Use of an enolase inhibitor pomhex for the treatment of fibrosis

Abstract

The present invention provides methods and uses for treating or preventing fibrosis and fibrotic diseases, including administering a composition containing an enolase inhibitor such as POMHEX.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 594,746, filed October 31, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Statement regarding federally funded research or development

[0004] This invention was completed with government support under license number HL121262 granted by the National Institutes of Health. The government holds certain rights to this invention. Background Technology

[0005] Fibrosis is a leading cause of morbidity and mortality in progressive fibrotic diseases such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc), and leads to organ dysfunction (Perelas et al., 2020, The Lancet Respiratory Medicine. 8:304-320; Henderson et al., 2020, Nature. 587:555-566; Herzog et al., 2014, 66:1967-1978). Currently, there are no effective treatments for fibrosis, as FDA-approved drugs slow disease progression but do not reverse it (Boleto et al., 2022, Therapeutic Advances in Musculoskeletal Disease. 14:1759720X211066686; King et al., 2014, New England Journal of Medicine). . 370:2083-2092; Richeldi et al., 2014, New England Journal of Medicine. 370:2071-2082; Khanna et al., 2016, The Lancet. 387:2630-2640. While lung transplantation is a viable option, it is only accessible to a small number of patients and is prohibitively expensive (Cheng, 2022, Stem Cell Research & Therapy). . 13:492; Cottin & Brown, 2019, Respiratory Research. 20:13; Laporta Hernandez et al., 2018, Medical Sciences. 6:68).

[0006] A major obstacle in developing effective treatment strategies is the incomplete understanding of disease mechanisms and fibrosis mediators. Further elucidation of these mechanisms may help in developing effective antifibrotic therapies that can halt and reverse the progression of fibrosis, thereby significantly reducing the burden associated with fibrotic disease.

[0007] Therefore, there is a need in the art for effective treatment strategies to treat and prevent fibrotic diseases. This invention addresses this unmet need. Summary of the Invention

[0008] In one aspect, the present invention relates to a composition for use in treating fibrosis in subjects who require it, said composition comprising an effective amount of POMHEX, or a POMHEX derivative, preferably POMHEX.

[0009] In one embodiment, the fibrosis or fibrosis-related disease or condition is selected from the group consisting of: pulmonary fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, familial pulmonary fibrosis, pulmonary arterial hypertension (PAH), radiation-induced pulmonary fibrosis, coal worker's pneumoconiosis, asbestosis, bleomycin lung, sarcoidosis, silicosis, acute lung injury, acute respiratory distress syndrome (ARDS), combined pulmonary fibrosis and emphysema (CPFE), asthma; cardiac fibrosis, vascular fibrosis, endocardial myocardial fibrosis (EMF), atherosclerosis, aortic valve sclerosis (AVS); skin fibrosis and wound healing diseases or conditions, hypertrophic scars, keloid scars, postoperative scars. Surgery), systemic scleroderma, localized scleroderma, morphine scleroderma, eosinophilic fasciitis; cirrhosis, hepatitis, metabolic dysfunction-associated steatohepatitis (MASH), congenital liver fibrosis, alcoholic liver disease, hepatitis C virus (HCV)- or hepatitis B virus (HBV)-induced liver fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis; renal fibrosis, fibrotic nephropathy (fibrotic nephropathy), IgA nephropathy, transplanted nephropathy, diabetic nephropathy, lupus nephritis, glomerulonephritis, focal segmental glomerulosclerosis (FSGS); ocular fibrosis, cystic fibrosis (cystic fibrosis, capsular fibrosis, capsule fibrosis) Fibrosis, conjunctival fibrosis, corneal fibrosis, retinal fibrosis, subretinal fibrosis, dry eye, macular edema, retinopathy, glaucoma, age-related macular degeneration (AMD); fibrosis caused by neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), multiple sclerosis or Alzheimer's disease; fibrosis caused by graft-versus-host disease (GVHD), subepithelial fibrosis, uterine fibrosis, Peyronie's disease, myelofibrosis, retroperitoneal fibrosis, renal systemic fibrosis, multifocal fibrosis, rheumatoid arthritis, tumor-associated fibrosis, radiation-induced fibrosis, chemotherapy-induced fibrosis, systemic sclerosis, and Sjogren's syndrome.

[0010] In one embodiment, the fibrosis or fibrosis-related disease or condition is pulmonary fibrosis, particularly idiopathic pulmonary fibrosis. In another embodiment, the subject with fibrosis has systemic scleroderma. In a further embodiment, the subject with fibrosis has hypertrophic scars or keloids. In yet another further embodiment, the subject with fibrosis has Peroni disease.

[0011] In one embodiment, a composition comprising POMHEX or a POMHEX derivative, preferably POMHEX, is administered together with a second agent or a second therapeutic agent, wherein the second agent or the second therapeutic agent is an antifibrotic agent.

[0012] In one embodiment, the subject is a non-primate mammal, preferably a dog, and most preferably a West Highland White Terrier. In another embodiment, the subject is a human subject.

[0013] In one aspect, the present invention relates to a method for reducing fibrosis, comprising contacting a group of cells or tissues with a composition that effectively reduces the amount of fibrosis in the cells or tissues, the composition comprising POMHEX or a POMHEX derivative, preferably POMHEX.

[0014] In one embodiment, the tissue is lung tissue. In some embodiments, the tissue is skin tissue. In one embodiment, the cells or tissue are maintained (held) in vitro. ex vivo In some embodiments, the cells or tissues are contained within a living subject. in vivo ).

[0015] In one embodiment, the composition comprises POMHEX.

[0016] In one embodiment, the present invention relates to a method of treating fibrosis, comprising administering to a subject suffering from fibrosis a composition comprising POMHEX or a POMHEX derivative, preferably POMHEX, in an amount effective in treating fibrosis in the subject.

[0017] In one implementation, the subject with fibrosis suffers from fibrosis or fibrosis-related diseases or conditions selected from the following groups: pulmonary fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis (IPF), familial pulmonary fibrosis, pulmonary arterial hypertension (PAH), radiation-induced pulmonary fibrosis, coal worker's pneumoconiosis, asbestosis, bleomycin-induced lung disease, sarcoidosis, silicosis, acute lung injury, fibrosing mediastinitis, acute respiratory distress syndrome (ARDS), pulmonary fibrosis with emphysema (CPFE), asthma; cardiac fibrosis, vascular fibrosis, endocardial myocardial fibrosis (EMF), atherosclerosis, aortic valve sclerosis (AVS); skin fibrosis and wound healing diseases or conditions, hypertrophic scars, keloids, postoperative scars, systemic scleroderma, localized scleroderma, morphea, eosinophilic fasciitis, palmar aponeurosis contracture (Dupuytren's Contracture); cirrhosis, hepatitis, metabolic dysfunction-associated steatohepatitis (MASH), congenital liver fibrosis, alcoholic liver disease, hepatitis C virus (HCV)- or hepatitis B virus (HBV)-induced liver fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis; renal fibrosis, fibrotic nephropathy, IgA nephropathy, transplanted nephropathy, diabetic nephropathy, lupus nephritis, glomerulonephritis, focal segmental glomerulosclerosis (FSGS); ocular fibrosis, sacral fibrosis, conjunctival fibrosis, corneal fibrosis, retinal fibrosis, retinal Subepithelial fibrosis, dry eye syndrome, macular edema, retinopathy, glaucoma, age-related macular degeneration (AMD); fibrosis caused by neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), multiple sclerosis, or Alzheimer's disease; fibrosis caused by graft-versus-host disease (GVHD), subepithelial fibrosis, uterine fibrosis, Peroni disease, myelofibrosis, retroperitoneal fibrosis, renal systemic fibrosis, multifocal fibrosis, rheumatoid arthritis, tumor-associated fibrosis, radiation-induced fibrosis, chemotherapy-induced fibrosis, systemic sclerosis, and Sjögren's syndrome.

[0018] In one embodiment, the subject has idiopathic pulmonary fibrosis. In one embodiment, the subject has proliferative scars or keloids. In one embodiment, the subject has systemic scleroderma. In one embodiment, the subject has Peroni disease.

[0019] In one embodiment, the composition comprises POMHEX.

[0020] In one embodiment, the method further includes administering an effective amount of a second therapeutic agent to the subject, wherein the second therapeutic agent is an antifibrotic agent.

[0021] In one embodiment, the subject is a non-primate mammal, preferably a dog, and most preferably a West Highland White Terrier. In another embodiment, the subject is a human subject.

[0022] In one aspect, the present invention relates to a composition comprising a first antifibrotic agent and a second different antifibrotic agent in a combined amount effective in treating fibrosis in a subject, wherein the first antifibrotic agent is POMHEX or a POMHEX derivative, preferably POMHEX.

[0023] In one aspect, the present invention relates to a therapeutic kit comprising a first antifibrotic agent and a second different antifibrotic agent in at least a first suitable container, wherein the first antifibrotic agent is POMHEX or a POMHEX derivative, preferably POMHEX. In one such embodiment, the first antifibrotic agent is contained in the first container, and the second different antifibrotic agent is contained separately in the second different container.

