[001] This application claims the benefit of priority of United States provisional application no. 63/476,065, filed December 19, 2022, the contents of which are incorporated by reference as if written herein in their entirety.

FIELD OF THE INVENTION

[002] The present invention relates to compounds for inhibition of plasminogen activator inhibitor 1 to treat a disease associated with gastrointestinal inflammation, such as IBD, colitis, diverticulosis, proctitis, autoimmune gastritis, autoimmune enteropathy, celiac disease, gastrointestinal manifestations of cystic fibrosis.

BACKGROUND

[003] The two distinct forms of inflammatory bowel diseases (IBD) - ulcerative colitis and Crohn’ s disease - have been sporadically observed since ancient times and have emerged as a growing problem in industrialized nations (PMID: 22001864). Since the middle of the twentieth century, the incidence of ulcerative colitis and Crohn’s disease has increased in the Western world, which includes North America, Europe, Australia, and New Zealand (PMID: 22001864). Currently, the prevalence of IBD in the Western world is up to 0.5% of the general population. In contrast IBD was relatively rare in developing nations. However, over the past few decades, newly industrialized countries in Asia, South America and the Middle East have documented the emergence of IBD (PMID: 23583432).

[004] IBD is a chronic, incurable disease with low mortality. Although IBD can be diagnosed at any age from infancy to elderly, the majority of new diagnoses are made in adolescence and early adulthood (PMID: 15168363). Thus, every year, newly diagnosed patients with IBD unremittingly expand the number of prevalent cases. This will lead to an exponential growth of the number of patients with IBD worldwide, which is called compounding prevalence in epidemiology. The compounding of prevalence of IBD is an important concept in health-care delivery because health-care systems might not be prepared for the staggering increase in the burden of IBD. [005] Crohn’s disease (CD) and ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), are chronic, relapsing, and tissue destructive lesions that are accompanied by the uncontrolled activation of effector immune cells in the mucosa. IBD occurs as a consequence of an abnormal immune response against the luminal microflora generating an unbalance between pro- and anti-inflammatory signaling. Cytokines released by T helper (Th)-l and Th- 17 cells, as interferon (IFN)-y, tumor necrosis factor (TNF)-a, interleukin- 12 (IL-12), IL-17, IL-21, IL-22 and IL-23 are dominant in CD, whereas the Th2 cytokines, as IL-5, IL-9 and IL- 13, are predominantly found in UC (PMID: 12042806). Classically, immune-modulating treatments of IBD have focused on adaptive immunity. Corticosteroids, which have been widely used to treat acute flares of IBD, suppress proinflammatory cytokines, such as TNF-a and IL-10, is (PMID: 21511881). Likewise, various immunomodulators that down-regulate the proinflammatory cytokines of T cells have been a well-established treatment for IBD. Of these treatments, the most widely used agents are thiopurines, that inhibit purine nucleotide synthesis and breaking of DNA in leukocytes via 6-thioguanine nucleotides (PMID: 1451710). Moreover, thiopurine suppresses CD4+ T cell activity and promotes T cell apoptosis in inflamed intestine (PMID: 12697733).

[006] The better understanding of the pathomechanism of IBD led to an increasingly rapid advent of novel biologies and small molecules available for the treatment of moderate-to-severe UC. The era of biological therapy began with an anti-TNF agents, first infliximab (chimeric antibody with a murine sequence) and then adalimumab (fully humanized antibody), which dramatically changed the treatment of refractory IBD, reduced surgeries for IBD and improved quality of life (PMID: 23896172). Blocking the TNF-a signal works through various mechanisms, including T cell apoptosis, inhibiting T cell differentiation, induction of Treg cells and macrophages, and barrier improvement (PMID: 12655295). In particular, since the introduction of anti-tumor necrosis factor alpha biologies (anti-TNFa) (ie, infliximab, adalimumab, golimumab), anti-integrins (vedolizumab) and, more recently, of anti- interleukins (ustekinumab), the natural history of the disease has started to change. Increasing rates of corticosteroid- free remission, mucosal healing, deep remission, and an ameliorated quality of life of UC patients have become possible (PMID: 28326566, PMID: 30694863, PMID: 30378141). Nevertheless, the treatment with biologies implicates various limitations, such as the primary non- responsiveness, a rather limited efficacy, the eventual secondary loss of response, and the risk of immunogenicity. Moreover, biologies have a parental administration, either intravenous or sub-cutaneous, which can impair their tolerance. Small molecules, administrated as oral agents, have the ambition of overcoming such limitations. Initially, tofacitinib, a Janus kinase (JAK) inhibitor, was the first small molecule drug approved by international regulatory authorities for the treatment moderate-to-severe UC (PMID: 28770978. Indeed, the cellular transduction pathway activated by JAK, intracellular tyrosine kinases, is involved in the pathogenesis of UC (PMID: 25110261, PMID: 24417533), and its pharmacological inhibition has been demonstrated as an effective treatment for UC (PMID: 28770978). Filgotinib is an oral JAK1 preferential inhibitor, that has been evaluated in several randomized controlled trials (RCTs) for both rheumatological disorders (e.g., psoriatic arthritis, ankylosing spondylitis) (PMID: 30360969, PMID: 30360970, PMID: 31334793) and IBD, and finally approved in patients affected by rheumatoid arthritis and moderately to severely active UC. In 2016 the randomized, double-blind, placebo-controlled Phase 2 study, the FITZROY study provided the first evidence on the efficacy and safety of filgotinib for the treatment of active moderate-to-severe Crohn’s disease (CD) (PMID: 27988142). Yet, there are still an unmet need to develop further therapy options for IBD patients.

[007] A possible therapeutic target is the TGF-P 1 signaling, which functions as a negative regulator of T cell immune responses and controls the activation, proliferation and differentiation of both immune and non-immune cells (PMID: 9597127). TGF-P dampens the function of effector T cells and macrophages, promotes the differentiation of regulatory T cells (Treg) and T helper (Th) 17 cells, stimulates collagen deposition by stromal cells and favors the margination of epithelial cells (PMID: 16648837). The activation of TGF-P receptor type I receptor by TGF-P is mediated from the membrane to the nucleus by Smad proteins (PMID: 12809600). Once activated TGF-PRI phosphorylates Smad2 and Smad3, thus promoting their association with Smad4 and translocation of the complex to the nucleus, where Smad proteins regulate expression of target genes (PMID: 9865691). Lack of TGF-P in mice led to the development of a multiorgan inflammatory disease involving the gut (PMID: 1436033). Moreover, transgenic mice, whose T cells cannot respond to TGF-P, developed inflammation in the gastrointestinal tract (PMID: 10714683). In murine models of IBD, inhibition of TGF-P signaling exacerbated colitis, while boosting its activity protected from the development of intestinal inflammation (PMID: 8676081). Monteleone et al. showed previously that Smad7, an inhibitor of TGF-P 1 signaling, is overexpressed in IBD mucosa and purified mucosal T cells, whereas inhibition of Smad7 antisense oligonucleotides restores TGF-P 1 signaling and enables TGF-pi to inhibit cytokine production (PMID: 11518725). In mice with trinitrobenzene sulfonic acid or oxazolone-induced colitis, there was increased production of non- functional TGF-P, decreased expression of phosphorylated Smad3 and elevated levels of Smad7. Oral administration of Smad7 antisense oligonucleotide to these mice reduced the colonic expression of Smad7, enhanced Smad3 phosphorylation and attenuated intestinal inflammation (PMID: 17087939).

