Treatment of Nonalcoholic Steatohepatitis (NASH)

Abstract

To provide novel compounds that target thrombocyte activity or aggregation capacity through cellular components for treatment of diseases associated with Non-Alcoholic Fatty Liver Disease (NAFLD), for treating non-alcoholic steatohepatitis (NASH), a progressed stage of NAFL (non-alcoholic fatty liver), in order to avoid development of liver cirrhosis and Hepatocellular Carcinoma (HCC), as well as pharmaceutical compositions comprising the compounds of the invention, and methods for screening new NASH therapeutics.SOLUTION: There is provided herein, an inhibitor of thrombocyte aggregation or activation for use in treatment and or prevention of non-alcoholic steatohepatitis (NASH), where the inhibitor is an inhibitor of Gp1b and the inhibitor is an antigen binding fragment of an anti-Gp1b antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention provides novel compounds that target the activity or aggregation ability of platelets by cellular components for the treatment of diseases associated with non-alcoholic fatty liver disease (NAFLD). The present invention provides these compounds for treating non-alcoholic steatohepatitis (NASH), an advanced stage of NAFL (non-alcoholic fatty liver), so as to avoid the development of liver cirrhosis and hepatocellular carcinoma (HCC). In addition, pharmaceutical compositions comprising the compounds of the present invention and methods for screening new NASH therapeutics are provided. [Background technology]

[0002] Lifestyle changes over the last few decades, such as high calorie intake (e.g., from high fat, high fructose and high glucose diets) combined with a sedentary lifestyle, have led to an increase in the incidence of overweight and metabolic syndrome, characterized by abdominal obesity, insulin resistance, hypertonia and dyslipidemia. The latest WHO cancer report predicts that cancer incidence will double within the next 20 years, with the majority of this attributable to modifiable risk factors such as high calorie intake, smoking and a sedentary lifestyle (Non-Patent Document 1). A strong association between obesity and cancer incidence is well established, and a body mass index (BMI) above 25 significantly increases the risk of developing some cancers (Non-Patent Document 2). The liver, the body's most important metabolic organ, is strongly affected by chronic conditions of high calorie intake, overweight, sedentary lifestyle and the resulting pathology (metabolic syndrome).

[0003] Nonalcoholic fatty liver disease (NAFLD), which includes several liver diseases including NAFL and NASH, is the most frequent liver disease worldwide and is a clinical manifestation of overweight and metabolic syndrome. NAFL is a chronic disease that can last for decades and is characterized by predominant macrovesicular steatosis of the liver. The prevalence of NAFL is increasing worldwide (Non-Patent Document 3). Currently, 90 million Americans and 40 million Europeans suffer from NAFLD. It is interesting to note that developing countries also show a strong rise in NAFLD cases, reflecting the consequences of industrialization and the associated "Western lifestyle." A significant number of NAFL patients develop nonalcoholic steatohepatitis (NASH), fibrosis, and subsequently hepatocellular carcinoma (HCC). At the same time, the total number of people suffering from NASH is increasing in the United States and Europe. As a result, obesity, steatosis and steatohepatitis have received increasing attention due to the rising incidence of HCC in Western countries (Non-Patent Document 4). In light of this, the most common etiology of HCC in industrialized countries has now switched from chronic viral infections (e.g., hepatitis B and C viruses) to obesity, making HCC the most rapidly increasing type of cancer in the United States, and a similar trend has been observed in Europe (Non-Patent Document 5).

[0004] Today, there is still a lack of detailed understanding of how chronic steatosis develops into NASH and which factors control the transition from NASH to HCC. At the same time, there is no efficient therapy to treat NASH, and treatment options for end-stage HCC are limited, extending the patient's life span by only 3 to 6 months (Non-Patent Document 6). It is becoming clear that multiple pathways are involved in the pathogenesis of NASH and its progression to advanced stages of liver disease. These pathways may vary in different cohorts of patients with NASH. Before embarking on a therapeutic modality, it is important to understand which pathways are involved in the development of NASH and NASH-driven HCC. Indeed, while NASH is associated with metabolic syndrome in most cases in Western countries, it can also appear at lower BMI in Asian countries, for example, and many patients do not appear to have insulin resistance. These observations suggest that additional genetic factors are potentially involved in the progression of the disease (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Stewart and Wild, 2014 [Non-Patent Document 2] Calle and Kaaks, 2004 [Non-Patent Document 3] Loomba et al., 2013 [Non-Patent Document 4] White et al., 2012 [Non-Patent Document 5] American Cancer Society, 2007 [Non-Patent Document 6] Villanueva et al., 2014 Summary of the Invention [Problem to be solved by the invention]

[0006] Clearly, there is a large unmet need for novel therapeutic approaches targeting the progression of NASH to cirrhosis and HCC. It is therefore an object of the present invention to provide novel therapeutic targets towards this goal. [Means for solving the problem]

[0007] The above problems are solved by modifying platelet activation and aggregation ability so as to inhibit the adverse effects of platelets on liver inflammation and hepatocyte metabolism in high-calorie diet.Accordingly, the present invention provides, in a first aspect, a platelet inhibitor for use in treating or preventing non-alcoholic fatty liver disease (NAFLD).