[0024] In one aspect, the present invention relates to the use of an effective amount of POMHEX, or a POMHEX derivative preferably POMHEX, in the preparation of a medicament for treating fibrosis in a subject who requires it. Attached Figure Description

[0025] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. The drawings illustrate presently preferred embodiments for the purpose of illustrating the invention. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0026] Figure 1 Include Figure 1 A to Figure 1 F depicts the results of an example experiment demonstrating that ENOBLOCK does not significantly reduce ECM gene expression in cell lysates of normal lung fibroblasts. Normal human lung fibroblasts ( n=3-4 The protein was pre-incubated with DMSO or ENOBLOCK (1.68 µM) for one hour, and then treated with TGF-β1 (10 ng / ml) for 72 hours to detect the expression of ECM proteins. Figure 1 A depicts the protein levels of COL1α1 detected in cell lysates of fibroblasts treated with ENOBLOCK; Figure 1 B depicts the protein levels of FN detected in cell lysates of fibroblasts treated with ENOBLOCK; Figure 1 C depicts the protein levels of MMP-1 detected in cell lysates of ENOBLOCK-treated fibroblasts; Figure 1D depicts the protein levels of MMP-3 detected in cell lysates of ENOBLOCK-treated fibroblasts; Figure 1 E depicts the protein levels of ENO monomers detected in cell lysates of ENOBLOCK-treated fibroblasts; and Figure 1 F depicts the protein levels of HMO-ENO detected in cell lysates of ENOBLOCK-treated fibroblasts. GAPDH was used as a loading control for the cell lysates. Quantitative analysis (top) and representative blots (bottom) are shown. Statistical analysis was performed using one-way ANOVA. There were no statistically significant differences in the ENOBLOCK-treated groups. Error bars represent mean + / - SEM.

[0027] Figure 2 Include Figure 2 A to Figure 2 D describes the results of an example experiment demonstrating that ENOBLOCK does not significantly reduce ECM gene expression in the conditioned medium of normal lung fibroblasts. Normal human lung fibroblasts ( n=3-4 The protein was pre-incubated with DMSO or ENOBLOCK (1.68 µM) for one hour, and then treated with TGF-β1 (10 ng / ml) for 72 hours to detect the expression of ECM proteins. Figure 2 A depicts the protein levels of COL1α1 in conditioned medium of ENOBLOCK-treated fibroblasts as measured by immunoblotting; Figure 2 B depicts the protein levels of FN in conditioned medium of ENOBLOCK-treated fibroblasts as measured by immunoblotting. Figure 2 C depicts the protein levels of MMP-1 in conditioned medium of ENOBLOCK-treated fibroblasts as measured by immunoblotting; and Figure 2 Figure D depicts the protein levels of MMP-3 in the conditioned medium of ENOBLOCK-treated fibroblasts as measured by Western blotting. Quantitative analysis (top panel) and representative blots are shown (bottom panel). Statistical analysis was performed using one-way ANOVA. No statistically significant differences were found among the ENOBLOCK-treated groups. Error bars represent mean + / - SEM.

[0028] Figure 3 Include Figure 3 A to Figure 3 E depicts the results of an example experiment demonstrating that ENO is required for the expression of YAP-1 and TWIST-1. Control plasmid (C) and ENO-containing plasmid (ENO) were transfected into normal lung fibroblasts (…). N=4 ) for 72 hours. Figure 3A depicts the protein levels of YAP-1 detected in lysates by immunoblotting. Normal human lung fibroblasts ( N=4-6 The protein was treated with rENO protein (4 µg) for 72 hours. Figure 3 B depicts an immunoblot used to measure the protein level of YAP-1 in cell lysates. Figure 3 C describes the use of contrast or ENO Normal lung fibroblasts were transfected with specific siRNA (siENO) (30 nM), stimulated with TGF-β1 (10 ng / ml) 24 hours later, and harvested for protein fractionation 72 hours later. Nuclear polar fractions were extracted, and YAP-1 protein levels were detected by Western blotting. Figure 3 D depicts an immunoblot used to measure the protein level of TWIST-1 (~80 kDa band) in cell lysates. Figure 3 E describes the use of contrast or ENO Normal lung fibroblasts were transfected with specific siRNA (siENO) (30 nM), stimulated with TGF-β1 (10 ng / ml) 24 hours later, and harvested for protein fractionation after 72 hours. Nuclear fractions were extracted, and TWIST-1 protein levels were detected by Western blotting. GAPDH and Tata-binding protein (TBP) or histone H3 (HisH3) were used as lysis buffer and nuclear fraction loading controls, respectively. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. Statistical analysis was performed using Student's t-test and one-way ANOVA (as applicable). p < 0.05, p < 0.01. Error bars are the mean plus / minus SD.

[0029] Figure 4 Include Figure 4 A to Figure 4 C depicts the results of an example experiment demonstrating that ENO and YAP-1 crosstalk modulates the fibrosis phenotype in vitro. Normal lung fibroblasts were used as a control or... ENO Cells were transfected with a specific siRNA (siENO) (30 nM) and stimulated with TGF-β1 (10 ng / ml) 24 hours later. Fibroblasts were harvested 72 hours later, and cytoplasmic fractions were extracted and subjected to Western blotting. Figure 4 A depicts a representative blot showing the protein levels of FN, COL1α1, CTGF (38 kDa isoform), PAI-1, and α-SMA in the cytoplasmic extract. Figure 4 B depicts a representative blot showing the protein levels of COL1α1, FN, and PAI-1 in conditioned medium for fibroblasts. Normal lung fibroblasts were compared with control or... YAP-1Specific siRNA (siYAP) (30 nM) was transfected for 24 hours, followed by stimulation with rENO (4 µg) for 48 hours. Fibroblasts were harvested 72 hours later, and cell lysates were subjected to Western blotting. Figure 4 C depicts a representative blot showing the protein levels of COL1α1 and PAI-1 in cell lysates. GAPDH was used as a loading control for lysates and cytoplasmic fractions. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. Statistical analysis was performed using one-way ANOVA. p < 0.05, p < 0.01. Error bars are the mean plus / minus SD.

[0030] Figure 5 Include Figure 5 A to Figure 5 C depicts the results of an example experiment demonstrating that POMHEX reduces ECM gene expression in normal lung fibroblasts. Normal human lung fibroblasts ( n=4-5 Preheat with DMSO or POMHEX (5µM) for one hour, then treat with TGF-β1 (10 ng / ml) for 48 and 72 hours to detect mRNA and protein levels, respectively. Figure 5 A describes genes associated with fibrosis. COL1A1 , COL1A2 , FN and ACTA2 The mRNA expression level, which is relative to the housekeeping gene B2M The protein levels of ECM proteins were measured using immunoblotting. Figure 5 B depicts a representative blot showing the protein levels of FN, COL1α1, COL1α2, CTGF, and α-SMA in cell lysates. Figure 5 C depicts representative blots showing the protein levels of FN, COL1α1, COL1α2, and MMP-1 in the conditioned medium. GAPDH and Ponceau S staining were used as loading controls for lysates and medium, respectively. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. Statistical analysis was performed using one-way ANOVA. p < 0.05, p < 0.01, p < 0.0001. Error bars are the mean plus / minus SD.

[0031] Figure 6 Include Figure 6 A to Figure 6D depicts the results of an example experiment demonstrating that POMHEX reduces ECM gene expression in lung fibroblasts from SSc patients. SSc lung fibroblasts were treated with DMSO or POMHEX (5-10 µM). n=3-4 mRNA expression and protein levels were measured at 48 and 72 hours, respectively. Figure 6 A describes the relationship between the butler gene and... B2M Measurement COL1A1 , COL1A2 , FN and ACTA2 mRNA expression levels. Figure 6 B depicts the protein levels of COL1α1, COL1α2, FN, α-SMA, and CTGF in cell lysates as measured relative to the housekeeping protein GAPDH. Figure 6 C depicts the protein levels of FN, COL1α2, MMP-1, and MMP-3 in fibroblast conditioned medium as measured relative to Ponceau S. Figure 6 D depicts COL1α1 in fibroblast conditioned medium at 10 µM POMHEX relative to Ponceau S. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. GAPDH and Ponceau S staining were used as loading controls for lysates and medium, respectively. Statistical analysis was performed using Student's t-test. p < 0.05, p < 0.01, p < 0.001. Error bars are the mean plus / minus SD.

