in 50 mM triethylammonium bicarbonate (TEAB) solution, labeled with TMT (Tandem Mass Tags) reagent, and reacted at room temperature (RT) for 1 hour. The reaction was stopped by adding 5% hydroxylamine. All peptides were then combined, dried, purified, and dissolved in 10 mM ABC solution. To separate the labeled peptides, the combined sample was loaded onto an Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ACQUITY UPLC Peptide CSH™ C18 column (130Å, 1.7μm, 1mm x 1500mm, Waters), and the labeled peptides were fractionated into 12 fractions over a total run time of 110 min.The gradient of buffer B was programmed as follows: 1% at 0 min, 3% at 1 min, 10% at 20 min, 45% at 90 min, 90% at 94 min, 90% at 103 min, 1% at 105 min, and 1% at 110 min, and the mobile phase consisted of buffer A (10 mM ABC) and buffer B (10 mM ABC in 90% ACN).

[0089]

[0090] [Example]

[0091] Example 1. Liver transplantation process

[0092] Orthotopic liver transplantation was performed in a manner similar to whole liver transplantation without venous bypass as previously described (Transplant Proc. 2017 Jun;49(5):1150-1152). Specifically, donor surgery was performed based on warm dessection. Custodiol was administered after systemic heparinization. 　 In situ perfusion via the aorta was performed using HTK solution (Essential Pharmaceuticals, Durham, NC, USA). Simultaneously, the recipient underwent hepatectomy, and the superior mesenteric artery was clamped to reduce the risk of intestinal obstruction. After anastomosing the suprahepatic and inferior vena cava and portal vein, the superior mesenteric artery was clamped. After reperfusion, the inferior subhepatic vena cava was anastomosed. The aortic conduit of the graft was anastomosed end-to-side to the recipient's infrarenal aorta. This conduit-to-conduit anastomosis was used for biliary tract reconstruction without a drainage tube. Internal stent insertion was considered when the common bile duct diameter was less than 2 mm.

[0093]

[0094] Example 2. Immunosuppressive therapy

[0095] The maintenance therapy course using humanized MD-3 is shown in Figure 1 (No-IS group, a group that did not receive immunosuppression; Con-IS group, a group that received conventional triple immunosuppression; and MD-3 group, a group that received conventional triple immunosuppression plus short-term MD-3 therapy). Specifically, two of the experimental subjects did not receive immunosuppression (No-IS group), and four subjects received conventional triple immunosuppression (prednisolone, tacrolimus, and an mTOR inhibitor) without induction therapy (Con-IS group). In addition, to identify the optimal MD-3 regimen, the induction and maintenance regimens of MD-3 were compared. Induction therapy is a short-term administration of high-dose immunosuppressants in the early stages after transplantation, and maintenance therapy refers to a combination therapy in which the transplant recipient receives immunosuppression over a long period of time. Five animals received MD-3 induction therapy (Induction MD-3) using chimeric MD-3, and four animals received MD-3 maintenance therapy (Maintenance MD-3) using humanized MD-3. The humanized and chimeric antibodies are described above. The MD-3 maintenance therapy process using humanized MD-3 is shown in Figure 1. MD-3 at 8 mg / kg was administered intravenously 13 times from 9 days before liver transplantation until 12 weeks after liver transplantation, and then once a month from the 16th week, for a total of 10 times until 24 months. In addition, MD-3 was administered once every 3 months from the 24th month, for a total of 2 times until 30 months. During this time, the existing triple therapy (prednisolone, tacrolimus, and sirolimus) was administered together until one month after liver transplantation.

[0096]

[0097] Example 3. Results of immunosuppressive therapy

[0098] The clinical outcomes according to immunosuppressive therapy in each group are shown in Table 1 below.

[0099]

[0100]

[0101] As shown in the table above, all subjects who did not receive immunosuppressive therapy (No-IS) died from acute rejection. Subjects who received conventional triple immunotherapy (Con-IS) also died from acute rejection, chronic rejection, or side effects of mTOR inhibitors. Subjects receiving MD-3 therapy showed differences depending on the specific MD-3 administration method. Subjects receiving MD-3 induction therapy did not show acute rejection but eventually died from chronic rejection, sepsis, or side effects of mTOR inhibitors, whereas most subjects receiving MD-3 maintenance therapy did not show acute or chronic rejection, and no subjects died. Comparing survival rates, all subjects in the No-IS and Con-IS groups died within a few days after transplantation, whereas subjects in the induction MD-3 group survived for more than 500 days, showing a significantly increased survival rate. Most subjects in the maintenance MD-3 group continued to survive after transplantation (Figure 2). These results demonstrate that MD-3 therapy is more effective in suppressing liver transplant rejection than conventional triple immunosuppressive therapy.

[0102]

[0103] Example 4. Results of inflammatory cytokine analysis

[0104] When analyzing inflammatory cytokines in liver tissue after liver transplantation, the MD-3 group showed lower levels of inflammatory cytokines compared to the No-IS and Control groups. In particular, the expression of IL-6, TNF-α, and MCP-1, which are closely related to the NASH mechanism, decreased, whereas the expression levels of immune regulatory molecules (IL-10, TGF-β, CTLA-4, and PD-1) did not show significant differences between the groups (Fig. 3).