[0008] The platelet inhibitor in the present invention is also referred to as an antiplatelet agent or a platelet aggregation inhibitor, and can be selected from any agent known in the art to inhibit platelet activity, proliferation or aggregation. Examples of such compounds are agents that inhibit platelet function by inhibiting aggregation or by platelet adhesion or granule secretion. Antiplatelet agents used in the present invention include, but are not limited to, various known nonsteroidal anti-inflammatory drugs (NSAIDS), such as aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac, sulfinpyrazone, piroxicam, and their pharmaceutically acceptable salts or prodrugs. In another embodiment, the antiplatelet agent is a IIb / IIIa antagonist (e.g., tirofiban, eptifibatide, and abciximab), a thromboxane-A2-receptor antagonist (e.g., ifetroban), a thromboxane-A2-synthetase inhibitor, a PDE-III inhibitor (e.g., dipyridamole), and pharmaceutically acceptable salts or prodrugs thereof. In another embodiment, the term antiplatelet agent (or platelet inhibitor) refers to an ADP (adenosine diphosphate) receptor antagonist, which in one embodiment is an antagonist of the purinergic receptors P2Yi and P2Yn. In one embodiment, the P2Yi2 receptor antagonist is ticlopidine, clopidogrel, or a combination thereof, and pharmaceutically acceptable salts or prodrugs thereof.

[0009] In some preferred embodiments of the present invention, there is provided an inhibitor of Gp1b, GPV, GPIX, Factor VIII, or Nbeal2 for use in the treatment or prevention of non-alcoholic fatty liver disease (NAFLD). The above protein designations are used with respect to the following protein names in parentheses: GpIb (glycoprotein Ib), GpV (glycoprotein V), GPIX (glycoprotein IX), Factor VIII (or Factor VIII; FVIII), and Nbeal2 (neurobeatin-like protein 2).

[0010] An "inhibitor of Gp1b, GPV, GPIX, factor VIII, or Nbeal2" is an antagonist of a mammalian homologue of Gp1b, GPV, GPIX, factor VIII, or Nbeal2, respectively, preferably human Gp1b, GPV, GPIX, factor VIII, or Nbeal2. As used herein, the term "inhibitor of Gp1b, GPV, GPIX, factor VIII, or Nbeal2" refers to a substance that affects a decrease in the amount or rate of expression or activity of Gp1b, GPV, GPIX, factor VIII, or Nbeal2. Such a substance can act directly, for example, by binding to Gp1b, GPV, GPIX, factor VIII, or Nbeal2 and by decreasing the amount or rate of expression or activity of Gp1b, GPV, GPIX, factor VIII, or Nbeal2. Gp1b, GPV, GPIX, factor VIII, or Nbeal2 antagonists can also reduce the amount or rate of expression or activity of Gp1b, GPV, GPIX, factor VIII, or Nbeal2, for example, by binding to Gp1b, GPV, GPIX, factor VIII, or Nbeal2, by binding to and modifying Gp1b, GPV, GPIX, factor VIII, or Nbeal2, for example by removing or adding a moiety, and by binding to and reducing the stability of Gp1b, GPV, GPIX, factor VIII, or Nbeal2, so as to reduce or prevent the interaction of Gp1b, GPV, GPIX, factor VIII, or Nbeal2 with a ligand. Gp1b, GPV, GPIX, factor VIII, or Nbeal2 antagonists can also act indirectly, for example, by binding to a regulatory molecule or gene region to modulate the function of the regulatory protein or gene region, affecting a decrease in the amount or rate of Gp1b, GpV, GpIX, factor VIII, or Nbeal2 expression or activity. Thus, Gp1b, GpV, GpIX, factor VIII, or Nbeal2 antagonists can act by any mechanism that results in a decrease in the amount or rate of Gp1b, GpV, GpIX, factor VIII, or Nbeal2 expression, stability, or activity.

[0011] The Gp1b, GpV, GpIX, factor VIII, or Nbeal2 antagonist may be a naturally occurring or non-naturally occurring macromolecule, such as, for example, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, a carbohydrate, or a lipid. Furthermore, the Gp1b, GpV, GpIX, factor VIII, or Nbeal2 antagonist may be an antibody, such as a monoclonal antibody, a humanized antibody, a chimeric antibody, a minibody, a bivalent antibody, a single chain antibody (scFv), a variable region fragment (Fv or Fd), a Fab or a F(ab)2, or an antigen-binding fragment thereof. The Gp1b, GpV, GpIX, factor VIII, or Nbeal2 antagonist may also be a polyclonal antibody specific for Gp1b, GpV, GpIX, factor VIII, or Nbeal2. Furthermore, Gp1b, GpV, GpIX, factor 8, or Nbeal2 antagonists can be partially or wholly synthetic derivatives, analogs, or mimetics of naturally occurring macromolecules or small organic or inorganic molecules.

[0012] An antibody Gp1b, GpV, GpIX, factor VIII, or Nbeal2 antagonist can be, for example, an antibody that binds to Gp1b, GpV, GpIX, factor VIII, or Nbeal2 and inhibits binding to a Gp1b, GpV, GpIX, factor VIII, or Nbeal2 ligand, or that alters the activity of a molecule that regulates the expression or activity of Gp1b, GpV, GpIX, factor VIII, or Nbeal2 such that the amount or rate of expression or activity of Gp1b, GpV, GpIX, factor VIII, or Nbeal2 is decreased. Antibodies useful in the methods of the invention can be naturally occurring antibodies, including monoclonal or polyclonal antibodies or fragments thereof, or non-naturally occurring antibodies, including, but not limited to, single chain antibodies, chimeric antibodies, bivalent antibodies, complementarity determining region grafted (CDR grafted) antibodies, and humanized antibodies or antigen-binding fragments thereof.

[0013] The nucleic acid Gp1b, GpV, GpIX, factor VIII, or Nbeal2 antagonist can be, for example, an antisense nucleotide sequence, an RNA, a DNA, an RNA / DNA, or an LNA molecule, or an aptamer sequence. The antisense nucleotide sequence can bind to a nucleotide sequence in a cell and regulate the level of expression of Gp1b, GpV, GpIX, factor VIII, or Nbeal2, respectively, or regulate the expression of another gene that controls the expression or activity of Gp1b, GpV, GpIX, factor VIII, or Nbeal2. Similarly, an RNA molecule, such as a catalytic ribozyme, can bind to and alter the expression of the Gp1b, GpV, GpIX, factor VIII, or Nbeal2 gene, or another gene that controls the expression or activity of Gp1b, GpV, GpIX, factor VIII, or Nbeal2. An aptamer is a nucleic acid sequence that has a three-dimensional structure that can bind to a molecular target.