[0032] Figure 7 Include Figure 7 A to Figure 7 H, depicting the results of an example experiment, demonstrates that POMHEX reduces YAP1 and HMW-ENO expression in human lung fibroblasts and SSc fibroblasts. Normal and SSc lung fibroblasts were treated with 5 µM POMHEX ( n=3-5 One hour after POMHEX treatment, normal lung fibroblasts were stimulated with TGF-β1 (10 ng / ml) and harvested 72 hours later for the detection of cell lysates and subcellular fractions. Figure 7 A depicts the protein levels of YAP-1 analyzed in cell lysates of normal lung fibroblasts. Figure 7 B depicts the protein levels of YAP-1 analyzed in cell lysates of SSc lung fibroblasts. Figure 7 C depicts the expression of YAP-1 as assessed in the cytoplasmic fractions of normal and SSc lung fibroblasts. Figure 7D depicts the expression of YAP-1 assessed in the nuclear fraction of normal and SSc lung fibroblasts. Normal human lung fibroblasts ( n=3 The cells were treated with TGF-β1 (10 ng / ml) for 72 hours and the nuclear fraction was extracted. Figure 7 E depicts the nuclear extract analyzed by immunoblotting of HMW-ENO protein expression. The extract was derived from a normal lung donor ( NL=5 Patients with SSc ( SScL=5 ) and patients with IPF ( IPF=4 Unstimulated lung fibroblasts were cultured for 72 hours. Figure 7 F depicts cell lysates for immunoblotting and measures protein levels of ENO and HMW-ENO. POMHEX-treated normal lung fibroblasts and POMHEX-treated SSc fibroblasts were harvested for immunoblotting. Figure 7 G depicts the protein levels of ENO and HMW-ENO in normal lung fibroblasts stimulated with TGF-β1 and treated with POMHEX. Figure 7 H depicts the protein levels of ENO and HMW-ENO in POMHEX-treated SSc fibroblasts as assessed by Western blotting. Quantitative analysis (top panel) and representative blots (bottom panel). GAPDH and Ponceau S, HisH3, or TBP were used as loading controls for lysates and nuclear extracts. Statistical analyses were performed using one-way ANOVA and Student's t-tests (as applicable). p < 0.05, p < 0.01, p < 0.001. Error bars are the mean plus / minus SD.

[0033] Figure 8 Include Figure 8 A to Figure 8 C depicts the results of an example experiment demonstrating that POMHEX reduces fibrosis in the lungs of mice. PBS ( n = 6), DMSO and PBS ( n =7), BLM and DMSO ( n =7)(1.2 mU / g), or BLM and POMHEX ( n =10) (10 mg / Kg) was administered intratracheally to male C57BL / 6J mice. Lung tissue was collected 14 days later. Figure 8 A depicts lung sections of mice treated with BLM or BLEO and POMHEX, stained with Masson's tricolor blue. Scale bar = 100 µM. Figure 8 B describes the relationship between the housekeeper gene and... B2m ECM gene measurement Col1a1 , Col1a2 , Fn , Acta2 and Eno The mRNA expression level. BLM = Bleomycin. Figure 8 C describes the total collagen content in mouse lungs measured by hydroxyproline assay. Statistical analysis was performed using one-way ANOVA. p < 0.05, p < 0.01, p < 0.001, p < 0.0001. Error bars are the mean plus / minus SD.

[0034] Figure 9 Include Figure 9 A to Figure 9 Figure C depicts the results of an example experiment demonstrating that ENO regulates TWIST-1 expression in vitro. Normal lung fibroblasts were transfected with either a control or ENO-specific siRNA (siENO) (30 nM) and stimulated with TGF-β1 (10 ng / ml) 24 hours later. Fibroblasts were harvested 72 hours later, and nuclear fractions were extracted and subjected to Western blotting. Figure 9 A depicts a representative blot showing the protein levels of ENO in the nuclear extract. Figure 9 B describes the stimulation of normal lung fibroblasts with rENO protein (4µg) for 72 hours. N=4-6 Immunoblotting was performed to measure the protein level of TWIST-1 (~25 kDa band) in cell lysates. Figure 9 C depicts representative blots showing TWIST-1 protein levels in chromatin fractions of ENO-silenced and TGF-β1-stimulated fibroblasts. GAPDH and HisH3 were used as loading controls for cell lysates and nuclear / chromatin extracts, respectively. Quantitative analysis (top panel) and representative blots (bottom panel). Statistical analysis was performed using Student's t-test and one-way ANOVA (as applicable). p < 0.05, p < 0.01. Error bars are the mean plus or minus SD.

[0035] Figure 10 The results of an example experiment are depicted, demonstrating that CTGF expression is downregulated in ENO-silenced fibroblasts. Normal lung fibroblasts were compared with control or... ENOSpecific siRNA (siENO) (30 nM) was transfected and stimulated with TGF-β1 (10 ng / ml) 24 hours later. Fibroblasts were harvested 72 hours later, and cytoplasmic fractions were extracted and immunoblotted. Representative blots show the protein level of CTGF (35 kDa isoform) in the cytoplasmic extract. GAPDH was used as a loading control for the cytoplasmic extract. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. Statistical analysis was performed using one-way ANOVA. Error bars are for mean + / - SD.

[0036] Figure 11 The results of an example experiment are depicted, demonstrating that YAP-1 was significantly silenced in normal lung fibroblasts. Normal lung fibroblasts were compared with a 30 nM control or... YAP-1 Specific siRNA (siYAP) was transfected for 24 hours, followed by stimulation with rENO (4 µg) for 48 hours. Fibroblasts were harvested after 72 hours, and cell lysates were immunoblotted. Representative blots show the protein levels of YAP-1 in the cell lysates. GAPDH was used as a loading control. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. Statistical analysis was performed using one-way ANOVA. p < 0.05, p < 0.001. Error bars are the mean plus / minus SD.

[0037] Figure 12 The results of an example experiment were described, demonstrating that the expression of the ENO isotype (~45 kDa) was similar in normal and patient-derived fibroblasts at steady state. Untreated lung donors ( NL=5 Patients with SSc ( SScL=5 ) and patients with IPF ( IPF=4 Lung fibroblasts were cultured for 72 hours, and then the cell lysates were immunoblotted, and the protein levels of ENO were measured. Representative blots show the steady-state levels of the ENO isotype (~45 kDa) in the cell lysates. GAPDH was used as a loading control. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. Statistical analysis was performed using one-way ANOVA. Error bars are for mean + / - SD.

[0038] Figure 13 The results of an example experiment were depicted, demonstrating that L-lactate / salt (L-Lactate) levels did not change significantly in ENO-silenced fibroblasts. Normal lung fibroblasts were compared with control or... ENOTransfection with specific siRNA (siENO) (30 nM) was performed 24 hours later, followed by stimulation with TGF-β1 (10 ng / ml), and harvesting of conditioned medium. L-lactate / salt levels were measured in the conditioned medium. Quantitative analysis (top figure) and representative blots (bottom figure) are presented. Statistical analysis was performed using one-way ANOVA. Error bars represent mean + / - SEM.

[0039] Figure 14 Include Figure 14 A to Figure 14 D depicts the results of an example experiment demonstrating that POMHEX effectively alleviates fibrosis in IPF fibroblasts. IPF lung fibroblasts were treated with DMSO or POMHEX (5µM). n=6 ). Figure 14 A describes what happened 48 hours later, relative to the butler's genes. B2M Measured COL 1A1 , COL1A2 , FN, ACTA2 and MMP-1 mRNA expression levels. Figure 14 B depicts the protein levels of FN, COL1α2, and α-SMA in fibroblast lysates analyzed by immunoblotting 72 hours later. Figure 14 C depicted the protein levels of FN and MMP-1 in fibroblast conditioned medium analyzed by immunoblotting after 72 hours. Figure 14 Figure D depicts the protein levels of ENO and HMO-ENO measured in cell lysates. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. GAPDH and Ponceau S S staining were used as loading controls for lysates and culture media, respectively. Statistical analysis was performed using Student's t-test. p < 0.05, p < 0.01, p < 0.001, p < 0.0001. Error bars represent the mean value + / - SEM.

[0040] Figure 15 The results of an example experiment are described, demonstrating that POMHEX reduces YAP-1 expression in IPF fibroblasts. IPF lung fibroblasts were treated with either DMSO or POMHEX (5 µM). n=6 YAP-1 protein levels were assessed by immunoblotting. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. GAPDH was used as a loading control. Statistical analysis was performed using Student's t-test. p < 0.01. Error bars represent the mean + / - SEM.

[0041] Figure 16 Include Figure 16 A to Figure 16 C describes the results of an example experiment demonstrating that HMW-ENO is a form of ENO that is triggered and translocated to the cell nucleus upon TGF-β1 stimulation. First, normal human lung fibroblasts ( n=3 The cells were treated with TGF-β1 (10 ng / ml) for 72 hours and nuclear fractions were extracted. Figure 16 A depicts nuclear fractions stimulated with TGF-β1 after incubation with anti-citrullinated antibody or mouse IgG isotype, and the binding of HMO-ENO was analyzed using Western blotting. Next, normal lung fibroblasts were treated with the citrullinated protease inhibitor AFM32a or GSK199 for 1 hour, followed by stimulation with TGF-β1 for 72 hours. Figure 16 B depicts cell lysates from AFM32a-treated cells analyzed by immunoblotting, and Figure 16 C depicts cell lysates from GSK199-treated cells analyzed by immunoblotting. Quantitative analysis (top panel) and representative blots (bottom panel). GAPDH and TBP were used as loading controls for lysates and nuclear extracts. Statistical analysis was performed using one-way ANOVA and Student's t-test (as applicable). p < 0.05. Error bars represent the mean + / - SEM.

[0042] Figure 17 Include Figure 17 A to Figure 17 C depicts the results of an example experiment demonstrating that delayed administration of POMHEX effectively reduced fibrosis in the lungs of mice. PBS ( n=8 ) or BLM ( n =6 (1.2 mU / g) was administered intratracheally to male C57BL / 6J mice. On day 8, mice treated with BLM ( n=7 POMHEX (10 mg / Kg) was administered to the PBS group. DMSO was administered to the PBS group. Lung tissue was collected 21 days later. Figure 17 A depiction of lung sections from mice treated with BLM or BLEO and POMHEX in Masson's tricolor blue. Scale bar = 100 µM. Figure 17 B describes the results of quantifying the total collagen content in mouse lungs using the hydroxyproline assay. Figure 17 C describes the relationship between the housekeeper gene and... B2m ECM gene measurement Col1a1 , Col1a2 , Fn and Eno mRNA expression levels. BLM = bleomycin. Statistical analysis was performed using one-way ANOVA. p < 0.05, p < 0.01, p < 0.001, p < 0.0001. Error bars represent the mean + / - SEM.