[008] The above preclinical studies on Smad7 in IBD led to the development of a pharmaceutical compound, named GED-0301 and later on mongersen, containing Smad7 ASO. The drug was formulated as an oral tablet protected by an external coating that allows the compound to be primarily released in the terminal ileum and right colon, the most frequently affected sites in CD. In a phase 1 open-label, dose-escalation study, GED-0301 was administered to patients with active, steroid-dependent/resistant CD, to assess the safety and tolerability of the drug. The compound was safe and well tolerated by all patients (PMID: 22252452). GED-0301 administration was associated with clinical benefit in all the patients. Subsequent pre-clinical and clinical studies showed that knockdown of Smad7 with GED-0301 associated with no intestinal fibrosis (PMID: 29668937, PMID: 22971085). Next, a phase 2, multicenter, double-blind placebo-controlled study was conducted to assess the efficacy of mongersen in patients with active, steroid dependent/resistant CD. Patients receiving 40 or 160 mg/day mongersen had significantly higher rates of remission than those treated with placebo, whereas there was no difference between the 10 mg/day mongersen and placebo. Later on, another phase 2, multicenter study confirmed the clinical efficacy of mongersen and demonstrated an endoscopic improvement in more than one third of the patients enrolled. (PMID: 28847751). Surprisingly, these promising results were not confirmed by a subsequent phase 3 clinical trial (PMID: 31850931). The reasons for these unexpected results, given the positive results of 2 phase 2 studies, remain unknown.

[009] Despite the failure of mongersen enhancing the TGF-|3 signaling remained a promising therapeutic target in IBD.

|010] Based on the above there is a huge unmet clinical need in IBD to develop novel drug candidates that would revolutionize the routine IBD clinical practice and can overcome the limitations of the current therapies. Although biologic therapies with anti-TNF are now the gold standard of IBD treatment, up to 40% of patients do not respond to the anti-TNF therapy (nonresponders) or lose response after initial improvement (loss of response) as described above (PMID: 24393836, PMID: 28275925). This increases the clinical and economic burden of IBD remarkably. Whereas despite the increased number of treatment options, it is still difficult to reach remission in IBD patients. Moreover, biologies have a parental administration, either intravenous or sub-cutaneous, that can impair their tolerance, which can be overcome by oral agents.

DESCRIPTION OF THE INVENTION

1. A compound according to Formula I:

Formula I wherein n= 0-10; i= 1-200;

Ri = H, Cl-10 alkyl or cycloalkyl, aryl, these ideally substituted with N, O, S, P, Se, Si, As or halides, and may form ideally a ring system, and may be glycosylated.

R ₂ = H, Cl-10 alkyl or cycloalkyl, aryl, these ideally substituted with N, O, S, P, Se, Si, As or halides, and may form ideally a ring system, and may be glycosylated.

X=H, OH, O-alkyl, O-aryl, NH2, NH-alkyl, NH-aryl, N-(alkyl)2, N-(aryl)2, SH, S- alkyl, S-aryl, alkyl, alkenyl, alkynyl, NH-NH ₂ , NH-NH-alkyl, NH-NH-aryl, NH-N- (alkyl) ₂ , NH-N-(aryl) ₂ ; and wherein the compound is selected from the group of pharmaceutically acceptable salt, enantiomers and diastereomers; and wherein the compound comprises pharmaceutically acceptable stereoisomers, enantiomers, diastereomers, racemic mixtures, polymorphs, tautomers, solvates, salts, esters, prodrugs or combinations thereof.

2. The compound of point 1, wherein Ri is an alkyl chain.

3. The compound of point 1, wherein Rl is a methyl group and R2 is an alkyl chain ideally substituted with O.

4. A compound according to formula II:

Formula II wherein

Ri = Cl-10 ideally substituted with O, S, P, Se, Si, As or halides, and may form ideally a ring system,

R2— Cl-10 alkyl or cycloalkyl, these ideally substituted with O, and may form ideally a ring system, and may be glycosylated, further; and wherein the derivatives of the compound is selected from the group of pharmaceutically acceptable salt, enantiomers and diastereomers; and wherein the compound comprises pharmaceutically acceptable stereoisomers, enantiomers, diastereomers, racemic mixtures, polymorphs, tautomers, solvates, salts, esters, prodrugs or combinations thereof.

5. The compound according to point 4 wherein Ri is an alkyl chain.

6. The compound according to point 5 wherein Rl is a methyl group and R2 is an alkyl chain ideally substituted with O.

7. A compound according to formula III:

Formula III wherein

Ri = Cl-10 ideally substituted with O, S, P, Se, Si, As or halides, and may form ideally a ring system,

R2— Cl-10 alkyl or cycloalkyl, these ideally substituted with O, and may form ideally a ring system, and may be glycosylated; and wherein the compound is selected from the group of pharmaceutically acceptable salt, enantiomers and diastereomers; and wherein the compound comprises pharmaceutically acceptable stereoisomers, enantiomers, diastereomers, racemic mixtures, polymorphs, tautomers, solvates, salts, esters, prodrugs or combinations thereof.

8. The compound according to point 7 wherein Ri is an alkyl chain.

9. The compound according to point 8 wherein RI is a methyl group and R2 is an alkyl chain ideally substituted with O.

10. The compound according to point 1 to point 9 wherein the compounds inhibit the activity of PAI- 1 allowing tPA to convert plasminogen into active plasmin, which activates MMPs.

11. The compounds according to point 1 to point 9 wherein the compounds inhibit PAI-1 at the concentration range of 1 pM to 1 mM.

12. The compounds according to point 1 to point 9 wherein the compounds effectively impair the transmural inflammation, mucosal ulceration and loss of crypt structures in colitis; and prevent the goblet cell depletion in colitis; and significantly reduce the colitis induced spleen weight increase and the colitis caused decrease of the colon length at 0.01-100 mg/bwkg dose.