[0014] The nucleic acid Gp1b, GpV, GpIX, factor 8, or Nbeal2 antagonist may also be a double-stranded RNA molecule for use in RNA interference. RNA interference (RNAi) is the process of sequence-specific gene silencing by post-transcriptional RNA degradation initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Double-stranded RNA (dsRNA) suitable for RNAi contains sense and antisense strands of about 21 adjacent nucleotides that correspond to the targeted gene forming 19 RNA base pairs, leaving a two-nucleotide overhang at each 3' end (Elbashir et al., Nature 411:494-498 (2001); Bass, Nature 411:428-429 (2001); Zamore, Nat. Struct. Biol. 8:746-750 (2001)). dsRNAs having about 25-30 nucleotides have also been successfully used in RNAi (Karabinos et al., Proc. Natl. Acad. Sci. USA 98:7863-7868 (2001)). dsRNAs can be synthesized in vitro and introduced into cells by methods known in the art. As already mentioned, antisense nucleic acids are RNA interference-inducing nucleic acids, including but not limited to siRNA, shRNA, miRNA, LNA constructs, preferably comprising sequences complementary to Gp1b, GpV, GpIX, factor 8, or Nbeal2 mRNA sequences. Such constructs are known in the art and can be easily made by those skilled in the art.

[0015] Yet another option is the use of CRISPR / Cas9 or similar gene editing approaches to introduce mutations into the Gp1b, GpV, GpIX, factor VIII, or Nbeal2 genes. Thus, such CRISPR gene editing constructs would be included in the term Gp1b, GpV, GpIX, factor VIII, or Nbeal2 antagonists to the extent that they are used to attenuate the expression, stability, or function of Gp1b, GpV, GpIX, factor VIII, or Nbeal2.

[0016] A preferred embodiment of the present invention relates to non-alcoholic steatohepatitis (NASH) as NAFLD of the present invention. Therefore, it is preferred in the present invention that the treatment of NAFLD includes a patient group that suffers from NASH but does not suffer from NAFL, and therefore patients who have already developed a more advanced form of NAFLD. Therefore, a suitable patient group to be treated with the inhibitor of the present invention is a patient that shows signs of fat and inflammation in the liver, and possibly damage. In some embodiments, the patient treated in the present invention is a patient that does not suffer from signs of cirrhosis or hepatocellular carcinoma (HCC). However, other embodiments provide that the patient treated by the present invention already shows signs of cirrhosis and / or HCC. Preferably, the inhibitor of the present invention is for use in a treatment that is the prevention of HCC in NASH patients at risk of developing cirrhosis and / or HCC.

[0017] In the present invention, patients at risk of developing NAFLD (preferably NASH) are preferred subjects for the benefit of the inhibitor for use in the present invention. Such at-risk patients are, for example, diabetic patients, obese patients, or patients suffering from metabolic syndrome or another metabolic disorder.

[0018] Most preferably, the subject (patient group) treated in the present invention does not have a condition selected from the group consisting of alcoholic liver damage, drug-induced liver damage, chronic active hepatitis, liver cirrhosis, liver cancer, hepatic steatosis and hepatocyte apoptosis.

[0019] The present invention provides for the alleviation or reduction of progression of NAFLD, particularly NASH, to be treated.Therefore, the present invention provides for the treatment of NAFLD, preferably NASH.The inhibitor of the present invention is preferably used to reduce, stall or reverse the progression of NASH to cirrhosis and / or the progression of NASH to hepatocellular carcinoma (HCC).Therefore, the treatment in the present invention is preferably the alleviation of NASH to NAFLD non-NASH state.

[0020] In another aspect of the present invention, there is provided a pharmaceutical composition for use in the treatment or prevention of non-alcoholic fatty liver disease (NAFLD) as described herein above, the pharmaceutical composition comprising an inhibitor of platelets or an inhibitor of Gp1b, GpV, GpIX, factor VIII, or Nbeal2 (as described herein above) and a pharma- ceutical acceptable carrier and / or excipient.

[0021] As used herein, the term "pharmaceutical acceptable carrier" is intended to include any solvent, solubilizer, filler, stabilizer, binder, absorbent, base, buffer, lubricant, sustained release vehicle, diluent, emulsifier, humectant, lubricant, dispersion medium, coating agent, antibacterial or antifungal agent, isotonicity agent and absorption delaying agent, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is intended. Supplements can also be incorporated into the composition. In certain embodiments, the pharmaceutical acceptable carrier comprises serum albumin.

[0022] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, such as intrathecal, intraarterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration.More preferably, the route of administration is the route that directly targets the liver of the subject to be treated.

[0023] In therapeutic applications, the composition is administered to a patient already suffering from NAFLD as described above in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. The appropriate dosage of the pharmaceutical composition is easily determined according to any one of several well-established protocols. For example, animal studies (e.g., mice or rats) are commonly used to determine the maximum tolerated dose of bioactive agent per kilogram of body weight. Generally, at least one of the animal species tested is a mammal. The results of animal studies can be extrapolated to determine dosages for use in other species, such as humans. What corresponds to an effective dosage also depends on the nature and severity of the disease or condition, and the overall health of the patient.

[0024] In prophylactic applications, for example, compositions containing Gp1b, GpV, GpIX, factor VIII, or Nbeal2 antagonists are administered to patients susceptible to or otherwise at risk of liver disease. Such amounts are defined as "prophylactically effective" amounts or doses. In this use, the exact amounts depend on the patient's health and weight.