[0043] Figure 18 Include Figure 18 A to Figure 18 D depicts the comparative results of the example experiment, demonstrating that POMHEX significantly reduced ECM gene expression in normal lung fibroblasts compared to ENOBLOCK. Normal human lung fibroblasts (…) were treated with DMSO, POMHEX (5 µM), or ENOBLOCK (1.68 µM). n=3-4 The cells were preheated for 1 hour and then treated with TGF-β1 (10 ng / ml) for 72 hours to detect the expression of ECM proteins. Figure 18 A depicts the protein levels of FN, COL1α1, and HMO-ENO detected in cell lysates of POMHEX-treated fibroblasts; and Figure 18 B depicts the protein levels of FN, COL1α1, and HMO-ENO detected in cell lysates of ENOBLOCK-treated fibroblasts. Figure 18 C depicts the protein levels of FN, COL1α1, and MMP-1 measured by immunoblotting in conditioned medium of POMHEX-treated fibroblasts; and Figure 18 Figure D depicts the protein levels of FN, COL1α1, and MMP-1 measured by Western blotting in conditioned medium of ENOBLOCK-treated fibroblasts. GAPDH and Ponceau S were used as loading controls for cell lysates and conditioned medium (POMHEX-treated fibroblasts), respectively. Quantitative analysis (top panel) and representative blots (bottom panel) are shown. Statistical analysis was performed using one-way ANOVA. p < 0.05, p < 0.01, p < 0.0001. Error bars represent the mean + / - SEM. Detailed Implementation

[0044] In one aspect, the present invention relates to methods and uses for treating, inhibiting, preventing, or reducing fibrosis. In one embodiment, the methods and uses include administering a composition comprising an enolase inhibitor, preferably an inhibitor of high molecular weight enolase (HMW-ENO), in the form of an enolase of about 70 kDa, which translocates to the cell nucleus during fibrosis. In a preferred embodiment, the enolase inhibitor is POMHEX or a POMHEX derivative, preferably POMHEX.

[0045] definition

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods, uses, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, uses, and materials are described.

[0047] As used herein, each of the following terms has the meaning relating to it in this section.

[0048] The articles “a” and “an” used in this article refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” refers to one or more elements.

[0049] The use of “about” when referring to measurable values ​​such as quantity, duration of time, etc., in this document is intended to cover variations of ±20%, ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0.1% from the specified value, as such variations are appropriate for performing the disclosed methods and uses.

[0050] When used in the context of organisms, tissues, cells, or components thereof, the term "abnormal" refers to those organisms, tissues, cells, or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those that exhibit "normal" (expected) corresponding characteristics. A characteristic that is normal or expected for one cell or tissue type may be abnormal for different cell or tissue types.

[0051] As used in this article, “relief” of a disease or condition such as fibrosis means a reduction in the frequency or severity of at least one sign or symptom of the disease or condition. A disease or condition is “relieved” if the severity of the sign or symptom of the disease or condition, the frequency with which the patient experiences the sign or symptom, or both are reduced.

[0052] As used in this article, “self” refers to biological material derived from the same individual that will later be reintroduced into that individual.

[0053] As used in this article, "allogeneic" refers to biological material derived from an individual of the same species as the individual into which the biological material was introduced, but which is genetically different.

[0054] The terms "cell" and "cell population" are used interchangeably and refer to multiple cells, i.e., more than one cell. The population can be a pure population containing only one cell type. Alternatively, the population can contain more than one cell type. In this invention, there is no limitation on the number of cell types that can be included in a cell population.

[0055] "Disease" is a state of health in an animal in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if the disease is not treated.

[0056] In contrast, an "illness" in animals is a state of health in which the animal is able to maintain homeostasis, but its health is not as good as it would be without the illness. Without treatment, an illness does not necessarily further deteriorate the animal's health.

[0057] The “effective amount” or “therapeutic effective amount” of a compound is an amount of the compound sufficient to provide a beneficial effect to a subject given the compound. The “effective amount” of a delivery medium is an amount sufficient to effectively bind or deliver the compound.

[0058] As used in this article, “endogenous” means any material that originates from or is generated within an organism, cell, tissue, or system.

[0059] As used herein, the term “exogenous” means any material introduced or produced from outside an organism, cell, tissue, or system.

[0060] As used in this article, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0061] As used herein, the term "inhibition" means the inhibition or blockage of at least about 10 percent of activity or function relative to a control. Preferably, the activity is inhibited or blocked by 50 percent, more preferably 75 percent, and even more preferably 95 percent compared to a control.

[0062] "Separated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "separated," but the same nucleic acid or peptide partially or completely isolated from its native coexisting material is "separated." Separated nucleic acids or proteins can exist in a substantially purified form or in non-native environments such as, for example, host cells.

[0063] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, suitable for the methods and uses described herein. In some non-limiting embodiments, the patient, subject, or individual is a human, dog, or cat, preferably a human.

[0064] "Parenteral" administration of the composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intraperitoneal (ip), or intrasternal injection, or infusion techniques.

[0065] A tissue-specific promoter is a nucleotide sequence that, when operatively linked to a gene-encoding gene or a polynucleotide specified by the gene, essentially only induces the production of the gene product in a cell if the cell is a cell of the tissue type corresponding to that promoter.

[0066] "Therapeutic" treatment is the treatment of subjects who exhibit pathological signs, with the aim of reducing or eliminating those signs.

[0067] As used in this article, “treating a disease or condition” means reducing the frequency with which a patient experiences symptoms of a disease or condition. The terms “disease” and “condition” are used interchangeably in this article.

[0068] As used in this article, “treatment” means reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a patient.

[0069] Scope: Throughout this disclosure, various aspects of the invention may be expressed in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as a strict limitation of the scope of the invention. Therefore, a description of a scope should be considered as specifically disclosing all possible sub-scopes within that scope and the individual numerical values. For example, a description of a scope such as 1 to 6 should be considered as specifically disclosing sub-scopes within that scope, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of how broad the scope may be.

[0070] "Isomers" or "stereoisomers" are compounds that have the same chemical composition but whose atoms or groups are arranged differently in space.

[0071] The term "prodrug" refers to a compound whose structure differs from that of a reference molecule, but which is chemically modified through specific cellular processes to ultimately retain the basic properties of the reference molecule or become the reference molecule.

[0072] As used herein, the terms “effective amount,” “pharmaceutical effective amount,” and “therapeutic effective amount” refer to an amount of a pharmaceutical agent sufficient to provide a desired biological or physiological outcome. This outcome may be the reduction and / or alleviation of a disease or its signs, symptoms, or cause, or any other desired alteration of a biological system. In any individual case, a person skilled in the art can determine the appropriate effective amount using routine laboratory methods.

[0073] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of a compound and is relatively non-toxic, meaning that the material can be given to an individual without causing adverse biological effects or interacting with any component of a composition containing it in a harmful manner.

[0074] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a given compound prepared from a pharmaceutically acceptable non-toxic acid (including inorganic acids, organic acids, their solvates, hydrates, or inclusion complexes). Examples of such inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, hexafluorophosphate, citric acid, gluconic acid, benzoic acid, propionic acid, butyric acid, sulfosalicylic acid, maleic acid, lauric acid, malic acid, fumaric acid, succinic acid, tartaric acid, stilbene sulfonic acid, pamoic acid, p-toluenesulfonic acid, and mesylic acid. Suitable organic acids can be selected from, for example, aliphatic, aromatic, carboxylic, and sulfonic acids, including formic acid, acetic acid, propionic acid, succinic acid, camphor sulfonic acid, citric acid, fumaric acid, gluconic acid, ethanesulfonic acid, lactic acid, malic acid, mucilage, tartaric acid, p-toluenesulfonic acid, glycolic acid, glucuronic acid, maleic acid, furfural acid, glutamic acid, benzoic acid, anthranilic acid, salicylic acid, phenylacetic acid, mandelic acid, pamoic acid, methanesulfonic acid, ethanesulfonic acid, pantothenic acid, benzenesulfonic acid (benzenesulfonate), stearic acid, sulfanilic acid, alginic acid, galacturonic acid, etc. Furthermore, pharmaceutically acceptable salts include, without limitation, alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium-dependent or potassium-dependent), and ammonium salts.

[0075] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as liquid or solid fillers, stabilizers, dispersants, suspending agents, diluents, excipients, thickeners, solvents, or encapsulating materials, relating to carrying or transporting compounds useful in this invention into or to a patient so that they can perform their intended function. Typically, such structures are carried or transported from one organ or body site to another. Each carrier must be "acceptable" in the sense of compatibility with other ingredients in the formulation, including compounds useful in this invention, and must not cause harm to the patient. Examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth gum; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic and compatible substances used in pharmaceutical formulations. As used herein, "pharmaceuticalally acceptable carrier" also includes any and all coatings, antimicrobial and antifungal agents, and absorption delayers, which are compatible with the activity of the compounds useful in the present invention and are physiologically acceptable to patients. Supplemental active compounds may also be incorporated into the composition. "Pharmaceuticalally acceptable carrier" may further include pharmaceutically acceptable salts of compounds useful in the present invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of this invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0076] As used herein, the term "potency" refers to the dose (ED) required to produce half of the maximum response. 50 ).