13. The compounds according to point 1 to point 9 wherein the compounds significantly reduce mRNA expression of TNF-a and IL-ip. The compounds according to point 1 to point 9 wherein the compounds have significant antiinflammatory effect due to the inhibition of PAI- 1 in inflammatory gastrointestinal diseases. The compounds according to point 1 to point 9 wherein the compounds promote the tissue remodelling and regeneration in chronic inflammatory diseases by decreasing the extent of the fibrotic tissue accumulation. The compounds according to point 1 to point 9 wherein the compounds decrease the expression of the fibrosis-related genes in human colonic fibroblasts in vitro. The compounds according to point 1 to point 9 wherein the compounds decrease the severity of chronic pancreatitis and chronic colitis. A method of treating a disease associated with gastrointestinal inflammation, such as IBD, colitis, diverticulosis, proctitis, autoimmune gastritis, autoimmune enteropathy, celiac disease, gastrointestinal manifestations of cystic fibrosis (acute, chronic and autoimmune pancreatitis, hepatitis, cholangitis, metabolic syndrome, ischemic stroke, coronary heart disease, venous thrombosis, and atherosclerosis, cystic fibrosis, chronic obstructive pulmonary disease, chronic kidney disease); and chronic inflammation associated and idiopathic fibrosis, such as chronic lung diseases, intestinal stenosis, liver cirrhosis and chronic kidney disease, idiopathic pulmonary fibrosis. A pharmaceutical composition comprising a compound of point 1 to point 9, which may be any of the compounds disclosed herein. Such pharmaceutical compositions may also comprise a pharmaceutically-acceptable diluent, carrier and/or excipient. In some embodiments, the pharmaceutical composition comprises more than one compound of the invention. In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients and/or adjuvants. In certain embodiments the pharmaceutical composition may further comprise one or more ingredients therapeutically effective for the same disease indication. The pharmaceutical composition of point 19 can be formulated: (a) for administration selected from the group consisting of oral, pulmonary, rectal, colonic, parenteral, intracisternal, intravaginal, intraperitoneal, ocular, otic, local, buccal, nasal, and topical administration; (b) into a dosage form selected from the group consisting of liquid dispersions, gels, aerosols, ointments, creams, lyophilized formulations, tablets, capsules; (c) into a dosage form selected from the group consisting of immediate release, controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; or (d) any combination of (a), (b), and (c).

21. The pharmaceutical composition of point 20 wherein the pharmaceutical composition can be formulated by adding different types of pharmaceutically acceptable excipients for oral administration in solid, liquid, local (powders, ointments or drops), or topical administration, and the like.

22. A method of treating disease associated with gastrointestinal inflammation and chronic inflammation associated fibrosis in a subject in need thereof, wherein said method comprises administration of a therapeutically effective amount of one or more compounds disclosed herein, to the subject, optionally in combination with one or more therapeutic agents, preferably by administering a therapeutically effective amount of compound according to point 1 to point 9 or variant thereof, to a human subject in need thereof.

23. A method of treating, reducing, inhibiting or controlling disease associated with gastrointestinal inflammation and chronic inflammation associated fibrosis in a subject, wherein said method comprises simultaneously, separately or sequentially administering to the subject, (i) one or more therapeutic agents, and, (ii) a therapeutically effective amount of one or more compounds disclosed herein or a pharmaceutical composition disclosed herein, preferably by administering a therapeutically effective amount of compound of point 1 to point 9 or variant thereof, or a pharmaceutical composition comprising any of compound point 1 to point 9 or a variant thereof, to a human subject in need thereof.

24. A method of treating, reducing, inhibiting or controlling at least one sign or symptom of disease associated with gastrointestinal inflammation and chronic inflammation associated fibrosis in a subject, wherein said method comprises administration of a therapeutically effective amount of one or more compounds disclosed herein or a pharmaceutical composition disclosed herein, to the human subject, optionally in combination with one or more therapeutic agents, wherein said sign or symptom is associated with disease associated with gastrointestinal inflammation. Preferably said method involves administering a therapeutically effective amount of a compound according to the invention described herein, such as compound point 1 to point 9 or variant thereof, or a pharmaceutical composition comprising any of compound point 1 to point 9 or a variant thereof, to a human subject in need thereof.

25. A method of treating, reducing, inhibiting or controlling at least one sign or symptom of disease associated with gastrointestinal inflammation and chronic inflammation associated fibrosis in a subject, wherein said method comprises simultaneously, separately or sequentially administering to the subject, (i) one or more therapeutic agents, and, (ii) a therapeutically effective amount of one or more compounds disclosed herein or a pharmaceutical composition disclosed herein, wherein said sign or symptom is associated with disease associated with gastrointestinal inflammation. Preferably said method involves administering a therapeutically effective amount of a compound according to the invention described herein, such as compound point 1 to point 9 or variant thereof, or a pharmaceutical composition of the invention, preferably comprising any of compound point 1 to point 9 or a variant thereof, to a human subject in need thereof.

26. A therapeutically effective amount of compound according to point 1 to point 9 is between about 1 ng and about 5 g, preferably between about 10 ng and 1g.

BRIEF DESCRIPTION OF DRAWINGS

[Oi l] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein) of which:

[012] Figure 1. shows the summary bar charts show PAI-1 activity after 1 hour incubation the plasma samples with different type of PAI-1 inhibitors.

[013] Figure 2 shows the summary bar charts show the percentage of PAI-1 activity after 1 hour incubation the plasma samples with different type of PAI- 1 inhibitors.

[014] Figure 3 shows that Compound IV (LAN-22-103) incubation in different concentration does not have toxic effect on HeLa cells in vitro. [015] Figure 4 shows the treatment protocol of DSS-induced colitis and Compound IV (LAN- 22-103) administration.

[016] Figure 5 shows the representative images of colon histology in DSS-induced colitis.

[017] Figure 6 shows the effect of Compound IV (LAN-22-103) treatment on spleen weight and colon length in DSS-induced colitis model in mice.

[018] Figure 7 shows the effect of Compound IV (LAN-22-103) treatment on spleen weight and colon length in DSS-induced colitis model in mice.

[019] Figure 8 shows the effect of Compound IV (LAN-22-103) on mRNA level of inflammatory cytokines.

[020] Figure 9 shows the effect of Compound IV (LAN-22-103) on protein level of inflammatory cytokines.

[021] Figure 10 shows the gene expression pattern of ACTA-2, FN1, and PAI-1 in human colonic fibroblasts. Relative gene expression fold changes (Fc) in the human colonic fibroblasts established from an IBD patient with active inflammation revealed that the TGFf> treatment induced marked increase in the gene expressions, which was impaired by Compound IV (LAN- 22-103).

[022] Figure 11 shows the quantification of the hydroxyproline concentration of the colonic tissue. Hydroxyproline is a major component of fibrillar collagen of all types and it’s concentration increases in the presence of tissue fibrosis. As summarized in the bar chart the DSS treatment significantly increased hydroxyproline concentration of the colonic tissue, which was significantly decreased by the treatment with Compound IV (LAN-22-103). Each datapoint represents an individual animal.

[023] Figure 12 shows the summary of the pancreas weight/body weight ratio. No significant increase was observed in the Compound IV (LAN-22-103) treated group, although we see a moderate improvement. Each datapoint represents an individual animal.