[0025] In both therapeutic and prophylactic treatments, the antagonist contained in the pharmaceutical composition can be administered in several doses or as a single dose until the desired response is achieved. Treatment is usually monitored, and further doses can be administered as necessary. The compounds of the present invention can be administered according to established dosing regimes whenever inactivation of p1b, GpV, GpIX, factor VIII, or Nbeal2 is required.

[0026] The daily dosage of the product may vary widely from 0.01 mg to 1000 mg per adult per day. Preferably, the composition contains 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1.0 mg, 2.5 mg, 5.0 mg, 10.0 mg, 15.0 mg, 25.0 mg, 50.0 mg, 100 mg, 250 mg and 500 mg of active ingredient for symptomatic adjustment of the dosage to the patient to be treated. The medicament usually contains about 0.01 mg to about 500 mg of active ingredient, preferably 1 mg to about 100 mg of active ingredient. An effective amount of the drug is usually supplied at a dosage level of 0.0002 mg / kg to about 20 mg / kg of body weight per day, in particular about 0.001 mg / kg to 10 mg / kg of body weight per day. It will be understood, however, that the specific dose level and frequency of dosing for any particular patient may vary and will depend upon a variety of factors including the activity, metabolic stability and length of action of the particular compound employed, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and any treatments the host is undergoing.

[0027] In the pharmaceutical composition of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active ingredient can be administered to animals and humans, alone or in combination with another active ingredient, in unit dosage form, in admixture with a conventional pharmaceutical support. Suitable unit dosage forms include oral route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal dosage forms, and rectal dosage forms.

[0028] Suitable unit forms for administration include oral administration forms such as tablets, gelatin capsules, powders, granules and solutions or suspensions to be taken orally, sublingual and buccal administration forms, aerosols, implants, forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration, and forms for rectal administration.

[0029] In the pharmaceutical composition of the present invention, the active ingredient is generally formulated as a dosage unit containing 0.5 mg to 1000 mg, preferably 1 mg to 500 mg, and more preferably 2 mg to 200 mg of the active ingredient per dosage unit for daily administration.

[0030] When preparing a solid composition in the form of a tablet, a wetting agent such as sodium lauryl sulfate can be added to the optionally micronized active ingredient, which is then mixed with a pharmaceutical vehicle such as silica, gelatin, starch, lactose, magnesium stearate, talc, gum arabic, etc. The tablets can be coated with sucrose, with various polymers or other suitable materials, or the tablets can be treated so as to have a prolonged or delayed activity and to continuously release a predetermined amount of the active ingredient.

[0031] Preparation in the form of gelatin capsules is obtained by mixing the active ingredient with a diluent, such as a glycol or glycerol ester, and pouring the resulting mixture into soft or hard gelatin capsules.

[0032] A preparation in the form of a syrup or elixir can contain the active ingredient together with a sweetener which is preferably calorie-free, methylparaben and propylparaben as preservatives, a flavoring, and an appropriate coloring.

[0033] The water-dispersible powders or granules can contain the active ingredient mixed with a dispersion medium or wetting agent, or a suspending agent, such as polyvinylpyrrolidone, and also with sweeteners or taste correctors.

[0034] Rectal administration is achieved using suppositories made with binders that melt at rectal temperature, such as cocoa butter or polyethylene glycols.

[0035] Parenteral, intranasal or intraocular administration is accomplished using aqueous suspensions, isotonic saline solutions or sterile and injectable solutions containing pharmacologically compatible dispersion media and / or wetting agents, such as propylene glycol, butylene glycol or polyethylene glycol.

[0036] Thus, aqueous solutions injectable by the intravenous route can be made using cosolvents, for example, alcohols such as ethanol or glycols such as polyethylene glycol or propylene glycol, and hydrophilic surfactants such as Tween™ 80. The active ingredient can be solubilized with triglycerides or glycerol esters to make oily solutions injectable by the intramuscular route.

[0037] Transdermal administration is accomplished using a multi-layered patch or reservoir in which the active ingredient is present in the form of an alcoholic solution.

[0038] Administration by inhalation is accomplished using aerosols containing, for example, sorbitan trioleate or oleic acid together with trichlorofluoromethane, dichlorotetrafluoroethane or any other biologically compatible propellant gas.

[0039] The active ingredient may also be formulated as microcapsules or microspheres, optionally with one or more carriers or additives.

[0040] Among the sustained release forms useful in the case of chronic treatment, implants can be used. These can be made in the form of an oily suspension or in the form of a suspension of microspheres in an isotonic medium.

[0041] Furthermore, the above problem is solved by a method for identifying whether a compound has activity for the treatment of NAFLD, preferably NASH (and also refers to specific medical indications as described above), the method comprising contacting a cell expressing a protein selected from the group consisting of Gp1b, GpV, GpIX, factor VIII, or Nbeal2 with a candidate compound and determining the expression, stability and / or activity of the protein compared to control cells expressing the protein and not contacted with the candidate compound, wherein a reduction in expression, stability and / or activity of the protein upon contact with the candidate compound indicates that the candidate compound has activity for the treatment of NAFLD.

[0042] The candidate compound is preferably selected from antisense nucleic acid, CRISPR-Cas9-like gene editing construct, antibody or its antigen-binding fragment, viral construct, small molecule, peptide, ribozyme, or recombinant protein. Generally, for the screening method of the present invention, any potential inhibitor of Gp1b, GpV, GpIX, factor 8, or Nbeal2 as defined herein may be used as a candidate compound.