[0077] As used in this article, the term "efficacy" refers to the maximum effect achieved in the test (Ei). max ).

[0078] "Measuring" or "measurement," or alternatively "detecting" or "detection," means assessing the presence, absence, quantity, or amount (which may be an effective amount) of a given substance in a sample, including deriving a qualitative or quantitative concentration level of such a substance, or otherwise assessing the value or classification of said substance or sample.

[0079] As used in this article, “related” means occurring concurrently with the development or manifestation of a disease, condition, or phenotype. Relatedness may be attributed to, but is not limited to: genes responsible for housekeeping functions; genes that are part of pathways involved in a particular disease, condition, or phenotype; and genes that indirectly contribute to the manifestation of a disease, condition, or phenotype.

[0080] describe

[0081] This invention is partly based on the discovery that inhibiting enolase reduces fibrosis. Therefore, in some embodiments, this invention is intended to treat fibrotic diseases.

[0082] In some embodiments, the present invention provides methods and uses for treating or preventing fibrosis or fibrosis-related diseases or conditions. In one embodiment, the methods and uses include administering a composition comprising an enolase inhibitor to a subject suffering from fibrosis or fibrosis-related diseases or conditions. Preferably, the enolase inhibitor is an inhibitor of high molecular weight enolase (HMW-ENO), in the form of an enolase of about 70 kDa, which translocates to the cell nucleus during fibrosis.

[0083] Enolase inhibitors include, but are not limited to, SF2312 and its derivatives (including Pom-SF2312), PhAH and its derivatives, 3-phospho-D-glyceric acid, POMSF, and HEX and its derivatives such as POMHEX. Further enolase inhibitors include monoamidated phosphates / salts or phosphonates / salts and prodrugs as described in U.S. Patent Application Publication No. 2023 / 0167142 A1, which is incorporated herein by reference. In a preferred embodiment, the enolase inhibitor is POMHEX, a derivative of POMHEX, or a pharmaceutically acceptable salt and hydrate thereof. POMHEX is a pivaloyloxymethyl (POM) prodrug of HEX. In one embodiment, the POMHEX derivative is a compound structurally derived from the POMHEX compound through one or more steps and maintains the ability of POMHEX to inhibit enolase, particularly the ability to inhibit higher molecular weight forms of enolase (HMW-ENO) in the cell nucleus, using an assay described herein, for example, in Example 2. In one embodiment, the POMHEX derivative comprises HEX, a prodrug of HEX, or a derivative of HEX. POMHEX and exemplary derivatives are preferably described in U.S. Patent No. 10,363,261 and also in U.S. Patent Application Publication No. 2022 / 0089620, each of which is incorporated herein by reference.

[0084] In one embodiment, the enolase inhibitor is a compound of the following formula:

[0085] Or pharmaceutically acceptable salts, wherein: R1 is hydrogen or acyl group. （C 12） Or substituted acyl group （C 12） ; R2 is a hydrogen or acyl group. （C 12） or substituted acyloxy group （C 12） ; X1 and X2 are each independently O, S, or NR. a ,in: R a Hydrogen, alkyl （C 6）, or substituted alkyl （C 6） ; R3 and R4 are each independently hydrogen or alkyl. （C 12） aryl （C 12） Aryl alkyl （C 12） , heteroaryl （C 12） heteroaryl （C 12） Or a substituted version of these groups; or a phosphate protecting group; or R3 and R4 taken together and being alkanediyl. （C 8） Or substituted alkyl diols （C 8） ; or —X3—R5; where: X3 is a covalent bond and an alkyl group. （C 8） , or substituted alkyl diols （C 8） ;and R5 is an acyl group. （C 18） alkoxy （C 18） —C(O)-alkoxy （C 18） acyloxy （C 18） or a substituted version of any of these groups; A1 is an alkyl diene. （C1-3） ;and Y1 represents hydrogen, amino, halogen, hydroxyl, phosphate ester (phosphate), alkyl （C 12） , or substituted alkyl （C 12） .

[0086] Regarding the above compounds, in various independent embodiments, R1 is hydrogen; R2 is an acyloxy group. （C 8） or substituted acyloxy group （C 8） R2 is hydrogen; and R3 is a phosphate ester protecting group, including R3 being a phosphate ester protecting group of the following formula: -alkyldiyl （C 6） -acyloxy （C 12） Or substituted alkyl diol （C 6） -acyloxy （C 12） And R3 is pivaloyloxymethyl (neovaleyloxymethyl).

[0087] In some embodiments of the above compounds, X1 and X2 are each O.

[0088] Regarding the above compounds, in various embodiments, R4 is a phosphate ester protecting group, including wherein R4 is a phosphate ester protecting group of the following formula: -alkyldiyl （C 6） -acyloxy （C 12） Or substituted alkyl diol （C 6） -acyloxy （C 12） And R4 is pivaloyloxymethyl.

[0089] Regarding the above compounds, in various independent embodiments, A1 is -CH2-, -CH2CH2-, or -CH2CH2CH2-; and Y1 is hydrogen.

[0090] In one embodiment, the enolase inhibitor is a compound of the following formula:

[0091] in: R2 is a hydrogen or acyl group. （C 12） or substituted acyloxy group （C 12） ; R3 and R4 are each independently hydrogen and alkyl groups. （C 12） Substituted alkyl groups （C 12） or phosphate ester protecting groups; and A1 is an alkyl diene. （C1-3） ;or

[0092] in: R1 is an acyl group. （C 12） Or substituted acyl group （C 12） ; R2 is an acyloxy group. （C 12） or substituted acyloxy group （C 12） ; R3 is an alkyl group. （C 12） Substituted alkyl groups （C 12） or phosphate ester protecting groups; R4 represents hydrogen or alkyl groups. （C 12） Substituted alkyl groups （C 12） or phosphate ester protecting groups; and A1 is an alkyl diene. （C1-3） ; Or any pharmaceutically acceptable salt.

[0093] In one embodiment, the enolase inhibitor is a compound of the following formula:

[0094] in: R2 is hydrogen; R3 and R4 are each independently hydrogen and alkyl groups. （C 12） Substituted alkyl groups （C 12） or phosphate ester protecting groups; and A1 is an alkyl diene.（C1-3） ; Or its pharmaceutically acceptable salt.

[0095] In one embodiment, the enolase inhibitor is a compound of the following formula: or

[0096] Or any of those pharmaceutically acceptable salts.

[0097] In one embodiment, the enolase inhibitor is a compound of the following formula: or

[0098] Or any of those pharmaceutically acceptable salts.

[0099] In one embodiment, the enolase inhibitor is a compound of the following formula: or

[0100] Or any of those pharmaceutically acceptable salts.

[0101] In some preferred embodiments, the enolase inhibitor is POMHEX of the following formula:

[0102] In one implementation, fibrosis or fibrosis-related diseases or conditions include, but are not limited to, pulmonary fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, familial pulmonary fibrosis, pulmonary arterial hypertension (PAH), radiation-induced pulmonary fibrosis, coal worker's pneumoconiosis, asbestosis, bleomycin-induced lung disease, sarcoidosis, silicosis, acute lung injury, fibrotic mediastinitis, ARDS, pulmonary fibrosis with emphysema, asthma; cardiac fibrosis, vascular fibrosis, endocardial myocardial fibrosis, atherosclerosis, and other related conditions. Arterial valve sclerosis; skin fibrosis and wound healing disorders or conditions, hypertrophic scars, keloids (scar tumors), postoperative scars, systemic scleroderma, localized scleroderma (including but not limited to morphea), eosinophilic fasciitis; liver (hepatic) cirrhosis, hepatitis, metabolic dysfunction-related steatohepatitis, congenital liver fibrosis, alcoholic liver disease, HCV- or HBV-induced liver fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis; kidney (renal) fibrosis, fibrotic nephropathy, IgA nephropathy Diseases including: transplant nephropathy, diabetic nephropathy, lupus nephritis, glomerulonephritis, focal segmental glomerulosclerosis (FSGS); ocular fibrosis, lenticule fibrosis, conjunctival fibrosis, corneal fibrosis, retinal fibrosis, subretinal fibrosis, dry eye syndrome, macular edema, retinopathy, glaucoma, age-related macular degeneration (AMD); fibrosis caused by neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), multiple sclerosis, or Alzheimer's disease; and graft-versus-host disease (GVHD). Fibrosis caused by VHD, subepithelial fibrosis, uterine fibrosis, Peroni disease, myelofibrosis (a type of blood cancer), retroperitoneal fibrosis (or Ormond's disease, a fibrotic disease of the retroperitoneal space, a body compartment containing the kidneys, aorta, renal canals and other structures), renal systemic fibrosis, multifocal fibrosis, rheumatoid arthritis, tumor-associated fibrosis, radiation-induced fibrosis, chemotherapy-induced fibrosis, systemic sclerosis, and Sjögren's syndrome.