[0105]

[0106] Example 5. Results of immune regulatory T cell analysis

[0107] After liver transplantation, Foxp3+CD4+ cells were isolated from liver tissue to identify changes in regulatory T cells (Treg) in each group. As a result, Treg cells in the MD-3 group increased compared to the control group, and this increase continued steadily for 24 months (Fig. 4). Treg cells decrease with the onset of NASH, and these results suggest that MD-3 may exhibit therapeutic effects in NASH through immunosuppression by inducing an increase in Treg cells.

[0108]

[0109] Example 6. Histological analysis results

[0110] All recipients in the No-IS group showed acute TCMR, while one monkey in the Con-IS group showed acute TCMR, and two showed chronic rejection with mild portal mononuclear infiltration, portal fibrosis, and loss of interlobular bile ducts. The other three monkeys in the MA-MD-3 group, including the longest-lived monkey that survived 1364 days after liver transplantation, showed no signs of rejection at final necropsy, and histologically, no changes in the portal tracts were evident, except for minimal lymphocytic infiltration in the portal stroma. In addition, there was no evidence of bile duct damage or endothelial inflammation, no liver lobules were visible, and immunohistochemical staining revealed no C4d or IgG deposition (Figures 5a and 5b). These results suggest that MD-3 may exert a therapeutic effect in NASH by suppressing inflammation in the liver tissue.

[0111]

[0112] Example 7. Liver function test results

[0113] Next, liver function tests were performed by group to compare the degree of liver function maintenance according to immunosuppressive therapy. As shown in Figure 6, a rapid increase in AST and ALT was observed in individuals in the Con-IS group who showed a graft rejection reaction. The group that received MD-3 induction therapy (Chimeric MD-3 IS group) generally maintained low levels of ALT and AST, but a tendency for ALT and AST to increase was observed in individuals who showed chronic rejection reactions. On the other hand, the group that received MD-3 maintenance therapy (Humanized MD-3 IS group) showed stable results with low levels of ALT and AST in all individuals. ALP, GGT (gamma glutamyl peptidase), T-BIL (total bilirubin), and CRP (C-reactive protein) also showed similar trends. In the Con-IS group, ALP and GGT increased rapidly in the early period after transplantation, and T-BIL and CRP levels also increased rapidly, confirming a rapid inflammatory response (Figures 7-8). The group that received MD-3 induction therapy had some individuals whose ALP, GGT, ALP, and GGT levels remained stable, and overall, the levels were relatively low compared to the Con-IS group, but a tendency for the above factors to increase over time was observed. On the other hand, the group that received MD-3 maintenance therapy all maintained stable low levels of ALP, GGT, ALP, and GGT, confirming that the liver function and inflammation levels were at normal levels. These results show that MD-3 can restore liver function to normal by suppressing the inflammatory response in liver tissue, and through this, it can be expected that MD-3 can have a therapeutic effect in NASH disease.

[0114]

[0115] Example 8. Results of multi-omics (proteomics) analysis

[0116] Proteomic analysis results confirmed that the GSTM3, GSTM2, and GSTA3 genes, which are involved in the glutathione metabolic process and biosynthetic process associated with NASH, showed statistically significant differences in the MD-3-administered group compared to Con-Is (Figures 9a and 9b). This suggests that MD-3 may exhibit therapeutic effects in NASH.

[0117]

[0118] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0119] The present invention relates to a pharmaceutical composition for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis. In the present invention, it was confirmed that an anti-ICAM-1 antibody effectively suppresses inflammatory responses in hepatocytes and stably maintains liver function when applied as an immunosuppressive therapy after liver transplantation, and also effectively regulates the expression of genes related to the mechanism of nonalcoholic steatohepatitis in liver tissue where inflammation is induced through administration of the anti-ICAM-1 antibody. Since it is expected that nonalcoholic steatohepatitis can be treated when the inflammatory response is regulated through targeting ICAM-1 in liver tissue, the anti-ICMA-1 antibody according to the present invention is expected to be effectively utilized as a therapeutic agent for nonalcoholic steatohepatitis, and thus has industrial applicability.

Claims

1. A pharmaceutical composition for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, comprising an anti-ICAM-1 antibody as an active ingredient.

2. In paragraph 1, A pharmaceutical composition characterized in that the above anti-ICAM-1 antibody comprises a CDR derived from a heavy chain variable region and a CDR derived from a light chain variable region of an antibody produced from a hybridoma deposited as KCLRF-BP-00264.

3. In paragraph 1, A pharmaceutical composition, characterized in that the anti-ICAM-1 antibody is an animal antibody, a chimeric antibody, or a humanized antibody.

4. In paragraph 1, A pharmaceutical composition, characterized in that the anti-ICAM-1 antibody is a full-length antibody, or scFv, (scFv)2, Fab, Fab', or F(ab')2 of the anti-ICAM-1 antibody.

5. A method for treating a subject suffering from nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, comprising administering to the subject a therapeutically effective amount of an anti-ICAM-1 antibody.

6. In paragraph 5, A therapeutic method, characterized in that the above anti-ICAM-1 antibody comprises a CDR derived from a heavy chain variable region and a CDR derived from a light chain variable region of an antibody produced from a hybridoma deposited as KCLRF-BP-00264.

7. Use of a composition comprising an anti-ICAM-1 antibody as an active ingredient for the treatment of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

8. Use for manufacturing a non-alcoholic fatty liver disease or non-alcoholic steatohepatitis treatment preparation comprising an anti-ICAM-1 antibody as an active ingredient.

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