[0043] The invention will now be further described in the following examples, but is not limited thereto, with reference to the accompanying figures and sequences. For purposes of the present invention, all references cited herein are hereby incorporated by reference in their entirety. [Brief description of the drawings]

[0044] [Figure 1] FIG. 1: Cholesterol-high lipid diet (CD-HFD) in mice recapitulates human NASH and NASH-induced HCC pathology. [Diagram 2] FIG. 1: CD-HFD recapitulates the inflammatory profile of human NASH. [Diagram 3] FIG. 1. Platelet proliferation and aggregation as a sign of activation in the liver of mice with NASH at 6 months on CD-HFD. [Figure 4]FIG. 1: Analysis of platelet activation and accumulation capacity analyzed by flow cytometry of peripheral blood. [Figure 5-1] FIG. 1 shows that treatment with the platelet aggregation inhibitor aspirin / clopidogrel (Asp / Clop) reduces the ability of platelets to aggregate, preventing NASH and abnormal hepatic lipid metabolism. [Figure 5-2] Same as above [Figure 6] FIG. 10: Reduction in the amount of intrahepatic immune cells in mice treated with Aps / Clp. [Figure 7] FIG. 1. Inhibiting platelet activity / aggregation reduces the number of HCC. [Figure 8-1] FIG. 10. COX1, COX2 inhibitors do not prevent steatosis or NASH development on CD-HFD. [Figure 8-2] Same as above [Figure 8-3] Same as above [Figure 9] Ticagrelor prevents the development of NASH and HCC. [Figure 10] Nbeal2- / - mice lack the development of markers indicative of NASH development. [Figure 11] FIG. 1 shows that GP1b− / − mice lack steatosis and NASH development. [Figure 12-1] FIG. 1. Antiplatelet therapy treats NASH and HCC in humans. [Figure 12-2] Same as above [Figure 13]Figure 1. Antiplatelet therapy reduces platelet activation / aggregation, cytokine release and platelet / immune cell interaction. (a) Levels of ADP in liver extracts of mice under ND, CD-HFD and CD-HFD / Asp-Clo (data pooled from two different independent experiments (n=10 mice per group)). *P<0.05, **P<0.01, Student's t-test. (b) Prothrombin measured by enzyme-linked immunosorbent assay (ELISA) in mouse liver extracts from different groups (representative of two independent experiments (n=5 mice per group)). **P<0.01, determined by Student's t-test. (c) Analysis of cytokines in liver extracts. Normalized amounts of liver protein extracts were analyzed in CD-HFD-fed mice treated with ND, CD-HFD and Asp-Clo (data pooled from two independent experiments, n>10 per group). Significance was determined by Student's t-test, *P<0.05, **P<0.01, ***P<0.001. (d) Representative confocal micrographs and quantification of platelet / B cell interactions in livers of ND, CD-HFD-fed and Asp-Clo-treated CD-HFD-fed mice (n=4 mice per group). Sinusoids are represented in blue, platelets in green and B cells in red. Scale bar represents 50 μm. Quantification of single platelet counts and platelets adjacent to B cells is shown in focus of view (FOV) (n=4 mice per group). Transparency of sinusoid rendering was set to 50% for visualization of intravascular events and to enhance image clarity. See also Movie S3 and Movie S4. (e) Representative confocal images from livers of ND, CD-HFD and Asp-Clo treated mice, the same mice as for (d), with platelet / CD8+T cell interactions visualized in the region of interest (ROI). Sinusoids are represented in blue, platelets in green and CD8+T cells in red. Scale bar represents 50 μm. Quantification of single platelet count and platelets / CD8+T cells is shown in focus of view (FOV) (same mice as for (d)).For a–e, two-tailed unpaired Student's t test was used for statistical analysis: *P<0.05, **P<0.01, ***P<0.001. EXAMPLES

[0045] Example 1: First research mouse model of human NASH and subsequent HCC development Indeed, this approach allowed us to study a chronic model of NASH in metabolic syndrome, inducing subsequent HCC in C57BL / 6 mice in the absence of chemical carcinogens or genetic mutations predisposing to NASH or HCC development. CD-HFD-fed mice exhibit several long-term pathologies observed in human patients: abdominal obesity, overweight, insulin resistance, liver damage, reactive oxygen species (ROS) production, fibrosis, hepatic mitochondrial damage, dyslipidemia, and NASH. Moreover, HCC developed 12 months after CD-HFD initiation and was histologically, genetically, and morphologically similar to human HCC (Figure 1). Cholesterol-high lipid diet (CD-HFD) in mice recapitulates human NASH and NASH-induced HCC pathology. (Figure 1A) Body weight development in male normal diet-fed (ND) and CD-HFD-fed C57BL / 6 mice. (Fig. 1B) Intraperitoneal glucose tolerance test performed in 6-month-old male ND and CD-HFD C57BL / 6 mice. (Fig. 1C) Quantification of serum aminotransferase (ALT) levels in male C57BL / 6 mice reflecting liver injury. (Fig. 1D) MRI analysis of livers in 6-month-old ND and CD-HFD C57BL / 6 mice. T1 (fast low-angle shot [FLASH]) OUT phase: dark color indicates steatosis. T2 TurboRare visualizes increased subcutaneous and abdominal fat as well as hepatic lipid accumulation (bright areas) in CD-HFD mice. (Fig. 1E) Gross and histopathological examination of livers from 12-month-old ND, HFD, or CD-HFD C57BL / 6 mice with arrows pointing to HCC. Scale bar: 5 mm. T: tumor. NT: nontumor. ****P=0.001. (F) Representative H&E staining of 12-month-old CD-HFD C57BL / 6 and human livers showing NASH. Accumulation of Mallory-Denk bodies (red arrows), ballooned hepatocytes (brown arrows), and neuroglial clusters (blue arrows) resembles human NASH pathology (right image). Scale bar: 50 μm.

[0046] This mouse model, which reproduces some pathophysiological aspects of human NASH, provides a basis for the present invention, allowing us to study the biology and development of NASH and the transition from NASH to HCC. Notably, using this mouse model, we demonstrate for the first time that CD8+ T cells and NKT cells become activated during metabolic syndrome and interact with hepatocytes via cytokines to alter hepatic lipid metabolism, leading to NASH and HCC. The same profile of CD8+ and NKT cell activation was found in human NASH liver, highlighting the clinical relevance of our model (Wolf et al., Cancer Cell, 2014).