[024] Figure 13 shows the quantification of the Crossmon’s Trichrome staining. The cerulein treatment significantly increased the % of the fibrotic tissue within the exocrine pancreas. This was significantly decreased by the treatment with Compound IV (LAN-22-103). Each datapoint represents an individual animal.

[025 ] Figure 14 shows the quantification of the hydroxyproline concentration of the pancreatic tissue. Hydroxyproline is a major component of fibrillar collagen of all types and it’s concentration increases in the presence of tissue fibrosis. As summarized in the bar chart the cerulein treatment significantly increased hydroxyproline concentration of the pancreatic tissue, which was significantly decreased by the treatment with Compound IV (LAN-22-103). Each datapoint represents an individual animal.

DETALED DESCRIPTION OF THE INVENTION

[026] The SERPINE1 gene provides instructions for making a protein called plasminogen activator inhibitor 1 (PAI-1). PAI-1 is involved in normal blood clotting (hemostasis) (PMID: 19085655). After an injury, clots protect the body by sealing off damaged blood vessels and preventing further blood loss. The PAI-1 protein blocks (inhibits) the action of other proteins called plasminogen activators (PA). These proteins, including urokinase plasminogen activator (u-PA) and tissue type plasminogen activator (t-PA), convert an inactive enzyme called plasminogen to its active form, plasmin. Plasmin is involved in fibrinolysis, which is the process of dissolving blood clots. By inhibiting the conversion of plasminogen to plasmin, and thereby preventing fibrinolysis, the PALI protein helps ensure that clots are only dissolved when they are no longer needed to stop bleeding. When the body does not have enough functional PAI1, the body’s ability keep blood clots intact is impaired. Plasminogen activator inhibitor type 1 deficiency is a rare bleeding disorder that causes excessive or prolonged bleeding due to blood clots being broken down too early (PMID: 9207454). PAI-1 mediated plasmin activation also regulates the normal extra cellular matrix (ECM) homeostasis (PMID: 1916049). PALI can be secreted by many cell types (e.g. endothelial-, epithelial- and immune cells) (PMID: 6438106). The expression of this cytokine is regulated by growth factors, hormones and inflammatory cytokines (e.g. TNF-a, IL-13, IL-6 and TGF-3) (PMID: 28520219). In pathophysiological condition, PALI expression is observed in many diseases. In metabolic disturbances (obesity, type 2 diabetes and metabolic syndrome) the higher expression of PALI was defined as well (PMID: 11916936). These diseases also promote the cardiovascular diseases (PMID: 31001415), where the upregulated PALI level is also defined. The increased level is a risk factor for cardiovascular diseases (PMID: 26896607). In IBD patients the risk for the development of thrombosis is three times higher (PMID: 24415858). In the active IBD PALI level is elevated highly and correlation with severity was observed (PMID: 30842312). One of the mechanisms by which PAI-l/tPA may affect colonic injury models appears to be through the cleavage and activation of TGF-0 which is known to be a potent anti-inflammatory and prorepair cytokine during colitis and colonic wounding, respectively (PMID: 22956684). Elevated PAI-1 may lead to ongoing chronic inflammation by dampening down this anti-inflammatory axis. Elevated PAI-1 may also signify a more severe level of colonic mucosal damage or fibrosis, which may correlate both with active disease and resistance to anti-TNF. Kaiko et al. conducted global transcriptome analysis to identify IBD-related pathways using colon biopsies, which highlighted the coagulation gene pathway as one of the most enriched gene sets in patients with IBD (PMID: 30842312). Using this gene-network analysis across 14 independent cohorts and 1800 intestinal biopsies, they found that, among the coagulation pathway genes PAI-1 expression was highly enriched in active disease and in patients with IBD who did not respond to anti-TNF biologic therapy and that PAI-1 is a key link between the epithelium and inflammation. Intestinal epithelial cells produced tPA, which was protective against chemical and mechanical-mediated colonic injury in mice. In contrast, PAI-1 exacerbated mucosal damage by blocking tPA-mediated cleavage and activation of anti-inflammatory TGF-[3, whereas the inhibition of PAI-1 reduced both mucosal damage and inflammation. It is probable that tPA/PAI- 1 as a protease/anti-protease coupling have pleiotropic effects and there are likely other mechanisms by which these molecules may impact colonic injury and inflammatory phenotypes. Indeed, studies of PAI-1 deficiency in other organ systems have shown that the PAl-l/tPA axis cleaves and activates other tissue factors, including growth factors unrelated to coagulation or fibrinolysis. For example, in the liver tPA increases (and PAI-1 inhibits) activation of hepatocyte growth factor to protect against liver damage (PMID: 17561000). In a recent study strong associations of SerpinEl with CEBPB (Bayesian network linking between the immune and epithelial compartments) and ITGA5 (by gene correlation analysis) was described in the colon of patients with IBD (PMID: 30842312).

[027] The present invention arises because novel artificial amino acid-based (peptide based) PAI- 1 inhibitors have surprisingly been found, and these artificial amino acid-based PAI-1 inhibitors are able to inhibit the PAI-1 even after oral administration. These artificial amino acid-based PAI-1 inhibitors are therefore expected to be useful for the treatment of diseases and conditions associated with increased PAI-1 activity in various inflammatory diseases, like IDB, CD, CU, COPD, chronic inflammation associated fibrosis in chronic lung diseases, intestinal stenosis, liver cirrhosis and chronic kidney disease, idiopathic fibrosis syndromes such as idiopathic pulmonary fibrosis etc. [028] The artificial amino acid-based PAI-1 inhibitors can increase the concentration of the mature TGF[3 and enhance the anti-inflammatory effect of the TGF[3 signaling pathway; thus all patients having a disease condition associated with acute or chronic inflammation, such as IBD, can be treated using the compounds and pharmaceutical compositions according to the invention.

[029] The application of the artificial amino acid-based PAI-1 inhibitors can increase the active protease content in the tissue, such as plasmin, or matrix metalloproteases, which can promote the tissue remodelling and regenation in chronic inflammatory diseases by decreasing the extent of the fibrotic tissue accumulation.

[030] Disclosed herein a compound according to Formula I:

Formula I wherein n= 0-10; i= 1-200;

Ri = H, Cl-10 alkyl or cycloalkyl, aryl, these ideally substituted with N, O, S, P, Se, Si, As or halides, and may form ideally a ring system, and may be glycosylated.

R ₂ — H, Cl-10 alkyl or cycloalkyl, aryl, these ideally substituted with N, O, S, P, Se, Si, As or halides, and may form ideally a ring system, and may be glycosylated.