[0103] In one embodiment, interstitial lung disease includes, but is not limited to, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, connective tissue disease-associated interstitial lung disease, coal worker's pneumoconiosis, asbestosis, acute lung injury, fibrotic mediastinitis, and ARDS. Pulmonary fibrosis also includes pulmonary hypertension. Pulmonary hypertension (PAH) is a specific subgroup of pulmonary hypertension (PH), characterized by elevated pressure in the pulmonary arteries and can be caused by a variety of diseases. Pulmonary hypertension is a pulmonary vascular disease involving fibrosis, meaning that these blood vessels have changed, leading to elevated pressure. Pulmonary fibrosis further includes pulmonary fibrosis with emphysema, a poorly understood fibrotic syndrome characterized by a chronic, progressive disease with a poor prognosis. Subepithelial fibrosis promotes airway remodeling and is also a key feature of asthma, and therefore can be treated with this invention. Indeed, pulmonary fibrosis is a common phenomenon in the pathogenesis of fatal and long-term asthma and is associated with disease severity and treatment resistance.

[0104] In one embodiment, skin fibrosis and wound healing diseases and conditions include, but are not limited to, hypertrophic scars, keloids (also known as “keloids”), postoperative scars, systemic scleroderma, and localized scleroderma, including but not limited to morphea, as described in more detail below. Skin fibrosis also includes eosinophilic fasciitis, which is part of the spectrum of localized scleroderma or morphea involving the subcutaneous tissue and fascia of the skin and is characterized by fascial thickening with eosinophilic tissue infiltration and peripheral eosinophilia. Eosinophils release TGF-β, which activates fibroblasts, leading to increased expression of type I collagen, fibronectin, and tissue inhibitor of metalloproteinase-1 (TMIP-1), thereby causing fibrosis. Skin fibrosis further includes palmar aponeurosis contracture, a fibrotic condition of the palmar fascia (the tissue beneath the skin of the palm and fingers).

[0105] In one embodiment, fibrosis or fibrosis-related diseases or conditions include, but are not limited to, cardiac fibrosis. In one embodiment, cardiac fibrosis is caused by cardiac injury. For example, in one embodiment, cardiac fibrosis is caused by injury, including but not limited to myocardial infarction, aortic stenosis, restrictive cardiomyopathy, systemic and pulmonary hypertension, or carcinoid heart disease. Cardiac fibrosis also includes vascular fibrosis, a broad pathological condition that occurs during vascular remodeling in cardiovascular dysfunction. Vascular fibrosis is characterized by endothelial matrix deposition and thickening of the vessel wall, and the RAAS and TGF-β / Smad signaling pathways have been reported to be involved. Cardiac fibrosis further includes endocardial myocardial fibrosis, a restrictive cardiomyopathy characterized by endocardial fibrosis of the right ventricle, left ventricle, or both at the apex and inflow tract, which can lead to high morbidity and mortality in certain patient populations. Cardiac fibrosis promotes other cardiovascular diseases, particularly atherosclerosis and aortic valve sclerosis, which are driven by persistently active resident fibroblasts, myofibroblasts, and smooth muscle cells, resulting in excessive extracellular matrix secretion and tissue thickening leading to serious pathological consequences.

[0106] In one implementation, fibrosis (“pathogenic fibrosis”) includes the formation or development of excessive fibrous connective tissue in an organ or tissue as an excessive repair or reaction process, as opposed to the formation of fibrous tissue (“healthy fibrosis”) as a normal component of an organ or tissue. All tissues and organs are susceptible to pathogenic fibrosis, with the liver, kidneys, and eyes being particularly prone, and the skin and lungs being especially susceptible.

[0107] In some cases, fibrotic diseases are characterized by fibroblast activation, increased production of collagen and fibronectin, and transdifferentiation into contractile myofibroblasts. This process typically lasts from months to years and can lead to organ dysfunction or death. Fibrosis-related diseases and conditions represent one of the largest groups of conditions for which there are no effective treatments, and therefore represent a significant unmet medical need. For patients with fibrosis, organ transplantation is often the only remedy; due to insufficient organ supply to meet demand, patients often die while waiting for a suitable organ. Pulmonary fibrosis itself can be a leading cause of death in scleroderma, lung diseases, idiopathic pulmonary fibrosis, radiation and chemotherapy-induced pulmonary fibrosis, and conditions caused by occupational exposure to particulate matter.

[0108] This invention can be practiced in any subject diagnosed with or at risk of developing fibrosis. Fibrosis is associated with many diseases and conditions. Subjects may be diagnosed with or at risk of developing interstitial lung disease, including idiopathic pulmonary fibrosis, scleroderma, radiation-induced pulmonary fibrosis, bleomycin-induced lung, sarcoidosis, silicosis, familial pulmonary fibrosis, autoimmune diseases, or any condition involving one or more fibrotic matrix molecular deposits, pathologically enhanced collagen accumulation, apoptosis, and rupture of alveolar septa with honeycomb-like structures. Subjects may be identified as having fibrosis or at risk of developing fibrosis due to exposure to asbestos, finely ground stone, silica, and metallic dust; due to administration of medications such as bleomycin, busulfan, phenytoin, and nitrofurantoin (which are risk factors for fibrosis); or due to radiation exposure (such as in head and neck cancer patients with salivary gland fibrosis). It is also envisioned that the compositions, methods, and uses of this invention can be used to treat organ fibrosis secondary to allogeneic organ transplantation, such as graft fibrosis. Non-limiting examples include fibrosis following kidney transplantation, fibrosis following heart transplantation, and fibrosis following liver transplantation.

[0109] In some embodiments, the methods and uses of the present invention are used to treat a variety of fibrosis or fibrosis-related diseases or conditions, the potential causes of which include myocardial infarction, sclerosis, hepatitis, etc. In other embodiments, the methods and uses of the present invention are used to treat a named disease or condition affecting multiple tissues. For example, renal systemic fibrosis is a syndrome involving fibrosis of the skin, joints, eyes, and internal organs. As another example, multifocal fibrosis is characterized by fibrotic lesions occurring simultaneously in multiple sites and includes mediastinal fibrosis and Riedle's thyroiditis.

[0110] In one implementation, liver (cirrhosis) diseases and conditions include, but are not limited to, hepatitis, including autoimmune hepatitis and metabolic dysfunction-associated steatohepatitis (MASH). Metabolic dysfunction-associated steatohepatitis (MASH), formerly known as nonalcoholic steatohepatitis (NASH), is a severe form of metabolic dysfunction-associated fatty liver disease (MASLD) (formerly known as nonalcoholic fatty liver disease (NAFLD)), where MASH indicates the presence of further liver inflammation and fibrosis, including advanced fibrosis, leading to an increased risk of cirrhosis and liver cancer. Liver fibrosis further includes congenital liver fibrosis and alcoholic liver disease. Excessive alcohol consumption leads to a wide range of liver diseases, including liver fibrosis, cirrhosis, and liver cancer. Furthermore, alcohol consumption accelerates liver fibrosis in patients with other types of liver disease, such as viral hepatitis and nonalcoholic fatty liver disease. Almost all clinical complications of alcoholic liver disease occur in patients with a confirmed diagnosis of fibrosis and cirrhosis, making fibrosis a key parameter for patient treatment and prognosis.

[0111] In another embodiment, liver (cirrhosis) diseases and conditions include, but are not limited to, HCV- or HBV-induced liver fibrosis. Removal or elimination of the causative agent, particularly control or cure of viral infection, has demonstrated that some types of liver fibrosis are reversible. However, reversal often occurs too slowly or too infrequently to avoid life-threatening complications, especially in advanced fibrosis. Therefore, this invention provides an important antifibrotic therapy for treating and / or preventing the progression of liver disease and the development of HCC in subjects infected with HCV- or HBV.

[0112] In further embodiments, liver (cirrhosis) diseases and conditions include, but are not limited to, primary sclerosing cholangitis and primary biliary cirrhosis. Primary sclerosing cholangitis (PSC) is a chronic, progressive cholestatic liver disease. PSC is characterized by inflammation, fibrosis, and narrowing of the intrahepatic or extrahepatic bile ducts, and is typically a progressive condition that leads to complications of cholestasis and liver failure. Primary biliary cholangitis (formerly known as primary biliary cirrhosis, PBC) is an autoimmune liver disease characterized by progressive destruction of the intrahepatic bile ducts, leading to cholestasis and liver fibrosis, which progresses to cirrhosis and liver failure.

[0113] This invention can be practiced in any subject diagnosed with or at risk of developing fibrotic skin disease, scleroderma. Scleroderma is a chronic autoimmune disease characterized by fibrosis (or hardening), vascular changes, and autoantibodies. There are two main types: localized systemic scleroderma and diffuse systemic scleroderma. Localized systemic scleroderma affects the hands, arms, and face. Patients with this form of scleroderma frequently experience one or more of the following complications: calcification, Raynaud's phenomenon, esophageal dysfunction, finger sclerosis, visceral organ fibrosis, and telangiectasia.