[0047] Opposite results have been published previously in the framework of short-term in vivo experiments on the role of immune cells in experimentally induced NASH (Martin-Murphy et al., 2014; Lynch et al., 2012; Bhattacharjee et al., 2014). Notably, these models lacked metabolic syndrome and were usually kept on the diet for only a few weeks. In contrast, the data based on long-term CD-HFD leading to obesity, metabolic syndrome and HCC in C57BL / 6 mice demonstrate that immune cells play a key role in inducing steatosis, NASH and NASH-driven HCC (Wolf et al., Cancer Cell, 2014). Moreover, we were able to demonstrate that the inflammatory profile of livers from CD-HFD mice seems to resemble that of human NASH livers, for example as far as inflammatory cells or cytokine expression in the liver is concerned (Figure 2).

[0048] In FIG. 2, from left to right: H&E, B220, CD3, F4 / 80, MHCII, and Ly6G. Inflammatory aggregates consisting of CD3+, F480+, and MHCII+ cells were observed. Scale bar: 50 μm. (B) CD8+T and NKT cells control the development of hepatic steatosis. Sudan red staining of liver sections (12 months old, indicated genotypes) demonstrated that depletion of B and T cells (Rag1− / − mice), or more specifically CD8+ and NKT cells (β2m− / − mice), was sufficient to strongly reduce the development of steatosis upon CD-HFD. Consistently, reductions in other parameters such as aminotransferases, hepatic triglycerides, etc. are seen (not shown). Scale bar: 50 μm. (C) Representative IHC of human non-diseased control liver and NASH patient liver for CD8+ T cells, CD3+CD57+ NKT cells, LTβ and LIGHT expression with arrows indicating positive cells. Scale bar: 50 μm. LIGHT expression analyzed by densitometric analysis of immune cells and mRNA levels was derived from human cryomaterial.

[0049] Example 2: Platelets are increased, activated and aggregated in NASH Figure 3 shows platelet increase and aggregation as a sign of activation in the liver of mice with NASH at 6 months on CD-HFD. (Figure 3A) Immunohistochemical images identifying platelets that are aggregated and increased in number. Platelets are stained with GPIb. (Figure 3B) Densitometric analysis of platelets shows a significant and large increase in numbers in the liver of CD-HFD-treated mice. Data not shown: Similar images and a similar increase in the number of GPIb can be observed in patients with NASH when compared to healthy patients.

[0050] Figure 4 shows the analysis of platelet activation and accumulation capacity by flow cytometry of peripheral blood. Flow cytometry analysis shows a significantly increased ability of platelets to be activated (top) and tendency to aggregate (bottom). By using various dilution curves of CRP or Thr, reproducible and consistent analysis of platelet response rate was observed.

[0051] Treatment with the platelet aggregation inhibitor Aspirin / Clopidol (Asp / Clop) reduces platelet aggregation capacity to prevent NASH and abnormal hepatic lipid metabolism (Figure 5). (Figure 5A) Aspirin / Clopidol treatment reduces the number of platelets in the liver (white bars: ND, black bars: CD-HFD, green bars: CD-HFD treated with Asp / Clop). (Figure 5B) Platelet activation status (studied by ex vivo assay) is significantly reduced upon Asp / Clop treatment on platelets taken from peripheral blood. (Figure 5C) Platelet aggregation capacity status (studied by ex vivo aggregation assay) is significantly reduced upon Asp / Clop treatment on platelets taken from peripheral blood. (Figure 5D) Asp / Clop CD-HFD-fed mice develop obesity similar to CD-HFD mice. (Figure 5E) Liver-to-body weight ratio is rescued upon Asp / Clop treatment at 12 months of age. (Fig. 5F) Liver damage measured by serum ALT levels is prevented in Asp / Clop-treated mice. (Fig. 5G) Triglycerides are reduced in serum of 12-month-old Asp / Clop-treated mice. (Fig. 5H) Serum cholesterol is reduced in 6- and 12-month-old Asp / Clop-treated mice. (Fig. 5I) Abnormal hepatic lipid metabolism and b-oxidation are restored in Asp / Clop-treated mice as analyzed by real-time PCR analysis of gene expression in the liver. * indicates significance. (Fig. 5J and Fig. 5K) MRI and histological analysis demonstrate that Asp / Clop treatment prevents the development of NASH. (Fig. 5J) Liver pigmentation, indicated by dark staining in the T1 (FLASH) OUT phase, is not detected as in CD-HFD. At the same time, abdominal and subcutaneous fat are still visible in Asp / Clop-treated mice. (FIG. 5K) Histopathological signs of NASH (eg, balloon-like hepatocytes, see also *) are not seen in Asp / Clop-treated mice.

[0052] Furthermore, a reduction in the amount of intrahepatic immune cells in Aps / Clp-treated mice was observed (Figure 6). (Figure 6A) Reduction of several immune cell types, including CD3+ T cells, F480+ cells and MHCII-expressing cells. B220+ immune cells are not altered. Furthermore, (Figure 6B) flow cytometry analysis shows a significant reduction in the number of CD8+ T cells, activation of CD44+CD8+ T cells, CD69+CD8+ T cells as well as the number of CD3+NH1.1+ NKT cells (white bars: ND, black bars: CD-HFD, green bars: CD-HFD treated with Asp / Clop).

[0053] Example 3: Inhibition of platelet activation / aggregation reduces the number of HCC In a mouse model of NASH, treatment with Asp / Clop resulted in a reduction in HCC incidence (Figure 7). Significant reduction in the number of HCC in Asp / Clop-treated mice (white circle: ND, black circle: CD-HFD, green circle: CD-HFD treated with low dose Asp / Clop, red circle: high dose Asp / Clop).