X=H, OH, O-alkyl, O-aryl, NH2, NH-alkyl, NH-aryl, N-(alkyl)2, N-(aryl)2, SH, S- alkyl, S-aryl, alkyl, alkenyl, alkynyl, NH-NH ₂ , NH-NH-alkyl, NH-NH-aryl, NH-N- (alkyl) ₂ , NH-N-(aryl) ₂ . [031] In an embodiment the derivatives of the compound of the present invention can be selected from the group of pharmaceutically acceptable salt, enantiomers and diastereomers.

[032] In an embodiment the compound of the present invention comprises pharmaceutically acceptable stereoisomers, enantiomers, diastereomers, racemic mixtures, polymorphs, tautomers, solvates, salts, esters, prodrugs or combinations thereof.

[033] In an embodiment Ri is an alkyl chain wherein the alkyl chain.

[034] In an embodiment the compound according to the present invention wherein Rl is a methyl group and R2 is an alkyl chain ideally substituted with O.

[035] A compound according to formula II:

Formula II wherein

Ri = Cl-10 ideally substituted with O, S, P, Se, Si, As or halides, and may form ideally a ring system,

R = Cl-10 alkyl or cycloalkyl, these ideally substituted with O, and may form ideally a ring system, and may be glycosylated, further.

[020] In an embodiment the derivatives of the compound of the present invention can be selected from the group of pharmaceutically acceptable salt, enantiomers and diastereomers.

[036] In an embodiment the compound of the present invention comprises pharmaceutically acceptable stereoisomers, enantiomers, diastereomers, racemic mixtures, polymorphs, tautomers, solvates, salts, esters, prodrugs or combinations thereof.

[037] In an embodiment Ri is an alkyl chain.

[038] In an embodiment the compound according to the present invention wherein Rl is a methyl group and R2 is an alkyl chain ideally substituted with O. [039] A compound according to formula III:

Formula III wherein

Ri = Cl-10 ideally substituted with O, S, P, Se, Si, As or halides, and may form ideally a ring system,

R2— Cl-10 alkyl or cycloalkyl, these ideally substituted with O, and may form ideally a ring system, and may be glycosylated.

[040] In an embodiment the derivatives of the compound of the present invention can be selected from the group of pharmaceutically acceptable salt, enantiomers and diastereomers.

[041] In an embodiment the compound of the present invention comprises pharmaceutically acceptable stereoisomers, enantiomers, diastereomers, racemic mixtures, polymorphs, tautomers, solvates, salts, esters, prodrugs or combinations thereof.

[042] In an embodiment Ri is an alkyl chain.

[043] In an embodiment the compound according to the present invention wherein RI is a methyl group and R2 is an alkyl chain ideally substituted with O.

[044] In an embodiment, the compounds of the present invention inhibit PAI-1 at the concentration range of 1 pM to 1 mM.

[045] In an embodiment, the compounds having Formula (I) to Formula (III) of the present invention effectively impaired the transmural inflammation mucosal ulceration and loss of crypt structures in colitis at 0.01-100 mg/bwkg dose.

[046] in an embodiment, the compounds having Formula (I) to Formula (III) of the present invention prevent the goblet cell depletion in colitis.

[047] In and embodiment, the compounds having Formula (I) to Formula (III) of the present invention significantly reduce the colitis induced spleen weight increase. [048] In an embodiment, the compounds having Formula (I) to Formula (III) of the present invention significantly reduce the colitis caused decrease of the colon length.

[049] In an embodiment, the compounds having Formula (I) to Formula (III) of the present invention significantly reduced mRNA expression of TNF-a and IL-ip, while IL-6 gene expression does not change.

[050] In an embodiment, the compounds having Formula (I) to Formula (III) of the present invention have strong immunomodulatory effect.

[051 ] In an embodiment, the compounds having Formula (I) to Formula (III) of the present invention promote the tissue remodelling and regeneration in chronic inflammatory diseases by decreasing the extent of the fibrotic tissue accumulation.

[052] In an embodiment, the compounds having Formula (I) to Formula (III) of the present invention decreases the expression of the fibrosis-related genes in human colonic fibroblasts in vitro.

[053] In an embodiment, the compounds having Formula (I) to Formula (III) of the present invention decrease the severity of chronic colitis.

[054] In an embodiment, the compounds having Formula (I) to Formula (III) of the present invention decreases the severity of chronic pancreatitis.

[055] In some embodiments of the invention, there is provided a method of treating a disease associated with gastrointestinal inflammation, such as IBD, colitis, diverticulosis, proctitis, autoimmune gastritis, autoimmune enteropathy, celiac disease, gastrointestinal manifestations of cystic fibrosis (acute, chronic and autoimmune pancreatitis, hepatitis, cholangitis, metabolic syndrome, ischemic stroke, coronary heart disease, venous thrombosis, and atherosclerosis, cystic fibrosis, chronic obstructive pulmonary disease, chronic kidney disease); and chronic inflammation associated and idiopathic fibrosis, such as chronic lung diseases, intestinal stenosis, liver cirrhosis and chronic kidney disease and idiopathic pulmonary fibsosis.

[056] In some embodiments, there is provided a pharmaceutical composition comprising a compound of the invention, which may be any of the compounds disclosed herein. Such pharmaceutical compositions may also comprise a pharmaceutically-acceptable diluent, carrier and/or excipient. In some embodiments, the pharmaceutical composition comprises more than one compound of the invention. In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients and/or adjuvants. In certain embodiments the pharmaceutical composition may further comprise one or more ingredients therapeutically effective for the same disease indication.

[057] The pharmaceutical composition disclosed herein can be formulated: (a) for administration selected from the group consisting of oral, pulmonary, rectal, colonic, parenteral, intracistemal, intravaginal, intraperitoneal, ocular, otic, local, buccal, nasal, and topical administration; (b) into a dosage form selected from the group consisting of liquid dispersions, gels, aerosols, ointments, creams, lyophilized formulations, tablets, capsules; (c) into a dosage form selected from the group consisting of immediate release, controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; or (d) any combination of (a), (b), and (c).

[058] In an embodiment, said compositions can be formulated by adding different types of pharmaceutically acceptable excipients for oral administration in solid, liquid, local (powders, ointments or drops), or topical administration, and the like.

[059] In some embodiments of the invention, there is provided a method of treating disease associated with gastrointestinal inflammation and chronic inflammation associated fibrosis in a subject in need thereof, wherein said method comprises administration of a therapeutically effective amount of one or more compounds disclosed herein, to the subject, optionally in combination with one or more therapeutic agents, preferably by administering a therapeutically effective amount of compounds having Formula (I) to (III) or variant thereof, to a human subject in need thereof.