[0114] In some embodiments, the methods and uses of the present invention are used to treat subjects. "Subjects" include, but are not limited to, humans and other primates, mammals and non-primates (including commercially relevant mammals and pets such as non-human primates, cattle, pigs, horses, sheep, cats and dogs), and research animals (such as rodents, rats and mice). Subjects may also be referred to as cattle, pigs, horses, sheep, cats, dogs and mice. In some embodiments, the subject to be treated is a domestic cat, known to be susceptible to idiopathic pulmonary fibrosis and particularly susceptible to renal fibrosis, leading to feline chronic kidney disease (CKD). In some embodiments, the subject to be treated is a West Highland White Terrier (or "West Highland White Terrier"), known to be particularly susceptible to pulmonary fibrosis and idiopathic pulmonary fibrosis, to the extent that the disease is also referred to as "West Highland White Terrier lung disease or WLD". In some preferred embodiments, the subject to be treated is a human subject.

[0115] Diffuse systemic scleroderma progresses rapidly and affects large areas of skin and one or more internal organs, often the kidneys, esophagus, heart, and / or lungs. Localized scleroderma, such as linear scleroderma and morphea, affects the skin but not internal organs.

[0116] Skin manifestations of scleroderma can be painful, may impair the use of the affected area (e.g., use of hands, fingers, toes, feet, etc.), and may cause disfigurement. Skin ulcers may occur, and such ulcers may be prone to infection or even gangrene. Ulcerative skin may be difficult to heal or heal slowly. Difficulty in healing skin ulcers may be particularly exacerbated in patients with impaired circulation (such as those with Raynaud's phenomenon). Lung involvement is a leading cause of death in patients with scleroderma, exhibiting high morbidity and mortality. In some embodiments, the compositions, methods, and uses of this disclosure are used to treat scleroderma, such as skin symptoms of scleroderma. In some embodiments, treating scleroderma includes treating skin ulcers, such as finger ulcers. The methods and uses of the present invention can be used to alleviate fibrotic and / or inflammatory symptoms of scleroderma in affected tissues and / or organs.

[0117] In addition to skin symptoms / manifestations, scleroderma can also affect the heart, kidneys, lungs, joints, and digestive tract. In some implementations, treating scleroderma involves treating disease symptoms in any or more of these tissues, such as by reducing fibrosis and / or inflammatory symptoms.

[0118] Lung problems are among the most serious complications of scleroderma and a leading cause of disease-related mortality. The two main lung conditions associated with scleroderma are pulmonary fibrosis and pulmonary hypertension. Patients with lung involvement may have one or both of these conditions. Pulmonary fibrosis associated with scleroderma is an example of pulmonary fibrosis that can be treated using the methods and uses of this invention.

[0119] Scleroderma involving the lungs can lead to scarring (pulmonary fibrosis). Approximately 70% to >90% of scleroderma patients develop this type of pulmonary fibrosis, although its progression is usually slow and symptoms vary in severity from patient to patient. For patients who do experience symptoms associated with pulmonary fibrosis, these symptoms include dry cough, shortness of breath, and decreased exercise capacity. About 16% of patients with some level of pulmonary fibrosis will develop severe pulmonary fibrosis. Patients with severe pulmonary fibrosis will experience a significant decline in lung function and alveolitis.

[0120] In some embodiments, the methods and uses of the present invention include the use of enolase inhibitors for the treatment of scleroderma (e.g., pulmonary fibrosis associated with scleroderma). The methods and uses of the present invention can be used to alleviate fibrotic symptoms of scleroderma in the lungs. For example, the methods and uses can be used to improve lung function and / or reduce the risk of death from scleroderma.

[0121] Kidney involvement is also common in patients with scleroderma. Kidney fibrosis associated with scleroderma is an example of kidney fibrosis that can be treated using the methods and uses of this invention.

[0122] Since renal fibrosis driven by abnormal accumulation of extracellular matrix is ​​the ultimate common pathway for almost all types of chronic repetitive damage in the kidneys and is therefore a hallmark of chronic kidney disease, the treatment of renal fibrosis using this invention also provides a range of treatments for fibrotic kidney diseases, including IgA nephropathy, transplanted nephropathy, diabetic nephropathy, and lupus nephritis. Other renal fibrotic diseases that can be treated with this invention include glomerulonephritis, a condition characterized by irreversible, progressive glomerular and tubulointerstitial fibrosis that can lead to chronic kidney disease and end-stage renal disease; and focal segmental glomerulosclerosis (FSGS), which presents as a morphological / histological pattern of damage identified in renal biopsy, characterized by focal (under light microscopy, less than 50% of all glomeruli affected) and segmental (less than 50% of the glomerular capillary plexus affected) fibrotic lesions within the glomeruli.

[0123] Harmful fibrosis, or ocular fibrosis, can easily occur in one or both eyes. Therefore, this invention can be used to treat various forms of ocular fibrosis, including capsular fibrosis, conjunctival fibrosis, corneal fibrosis, retinal fibrosis, and subretinal fibrosis, which are among the causes of a variety of prominent fibrosis or fibrosis-related eye diseases or conditions, including but not limited to dry eye, macular edema and diabetic macular edema, retinopathy and diabetic retinopathy, glaucoma, and especially macular degeneration. In macular degeneration, particularly age-related macular degeneration (AMD) or neovascular age-related macular degeneration, retinal and subretinal fibrosis is known to lead to progressive vision loss, ultimately resulting in irreversible blindness.

[0124] This invention can also be used to treat ocular fibrosis in further fibrosis or fibrosis-related ocular diseases or conditions, including proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), failed glaucoma filtration surgery, corneal scarring, conjunctival scarring, choroidal neovascularization, posterior capsule opacification (PCO), congenital extraocular muscle fibrosis (CFEOM), congenital cataracts, Stargardt's disease, Usher syndrome, retinitis pigmentosa, Stickler syndrome, ocular mucosal pemphigoid (OMMP), Stevens-Johnson syndrome, and other similar conditions. Sun syndrome (SJS), uveitis, epiretinal membrane, aniridia, Axenfeld-Rieger syndrome, Fuchs endothelial dystrophy, Bietti lens dystrophy, Leber congenital amaurosis, Marfan syndrome with ocular manifestations, Ehlers-Danlos syndrome with ocular manifestations, Alport syndrome with ocular manifestations, Fabry disease with ocular manifestations, neurofibromatosis type 1 with ocular manifestations, tuberous sclerosis complex with ocular manifestations, and Von Hippel-Lindau disease with ocular manifestations.

[0125] In a further embodiment, fibrosis or fibrosis-related diseases or conditions include those in which fibrosis coexists with neurodegenerative diseases. Therefore, the present invention can be used to treat various forms of neurodegenerative fibrotic diseases, including but not limited to amyotrophic lateral sclerosis (ALS, also known as motor neuron disease (MND) or Lou Gehrig's disease), multiple sclerosis, or Alzheimer's disease. Fibrosis (particularly liver fibrosis) has been reported to be associated with cognitive decline in patients with Parkinson's disease; therefore, the present invention can also be used to treat Parkinson's disease.

[0126] Reproductive tissues and organs are more prone to unwanted fibrosis. Therefore, this invention can be used to treat various fibrosis or fibrosis-related reproductive diseases or conditions, including but not limited to uterine fibrosis, prostatic fibrosis, Peroni's disease, balanitis, and lichen sclerosus, most particularly Peroni's disease.

[0127] The urinary system tissues and organs are also prone to abnormal fibrosis. Therefore, this invention can be used to treat various fibrosis or fibrosis-related urinary system diseases or conditions, including but not limited to bladder neck contracture, interstitial cystitis (also known as bladder pain syndrome), vesicoureteral reflux (VUR), ureteropelvic junction obstruction (UPJO), ureteral stricture, urethral stricture, urethral diverticulum, urethral caruncle, urethral prolapse, and urethral fistula.

[0128] In some embodiments, the methods and uses of the present invention include the use of enolase inhibitors to reduce fibrosis in a group of cells or tissues. In one embodiment, the tissue is cardiac tissue, skin (skin) tissue (especially keloid tissue), liver tissue, kidney tissue, eye tissue, nerve tissue, reproductive tissue (especially Peronis disease tissue), or urinary tissue, preferably lung tissue or skin tissue. In some embodiments, the cells are fibroblasts, mesenchymal fibroblasts, myofibroblasts, fibroblasts, epithelial cells, endothelial cells, neutrophils, monocytes, macrophages, T cells, mesenchymal cells, lung cells, cardiomyocytes, skin cells, hepatocytes, hepatic stellate cells (HSCs), renal tubular epithelial cells, astrocytes, or cancer cells, most particularly fibroblasts and myofibroblasts. In some embodiments, the methods and uses of the present invention include the use of enolase inhibitors to reduce fibrosis in a group of isolated cells or tissues. In some embodiments, the methods and uses of the present invention include the use of enolase inhibitors to reduce fibrosis in a group of cells or tissues in vivo in a subject.

[0129] Dosage

[0130] In some embodiments, the methods and uses of the present invention comprise administering to a subject who has been diagnosed with, is suspected of having, or is at risk of developing fibrosis or fibrosis-related diseases or conditions an effective amount of a composition comprising an enolase inhibitor (preferably an HMW-ENO enolase inhibitor, such as POMHEX or a derivative thereof). In some embodiments, the composition is contacted with cells or tissues that already have fibrosis or are at risk of developing fibrosis. In one embodiment, the composition is systemically administered to the subject.