[0054] Example 4: COX1 and COX2 are not involved in the development of NASH Sulindac, a COX1, COX2 inhibitor, does not prevent steatosis or NASH development on CD-HFD (Figure 8). (Figure 8A) CD-HFD-fed mice develop obesity similar to sulindac CD-HFD mice (white bars: ND, black bars: CD-HFD, red bars: CD-HFD treated with sulindac). (Figure 8B) Liver-to-body weight ratio is not restored upon sulindac treatment at 12 months of age. (Figure 8C) Liver damage, as measured by ALT levels in serum, is not prevented in sulindac-treated mice. (Figure 8D) Triglycerides are not significantly reduced in serum of sulindac-treated mice at 6 or 12 months of age. (Figure 8E) Serum cholesterol is not significantly reduced in Asp / Clop-treated mice at 6 and 12 months of age. (Figure 8F) Sulindac-treated CD-HFD-fed mice are insulin resistant similar to CD-HFD-fed mice, as studied by intraperitoneal glucose tolerance test. (FIG. 8G and FIG. 8H) MRI and histological analyses demonstrate that sulindac treatment does not prevent the development of NASH. (FIG. 8G) Lipid deposition in the liver, indicated by dark staining in the T1 (FLASH) OUT phase, is detected similarly to CD-HFD. At the same time, abdominal and subcutaneous fat is visible in sulindac-treated CD-HFD-fed mice as in CD-HFD-fed mice alone. (FIG. 8H) Histopathological signs of NASH, indicated by H / E staining (e.g., balloon-like hepatocytes, see also *), are present in sulindac-treated CD-HFD-fed mice. (FIG. 8I) Abnormal hepatic lipid metabolism and b-oxidation are not restored in sulindac-treated mice as analyzed by real-time PCR analysis of gene expression in the liver. * indicates significance. (FIG. 8J) Lipid deposition (indicated by Sudan red) is not reduced in 12-month sulindac-treated CD-HFD mice as shown by histological and densitometric analyses.

[0055] Example 5: The platelet aggregation inhibitor ticagrelor inhibits the development of NASH and HCC Ticagrelor prevents the development of NASH and HCC (Figure 9). (Figure 9A) CD-HFD-fed mice develop obesity similar to ticagrelor-treated CD-HFD mice (white bars: ND, black bars: CD-HFD, green bars: CD-HFD treated with ticagrelor). (Figure 9B) Liver damage measured by serum ALT levels is significantly reduced in ticagrelor-treated mice. (Figure 9C) Triglycerides tend to be reduced in the serum of 12-month-old ticagrelor-treated mice. (Figure 9D) Serum cholesterol is significantly reduced in 6-month-old and 12-month-old ticagrelor-treated mice. (Figure 9E) Abnormal hepatic lipid metabolism and b-oxidation are restored in ticagrelor-treated mice as analyzed by real-time PCR analysis of gene expression in the liver. * indicates significance. (FIG. 9F) Histopathological signs of NASH as shown by H / E staining (e.g., balloon-like hepatocytes, see also *) are absent in ticagrelor-treated CD-HFD-fed mice. (FIG. 9G) Lipid deposition (as shown by Sudan red) is reduced in 12-month sulindac-treated CD-HFD mice as shown by histological and densitometric analysis. (FIG. 9H) HCC is absent in ticagrelor-treated mice.

[0056] Example 6: Nbeal2 knockout inhibits NASH and prevents HCC Nbeal2- / - mice lack the development of markers indicative of NASH onset (Figure 10). (Figure 10A) CD-HFD-fed C57BL / 6 mice develop obesity similar to CD-HFD-fed Nbeal2- / - mice (white bars: ND, black bars: CD-HFD, blue bars: Nbeal2- / - mice receiving CD-HFD). (Figure 10B) Liver damage, as measured by ALT levels in serum, is significantly reduced in CD-HFD-fed Nbeal2- / - mice. (Figure 10C) Triglycerides are significantly reduced in serum of 6-month-old CD-HFD-fed Nbeal2- / - mice. (Figure 10D) Serum cholesterol is significantly reduced in 6-month-old CD-HFD-fed Nbeal2- / - mice. (FIG. 10E) CD-HFD-fed Nbeal2− / − mice are insulin resistant as CD-HFD-fed mice, as studied by intraperitoneal glucose tolerance test. (FIG. 10F) Abnormal hepatic lipid metabolism and b-oxidation are partially restored in CD-HFD-fed Nbeal2− / − mice, as analyzed by real-time PCR analysis of gene expression in liver. * indicates significance.

[0057] Example 7: Gp1b knockout inhibits NASH and prevents HCC GP1b- / - mice lack steatosis and NASH development (Figure 11). (Figure 11A) CD-HFD-fed C57BL / 6 mice develop obesity similar to CD-HFD-fed Gp1b- / - mice (white bars: ND, black bars: CD-HFD, yellow bars: Gp1b- / - mice receiving CD-HFD). (Figure 11B) Liver damage measured by ALT levels in serum is significantly reduced in CD-HFD-fed Gp1b- / - mice. (Figure 11C) Triglycerides are strongly reduced in serum of 6-month-old CD-HFD-fed Gp1b- / - mice. (Figure 11D) Serum cholesterol is significantly reduced in 6-month-old CD-HFD-fed Nbeal2- / - mice. (FIG. 11E) Abnormal hepatic lipid metabolism and b-oxidation are partially restored in CD-HFD-fed Gp1b- / - mice as analyzed by real-time PCR analysis of gene expression in the liver. * indicates significance. (FIG. 11F and FIG. 11G) Steatosis and NASH (e.g., balloon-like hepatocytes) are not seen in Gp1b- / - mice. Quantification of Sudan red-positive lipid droplets supports our data. (FIG. 11H) Activation and aggregation capacity are significantly reduced upon ticagrelor treatment.