[060] In some embodiments of the invention, there is provided a method of treating, reducing, inhibiting or controlling disease associated with gastrointestinal inflammation and chronic inflammation associated fibrosis in a subject, wherein said method comprises simultaneously, separately or sequentially administering to the subject, (i) one or more therapeutic agents, and, (ii) a therapeutically effective amount of one or more compounds disclosed herein or a pharmaceutical composition disclosed herein, preferably by administering a therapeutically effective amount of compounds having Formula (I) to (III) or variant thereof, or a pharmaceutical composition comprising any of compounds having Formula (I) to (III) or a variant thereof, to a human subject in need thereof. [061 ] In some embodiments of the invention, there is provided a method of treating, reducing, inhibiting or controlling at least one sign or symptom of disease associated with gastrointestinal inflammation and chronic inflammation associated fibrosis in a subject, wherein said method comprises administration of a therapeutically effective amount of one or more compounds disclosed herein or a pharmaceutical composition disclosed herein, to the human subject, optionally in combination with one or more therapeutic agents, wherein said sign or symptom is associated with disease associated with gastrointestinal inflammation. Preferably said method involves administering a therapeutically effective amount of a compound according to the invention described herein, such as compounds having Formula (I) to (III) or variant thereof, or a pharmaceutical composition comprising any of compounds having Formula (I) to (XV) or a variant thereof, to a human subject in need thereof.

[062] In some embodiments of the invention, there is provided a method of treating, reducing, inhibiting or controlling at least one sign or symptom of disease associated with gastrointestinal inflammation and chronic inflammation associated fibrosis in a subject, wherein said method comprises simultaneously, separately or sequentially administering to the subject, (i) one or more therapeutic agents, and, (ii) a therapeutically effective amount of one or more compounds disclosed herein or a pharmaceutical composition disclosed herein, wherein said sign or symptom is associated with disease associated with gastrointestinal inflammation. Preferably said method involves administering a therapeutically effective amount of a compound according to the invention described herein, such as compounds having Formula (I) to (III) or variant thereof, or a pharmaceutical composition of the invention, preferably comprising any of compounds having Formula (I) to (III) or a variant thereof, to a human subject in need thereof.

[063] A therapeutically effective amount, as disclosed herein, may be between about 1 ng and about 5 g, preferably between about 10 ng and 1g.

[064] In some embodiments, there is provided a kit comprising a compound, pharmaceutical composition of the invention. In some embodiments, the kit also comprises one or more therapeutic agents. In these embodiments, each of the compound, pharmaceutical composition, and the one or more therapeutic agents, may be provided in separate vials or compartments. In some embodiments, the kit further comprises instructions for the administration of the compound, pharmaceutical composition, and optionally the one or more therapeutic agents. EXAMPLES

Example 1

Synthesis of Compounds having Formulae (I) to (III)

[065] The peptide chain was elongated on TentaGel R RAM resin (0.19 mmol g ¹ ) with a Rink amide linker on a 0. 1 mmol scale manually with Fmoc protection scheme. The coupling was performed in two steps. In the first step, 3 equivalents of Fmoc -protected amino acid, 3 equivalents of the uronium coupling agent O-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (HATU) and 6 equivalents of N,N- diisopropylethylamine (DIPEA) were used in N,N-dimethylformamide (DMF) as solvent with shaking for 3 h. The second coupling was performed with 1 equivalent of amino acid, 1 equivalent of HATU and 2 equivalents of DIPEA. After the coupling steps, the resin was washed 3 times with DMF, once with MeOH and 3 times with DCM. No truncated sequences were observed under these coupling conditions. Deprotection was performed with 2% DBU and 2% piperidine in DMF in two steps, with reaction times of 5 and 15 min. The resin was washed with the same solvents as described previously. The cleavage was performed with TFA/water/DL-dithiothreitol (DTT)/TIS (90:5:2.5:2.5) at 0 °C for 1 h. The purification was carried out by RP-HPLC, using a Phenomenex Luna C18 100 A 10 pm column (10 mm x 250 mm). The HPLC apparatus was made by JASCO. The solvent system used was as follows: 0.1% TFA in water; 0.1% TFA in 80% acetonitrile in water; a linear gradient was used during 60 min, at a flow rate of 4.0 mL min ¹ , with detection at 206 nm. The purities of the fractions were determined by analytical RP-HPLC-MS using an Agilent 1200HPLC system equipped with a Bruker HCT II ion trap MS with a Phenomenex Luna C18 100 A 5 pm column (4.6 mm x 250 mm) ¹²³ and the pure fractions were pooled and lyophilized. The purified peptides were characterized by MS, Bruker HCT II ion trap mass spectrometer equipped with an electrospray ion source.

[066] Compound I (LAN-22- 100)

MW: 504.541 [067] Compound II (LAN-22-101) MW: 516.55

[071] Compound VI (LAN-22-105)

MW: 530.27

Example 2

PAI-1 activity of Formulae I-III

[072] The PALI inhibitors were tested by an in vitro PALI activity assay (Abeam, ab283368) and human, healthy, citrate treated plasma sample was used. Blood samples were centrifuged for 15 minutes at 2000 g at 4°C to remove cells and platelets and the samples were stored at - 80°C until for use. Before the PALI activity assay, plasma samples were incubated with the PALI inhibitors in different concentrations (1, 10, 100 pM and 1 mM) for 1 hour on room temperature. The experiment was performed by the manufacturer’s protocol. First, the samples were incubated with a known amount of tPA (tissue Plasminogen Activator) which will result PALI and tPA inactive complex. Next, the remaining free tPA cleaved the plasminogen to plasmin, which in turn hydrolyzes the plasmin chromogenic substrate thereby releasing chromophore. The absorbance was measured on OD 405 nm in the end-point mode (BMG Labtech, CLARIOstar). The released chromophore was inversely proportional to the PALI activity in the samples. For the evaluation the PALI standard curve was calculated, and the samples were corrected by the background control.

[073] Based on the results of 4 different PALI inhibitors, Compound IV (LAN-22-103) was the most effective in the reduction of PALI activity (Figure 1 and 2).

Table 1. show the average PALI activity of 3 biological parallel samples after 1 hour incubation of different type and concentration of PAI-1 inhibitor.

Control 1 uM 10 uM 100 uM hnM

LAN-22 100 18.94+0.038 15.57+0.012 14.2+0.038 17.22+0.135 17.44+0.063

LAN-22-101 18.94+0.038 17.87+0.077 19.78+0.062 19.73+0.043 17.2+0.049 I. \N-22- 102 18.94+0.038 18.74+0.027 19.46+0.008 I7. I 4±O.O66 I7.9±O.O27

I.AN-22 103 18.94+0.038 I 5.81+O.()O8 15.58±0.033 16.07+0.052 14.86+0.021

Example 3

Measurement of cell viability

[074] To test the possible cytotoxic effect of Compound IV (LAN-22-103), IxlO ⁴ HeLa cells were plated into the wells of an F-bottom cell culture 96-well white microplate (Greiner Bio- One, Hungary). After 2 days, Compound IV (LAN-22-103) containing (10-100-1000 pM) media was added and cells were incubated for 30 min at 37°C. Non-treated HeLa cells served as control group. Cell culture medium without cells was used as blank during the measurement. Cell culture medium plus dH2O (10%) was applied as vehicle control. We determined the intracellular ATP content, which is proportional to the number of viable cells, using the CellTitre-Glo 3D (Promega) luminometric assay on a CLARIOStar plate reader. For the calculation 4-parameter fit was used on signal curve, in which measured values was blank corrected and converted into nM ATP. To define cell viability, obtained values in case of nontreated control cells were considered as one hundred percent and the treated samples were compared to these values (% = (test value/control value) *100).

[075] Our results show that incubation of HeLa cells with different concentration of Compound IV (LAN-22-103) for 30 min did not cause significant reduction of cell viability (Figure 3)

Example 4

3% DSS induced colitis mice model

[076] To test the effect of Compound IV (LAN-22- 103) on the severity of inflammatory bowel disease, 3% DSS-induced colitis model was performed. At the beginning of the experiment all mice were signed. The 3% DSS (dextran sulfate sodium, MP Biomedicals, 160110) solution was diluted in dfLO. 5 ml of 3% DSS solution or tap water (as control) was used per mouse per day. In the treated groups, DSS was administered for four times, in every second days. Between the DSS-treatment, all mice were got tap water.

[077] Oral administration of Compound IV (LAN-22-103) was began in the same time of the third DSS treatment. Compound IV (LAN-22-103) was diluted in AccuGene H2O for 5 mg/ml stock solution. Animals received a daily dose of the 10 mg/bwkg Compound IV (LAN-22-103) or sterile AccuGene H2O once a day from the day 5 to 8. 200 pl dilution reagent was used as a vehicle. Stainless steel feeding needle (Instech Laboratories, FTSS-22S-25) was applied for the oral administration. Figure 4 shows the treatment protocol.

[078] During the experiment the body weight, DSS caused symptoms and leftover of the solution were monitorized every day. For terminal anesthesia of the mice ketamine/xylazine coktail (12.5 and 125 mg/bwkg) was used and sacrificed through exsanguination through the heart. Blood samples were centrifuged, and serum samples were stored on -80°C until for use. To evaluate the severity of colitis, spleen weight and colon length were defined. The distal part of the colon was separated for three sections. One piece was put into 4% formaldehyde for the histological analysis (hematoxylin-eosin and periodic acid-Schiff (PAS) staining), and the other two parts were taken to RNA Later (Sigma, R0901) for further examination. Fecal samples were also collected from the colon. During the histologic evaluation, inflammatory cell infiltration, epithelial and mucosal architecture changes were scored from 0 to 3 and the summary of these two scores was represented. In case of PAS staining, the severity of goblet cells depletion was evaluated (0: no depletion; 1 : minimal depletion (<20%); 2: mild (21 -35%); 3: moderate (36-50%); 4: severe (>50%).

[079] Representative H&E images of colon and summary bar charts show that DSS caused excessive inflammation, epithelial erosion and goblet cells depletion which was significantly reduced after Compound IV (LAN-22-103) treatment (Figure 5 and 6).

[080] Increased spleen weights generally correlate with the extent of inflammation. Summary bar charts show that administration of DSS significantly increased the spleen weight compared to control, and significant reduction was observed after Compound IV (LAN-22-103) treatment (Figure 6). Similar result was detected in case of colon length. DSS administration caused a shortened colon, and Compound IV (LAN-22-103) treatment significantly reduced this effect of DSS (Figure 7).

Example 5

Gene expression analysis by qRT-PCR

[081] The levels of inflammatory cytokines were elevated during colitis. To test the effect of Compound IV (LAN-22-103) on the mRNA level of inflammatory cytokines, total mRNA was isolated from homogenized tissue with NucleoSpin RNA Plus Kit (Macherey-Nagel, 740984) was used according to the instructions of the manufacturer. mRNA concentrations were measured by Nanodrop One (Thermo Scientific, ND-ONE-W). iScript cDNA Synthesis Kit (Bio-Rad, 1708891) was used for the reverse transcription of mRNA samples. The cDNA concentration was 50 ng/pl at the tissue samples. The primers were designed in the NCBI Primer-BLAST (Table X.). All primers were designed to the exon-exon junction and all PCR products were less than 200 bp. Amplicons were detected by a CFX Connect 96 (Bio-Rad, 1855201SP) using SYBR Green (Bio-Rad, 1725274). Poll2 and Rps23 were used as housekeeping genes for normalization and the Livak Method was applied to the evaluation. The gene expression of control was applied to define the relative gene expression fold changes of treated subjects.

[082] Our results demonstrated that DSS induced significant elevation of mRNA level of main inflammatory cytokines, such as TNF-a, IL- 10 and IL-6 and EPL071 administration was significantly reduced mRNA level of TNF-a and IL-10 (Figure 8). However, IL-6 level did not change after Compound IV (LAN-22-103) treatment.

Example 6

Measurement of tissue cytokine level by using ELISA

[083] To examine the effect of Compound IV (LAN-22-103) on the protein level of inflammatory cytokines, total protein was isolated from the tissue samples. For total protein isolation the colonic tissue samples were minced into small pieces in 0.5 ml RIPA lysis buffer (Millipore, 20-188) containing tube. EASYpack Protease Inhibitor Cocktail was added to the samples and they were homogenized with a Branson Sonifier SFX150 on ice. After that the samples were centrifuged, the supernatant was transferred, snap-freezed and stored at -80°C until use.

[084] The total protein concentrations were obtained by Pierce BCA Protein Assay method (Thermo Scientific, 23225). This experiment was performed by the company’s instruction. The BCA working solution was mixed with the standards or the samples and they were loaded into the 96-well plate (Greiner, 655101). After 30 minutes incubation, the absorbances were measured at 562 nm in the plate reader. For the calculation 4-parameter fit was used and the measured values were blank corrected. The total protein levels were used to normalizing the cytokines protein concentrations. [085] Traditional ELISA was performed to define the concentration of IL-6. In this assay, a 96-well plate with immobilized, specific mouse IL-6 antibody was used. The experiment was applied by the company’s instructions.

[086] The results showed that Compound IV (LAN-22-103) treatment decreases the mucosal concentration of IL-ip and IL-6 (two major cytokines in IBD (Figure 9).

Example 7

Measurement of gene expression of fibrosis-associated proteins

[087] Human colonic fibroblasts were isolated from biopsy samples obtained from an active IBD patient as described previously (PMID: 37227782). The human colonic fibroblasts were treated with 5 ng/mL TGFP for 72 hours to maintain In the Compound IV (LAN-22-103)- treated cell cultures 10 M Compound IV (LAN-22-103) was administrated for 48 hours (the Compound IV (LAN-22-103) was initiated 24 hours after the beginning of the TGFP treatment). Gene expression analyses were performed as previously described (PMID: 31409889). Statistical analysis and strength of significance were performed with GraphPad Prism software. All data are expressed as means ± SEM. Shapiro-Wilk normality test was applied. Both parametric (Unpaired t-test or one-way analysis of variance with Tukey’s multiple comparisons test) and nonparametric (Mann- Whitney test and Kruskal -Wallis test) tests were used based on the normality of data distribution. P value below 0.05 was considered statistically significant.