[0058] Example 8: Antiplatelet therapy in humans Figure 12A shows the enzyme aspartate transaminase (AST) and alanine transaminase (ALT) levels of patients before study inclusion and after 6 months of dual antiplatelet therapy (DAPT) with aspirin and clopidogrel (n=148). Figure 12B shows MRI and ultrasound examinations of patients receiving ASA or DAPT before treatment and after 6 months of treatment. Quantification of liver fat deposition in human patients as assessed by MRI is shown in Figure 12C. The experiment shows the significant effect of DAPT in treating NASH.

[0059] Example 9: Antiplatelet therapy attenuates immune cell activity Antiplatelet therapy reduces chemokines and cytokines, and reduces ADP and prothrombin levels in the liver, as shown in Figures 13A-C. Furthermore, antiplatelet therapy was observed to reduce immune cells in the liver by blocking platelet-immune cell interactions (Figures 13D and E). [Explanation of symbols]

[0060] Drawing translation Figure 1 Body weight (g) age (months) blood glucose (mmol / l) time (minutes) T1 (FLASH)OUT phase T1 (FLASH)OUT phase Murine NASH Mouse NASH human NASH Figure 2 non-diseased human NASH CD8 + T-cells / mm 2 CD8 + T cells / mm 2 CD3 + CD57 + cells / 20x view filed CD3 + CD57 + Cell / 20x field of view LTβ + cells / mm 2 LTβ + cells / mm 2 LIGHT mRNA expression Figure 3 Platelet counts (HPF) Platelet counts (HPF) 12 months Figure 4 P-Selectin-FITC (MFI) P-Selectin-FITC (MFI) Rest Figure 5 Thrombocytes / gr Platelets / gr Liver Blood blood Rest Light transmission Body weight (g) age (months) Liver / body weight(%) Figure 5 (continued) triglycerides μg / mg 6 months 12 months Cholesterol mg / dl T1 (FLASH)IN Phase T1 (FLASH)IN phase T1 (FLASH)OUT Phase T1 (FLASH)OUT phase H / E(10x) H / E(10x) H / E(20x) H / E(20x) H / E(40x) H / E(40x) Figure 6 total # ofcells / liver Figure 8 body weight [g] age [months] Age in months Sulindac liver / body weight[%] 6 months 12 months triglycerides [μg / mg] Triglycerides (μg / mg) cholesterol [mg / dl] Cholesterol (mg / dl) blood glucose [mmol / l] Blood glucose (mmol / l) Figure 8 (continued) T1 (FLASH)IN Phase T1 (FLASH)IN phase CD-HFD / Sulindac CD-HFD / Sulindac H / E(10x) H / E(10x) H / E(20x) H / E(20x) H / E(40x) H / E(40x) Figure 8 (continued) CD-HFD / Sulindac CD-HFD / Sulindac 6 months 12 months Sudan Red area(%) Sudan Red area(%) Figure 9 Body weight (g) Ticagrelor age (months) triglycerides μg / mg Cholesterol mg / dl CD-HFD / Ticagrelor CD-HFD / Ticagrelor H / E(10x) H / E(10x) H / E(20x) H / E(20x) H / E(40x) H / E(40x) 6 months Sudan red area(%) Sudan Red area(%) Figure 10 Body weight (g) age (months) triglycerides μg / mg Cholesterol mg / dl blood glucose (mmol / l) Figure 11 Body weight (g) age (months) triglycerides μg / mg Cholesterol mg / dl H / E 10x H / E 10x H / E 20x H / E 20x H / E 40x H / E 40x 6 months Sudan red area(%) Sudan Red area(%) Pselectin-FITC (MFI) Pselectin-FITC (MFI) Rest Figure 12 Study inclusion After 6M of DAPT Right lobe Light lobe left lobe Ultra sound Figure 12 (continued) Liver fat reduction (%) No antiaggregation (antiaggregation) After 6 months of Aspirin Figure 13 Prothrombin (ng / mg) Concentration (pg / ml) PLT count (FOV) B cells adjacent to PLT (number per FOV)

Claims

1. A pharmaceutical for treating or preventing non-alcoholic steatohepatitis (NASH), comprising an inhibitor of platelet aggregation or activation, wherein the inhibitor is an inhibitor of Gp1b, and the inhibitor is an antigen-binding fragment of an anti-Gp1b antibody.

2. The pharmaceutical composition of claim 1, wherein the treatment is the alleviation or reduction of progression of NASH.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the treatment is reducing, stalling or reversing the progression of NASH to cirrhosis.

4. The medicament according to any one of claims 1 to 3, wherein the treatment is prevention of hepatocellular carcinoma (HCC) in NASH patients at risk of developing cirrhosis and / or HCC.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the treatment is carried out in a patient at risk of developing NASH, the patient being a diabetic patient, an obese patient, or a patient suffering from metabolic syndrome or another metabolic disorder.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the patient to be treated does not have a condition selected from the group consisting of alcoholic liver damage, drug-induced liver damage, chronic active hepatitis, hepatic steatosis and hepatocellular apoptosis.

7. The method of claim 6, wherein the patient suffers from inflammatory fatty liver.

8. The pharmaceutical agent according to any one of claims 1 to 7, wherein the antigen-binding fragment of the anti-Gp1b antibody is selected from the group consisting of a minibody, a single-chain antibody (scFv), a variable region fragment (Fv or Fd), Fab and F(ab)2.

9. A method for identifying whether a compound has activity for treating NASH, comprising contacting a cell expressing Gp1b protein with a candidate compound and determining the expression, stability and / or activity of the protein compared to control cells expressing the protein and not contacted with the candidate compound, wherein a reduction in expression, stability and / or activity of the protein upon contact with the candidate compound indicates that the candidate compound has activity for treating NASH, and wherein the candidate compound is an antigen-binding fragment of an anti-Gp1b antibody.

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