[088] To study the effect of Compound IV (LAN-22-103) on the profibrotic gene expression pattern, we isolated and cultured human colonic fibroblasts from the colonic biopsy samples of an IBD patient with active disease. To maintain the proinflammatory microenvironment, the fibroblast cultures were treated with 5 ng/mL TGFP for 72 hours. The control cultures were treated with vehicle control. Our results demonstrated that the TGF treatment induced marked increase in the expression of the profibrotic genes (ACTA2: alpha 2 smooth muscle actin; FN 1 : fibronectin 1 ; PALI: plasminogen activator inhibitor 1). In contrast, in the cell cultures, which were treated with 10 pM Compound IV (LAN-22-103) for 48 hours (the Compound IV (LAN- 22-103) was initiated 24 hours after the beginning of the TGFP treatment), the expression of the three investigated genes were significantly decreased (Figure 10). Example 8

3% DSS-induced chronic colitis mice model

[089] 8-12-week-old FVB/N mice weighing 20-25 g were utilized in our experiments. The mice were kept at a constant room temperature of 22-24°C with a 12-hour light-dark cycle and free access to food and drink. The gender ratio was 1:1. VRF1 (P) standard rodent food and standard bedding were used. The treatments were carried out during the light cycle. The 3% DSS (dextran sulfate sodium, MP Biomedicals, 160110) solution was diluted in dH2O. 5 ml of 3% DSS solution was used per mouse per day and the control mice received tap water without DSS in a same amount. The treated mice received 3 cycles of DSS treatment as detained below. One DSS treatment cycle consisted of 8 days, in which mice received DSS every other day (4 times). The DSS cycles were disrupted by H2O cycles in which all animals received tap water for 8 days. Compound IV (LAN-22-103) was diluted in AccuGene H2O (Lonza, BE51223) for 5 mg/ml stock solution. Animals received a daily dose of 10 mg/bwkg Compound IV (LAN- 22-103) or vehicle daily from the day 26 to 35 in 200 pl final volume in oral gavage. Stainless steel feeding needle (Instech Laboratories, FTSS-22S-25) was used for the gavage administration. Animals were sacrificed after the third DSS cycle on the day 36. During the experiment the mice were constantly monitored for the body weight loss, and for signs of DSS caused symptoms. The leftover of the drinking solutions was recorded every day. Intraperitoneal xylazine and ketamine (125 and 12.5 mg/bwkg) was used for terminal anesthesia and the blood sample was taken by intracardiac puncture. The whole blood was transferred to a serum clot activator tube (Greiner, 454098) and incubated for 30 minutes at room temperature. The centrifuge step was performed for 20 minutes at 2000 g at 4°C, and the serum part was moved in a new tube and stored on -80°C until for use. The distal part of the colon was separated into three sections. One piece was put into 4% formaldehyde for the paraffin embedding and the other two portions and remaining colon were taken to RNA Later (Sigma, R0901) separately for further examination. The fecal samples were also collected from the colon. All sample types were stored at -80°C until for use. or histologic evaluation, a part of the distal colon was removed and stored at 4°C in 4% formaldehyde. Paraffin-embedded sections of the colon were stained with hematoxylin-eosin and periodic acid-Schiff staining following standard protocol. Hydroxyproline concentration was determined by an end-point colorimetric method using the Hydroxyproline Assay Kit. [090] To quantify the tissue fibrosis the hydroxyproline concentration was measured. As shown on the bar chart, Compound IV (LAN-22-103) treatment significantly decreased the hydroxyproline concentration of the colonic tissue in chronic DSS-induced colitis (Figure 11). This was also confirmed hy histological analysis, which showed decreased colonic fibrosis, decreased severity of the chronic transmural inflammation, decreased loss of crypt structures and depletion of the goblet cells in the Compound IV (LAN-22-103) treated group.

Example 9

Cerulein-induced chronic pancreatits mice model

[091] 8-12-week-old FVB/N mice weighing 20-25 g were utilized in our experiments. The mice were kept at a constant room temperature of 22-24°C with a 12-hour light-dark cycle and free access to food and drink. The gender ratio was 1:1. VRF1(P) standard rodent food and standard bedding were used. The treatments were carried out during the light cycle. Recurrent acute pancreatitis and chronic pancreatitis were induced by repetitive intraperitoneal (i.p.) cerulein injections (Szabo V et al., ICI Insight. 2023 Jul 10;8( 13):el 67645.). The mice received 5 series of 8 hourly physiological saline (p.s., control group) or 50 pg/bwkg cerulein injections every third day. 10 mg/bwkg Compound IV (LAN-22-103) or vehicle was administered daily in oral gavage on the last 6 consecutive days (after 3 episodes of RAP). 24 hours after the last cerulein injections, the mice were sacrificed. The severity of CP was assessed by determining the pancreas weight/body weight ratio, histological parameters and by biochemical assays. Hydroxyproline concentration was determined by an end-point colorimetric method using the Hydroxyproline Assay Kit. Statistical analysis and strength of significance were performed with GraphPad Prism software. All data are expressed as means ± SEM. Shapiro-Wilk normality test was applied. Both parametric (Unpaired t-test or one-way analysis of variance with Tukey’s multiple comparisons test) and nonparametric (Mann- Whitney test and Kruskal- Wallis test) tests were used based on the normality of data distribution. P value below 0.05 was considered statistically significant.

[092] We observed no significant increase in the pancreas weight/body weight ratio of cerulein+ Compound IV (LAN-22-103)-treated mice compared to the cerulein-treated group (Figure 12), although there was a moderate increase in this parameter upon Compound IV (LAN-22-103) treatment. Notably, the lack of effect could be explained by changes in other parameters as well, such as the impaired weight loss of the Compound IV (LAN-22-103)- treated mice (data not shown). To further quantify the extent of tissue fibrosis and the effect of Compound IV (LAN-22-103) Crossmon’s Trichrome staining was performed. Our results showed that the cerulein treatment remarkably increased the extent of fibrotic tissue within the exocrine pancreas, whereas the extent of fibrosis in the Compound IV (LAN-22-103) treated animals with chronic pancreatitis was not pronounced (Figure 13). Another independent biochemical method to quantify tissue fibrosis is the measurement of the hydroxyproline concentration, which is a major component of fibrillar collagen of all types and it’s concentration increases in the presence of tissue fibrosis. As shown on the bar chart, Compound IV (LAN-22-103) treatment significantly decreased the hydroxyproline concentration of the pancreatic tissue in chronic pancreatitis (Figure 14).