[0131] The methods and uses of this invention may include administering enolase inhibitors to patients or subjects in need in various ways. Routes of administration include oral administration, inhalation, intraoperative, intravenous, intravascular, intramuscular, subcutaneous, intracerebral, intraperitoneal, soft tissue injection, surgical implantation, arthroscopic placement, and percutaneous insertion, such as direct injection, cannulation, or catheter insertion. Any administration may be a single application of the composition of this invention or multiple applications. Administration may be to a single site in the individual to be treated or to more than one site. Multiple administrations may be performed substantially simultaneously or separately in time.

[0132] In some embodiments of the methods and uses, the composition is administered during surgical removal or debulking of a tumor or diseased tissue (e.g., fibrotic tissue). For example, in patients undergoing surgical treatment for diseased tissue or tumors, the composition may be administered to the site for further treatment of the tumor, fibrosis, acute lung injury, or a combination thereof.

[0133] In some embodiments, the composition containing an enolase inhibitor can be administered in a manner suitable for treating (or preventing) the disease. The amount and frequency of administration will be determined by factors such as the subject's condition, as well as the type and severity of the subject's disease, although the appropriate dosage can be determined through clinical trials.

[0134] When instructing on a “therapeutic dose,” physicians can take into account individual differences in patients (subjects), such as age, weight, disease type, disease severity, and condition, to determine the precise amount of the composition of the present invention.

[0135] Enolase inhibitors can be administered in any convenient manner, including by inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to patients orally, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (iv), or intraperitoneally.

[0136] In some embodiments, methods and uses for treating fibrosis or fibrosis-related diseases or conditions include administering an enolase inhibitor (preferably an HMW-ENO enolase inhibitor, such as POMHEX or a POMHEX derivative) as part of a treatment regimen to a subject suffering from fibrosis, together with one or more other drugs, biologics, or therapeutic interventions suitable for treating fibrosis. In some embodiments, the additional drug, biologic, or therapeutic intervention is appropriate for specific symptoms associated with fibrosis. In such combined treatment embodiments, the methods and uses of the present invention include administering to a subject suffering from fibrosis at least a first composition comprising an effective amount of at least a first therapeutic agent or antifibrotic agent, wherein the at least first therapeutic agent or antifibrotic agent is an enolase or HMW-ENO enolase inhibitor, preferably POMHEX or a POMHEX derivative; and further administering to the subject (optionally, in at least a second composition) an effective amount of at least a second therapeutic agent or antifibrotic agent, wherein the at least second therapeutic agent or antifibrotic agent is at least one other drug, biologic, or therapeutic intervention effective for treating fibrosis, i.e., at least a second different antifibrotic agent.

[0137] For example, enolase inhibitors can be administered as part of a treatment regimen along with one or more immunosuppressants, such as methotrexate, cyclophosphamide, azathioprine, pirfenidone, nintedanib, tocilizumab, and mycophenolate mofetil. In some embodiments, suitable adjunctive medicaments, biologics, or therapeutic interventions for treating fibrosis are C-terminal endostatin peptides of 48 to 53 amino acids in length, as described in U.S. Patent Nos. 8,507,441, 8,716,232, or 10,709,769; or C-terminal endostatin peptides of 9 amino acids in length, as described in U.S. Patent Nos. 9,365,616, 9,556,252, or 10,172,923; or C-terminal endostatin peptides of 36 amino acids in length, as described in U.S. Patent Application Publication No. US 2021 / 0008173; or C-terminal endostatin peptides of 14 to 34 amino acids in length, as described in Table 1 of PCT Patent Application Publication No. WO 2023 / 039399, each of which is incorporated herein by reference.

[0138] As a further example, enolase inhibitors can be administered as part of a treatment regimen along with one or more agents (such as nifedipine, amlodipine, diltiazem, felodipine, or nicardipine) designed to increase blood flow (such as blood flow to an ulcerated finger). As a further example, enolase inhibitors can be administered as part of a treatment regimen along with one or more agents (such as d-penicillamine, colchicine, PUVA, relaxin, and cyclosporine) designed to reduce skin fibrosis. As a further example, enolase inhibitors can be administered as part of a treatment regimen along with steroids or bronchodilators.

[0139] This invention covers the administration of enolase inhibitors, such as POMHEX or derivatives thereof, to treat or prevent fibrosis or fibrotic diseases and conditions. To practice the methods and uses of this invention, those skilled in the art will understand how to formulate and administer suitable compositions of this invention to subjects based on the disclosure provided herein. This invention is not limited to any particular method of administration or treatment regimen.

[0140] Dosage and formulation (pharmaceutical composition)

[0141] In some embodiments, the present invention envisions treating fibrosis or fibrosis-related diseases or conditions in mammals by administering a therapeutic agent (e.g., an enolase inhibitor). In one embodiment, the therapeutic agent is POMHEX or a derivative thereof.

[0142] Administration of the therapeutic agents according to the invention can be continuous or intermittent, depending on factors such as the recipient's physiological condition, whether the purpose of administration is therapeutic or preventative, and other factors known to a skilled practitioner. Administration of the agents of the invention can be substantially continuous over a pre-selected time period or can be a series of intermittent doses. Local and systemic administration are considered. The dosage will vary depending on various factors, including but not limited to the selected composition, the specific disease, the mammal's weight, physical condition and age, and whether prevention or treatment is desired. Clinicians can readily determine such factors using animal models or other testing systems well known in the art.

[0143] One or more suitable unit dosage forms of the therapeutic agents of the present invention can be administered via a variety of routes, including parenteral, intravenous, intraperitoneal, subcutaneous, inhalation, and intramuscular routes, as well as by direct injection into diseased tissues. Where appropriate, the formulation can be conveniently available in discrete unit dosage forms and can be prepared by any method well known in pharmaceutical science. Such methods may include the steps of associating the therapeutic agent with a liquid carrier, a solid matrix, a semi-solid carrier, a subdivided solid carrier, or a combination thereof, and then, if necessary, introducing the product into or forming the desired delivery system.

[0144] In various embodiments, pharmaceutical compositions useful in the methods and uses of the invention can be administered, for example, systemically, subcutaneously, parenterally, or topically, such as in oral formulations, inhaled formulations (including solids or aerosols), and by topical or other similar formulations. In addition to suitable therapeutic compositions, such pharmaceutical compositions may also contain pharmaceutically acceptable carriers and other ingredients known to enhance and promote drug delivery. Other possible formulations (e.g., nanoparticles, liposomes, resealed red blood cells, and immunologically based systems) can also be used to administer their suitable modulators according to the methods and uses of the invention.

[0145] When the therapeutic agents of the present invention are prepared for administration, they are preferably combined with pharmaceutically acceptable carriers, diluents, or excipients to form a pharmaceutical formulation or unit dosage form. The total active ingredient in such formulations accounts for 0.1% to 99.9% of the formulation weight. "Pharmaceutically acceptable" refers to carriers, diluents, excipients, and / or salts that are compatible with other components in the formulation and harmless to the recipient. The active ingredient for administration may be in the form of a powder or granules; or in the form of a solution, suspension, or emulsion.

[0146] Pharmaceutical formulations containing the therapeutic agents of the present invention can be prepared using well-known and readily available ingredients through procedures known in the art. The therapeutic agents of the present invention can also be formulated into solutions suitable for parenteral administration, for example, via intramuscular, subcutaneous, or intravenous routes.

[0147] The pharmaceutical formulation of the therapeutic agent of the present invention may also be in the form of an aqueous solution, an aqueous solution, or a dispersion, or alternatively in the form of an emulsion or suspension.

[0148] Therefore, therapeutic agents can be formulated for parenteral administration (e.g., by injection, such as bolus or continuous infusion) and can be available in unit dosage forms in ampoules, pre-filled syringes, small-volume infusion containers, or multi-dose containers with added preservatives. The active ingredient can be in the form of a suspension, solution, or emulsion in an oily or aqueous carrier and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the active ingredient can be in powder form, obtained by aseptic separation of sterile solids or lyophilization from a solution, to be formulated with a suitable carrier (e.g., sterile, pyrogen-free water) prior to use.

[0149] In some embodiments, the compositions of the invention are administered by inhalation. In some embodiments, the invention can be conveniently delivered from an inhaler, nebulizer, or other convenient device that pressurizes or delivers aerosol sprays. Pressurized packaging may contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. In some embodiments, the invention can be in the form of a dry powder composition, for example, a powder mixture of the compound with a suitable powder matrix (such as lactose or starch). The powder composition may be present in unit dosage forms, packaged, for example, in capsules or cartons, or in packages such as gelatin or blister packs, and the powder can be administered using an inhaler or inhaler. When dispersed, the powdered or aerosol formulation preferably has an average particle or droplet size of about 0.1 nanometers to about 2000 micrometers, and may further contain one or more additional ingredients described herein.

[0150] It should be understood that the amount of active ingredient contained in a single aerosol dose of each dosage form does not necessarily constitute an effective amount for treating a specific indication or disease, as the necessary effective amount can be achieved by administering multiple dose units. Furthermore, an effective amount can be achieved by using a smaller dose than that in the dosage form, either alone or in a series of administrations.

[0151] The pharmaceutical formulations of the present invention may include (optionally) pharmaceutically acceptable carriers, diluents, solubilizers or emulsifiers, and salts well known in the art. Specific, non-limiting examples of carriers and / or diluents useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate-buffered saline solutions with a pH of 7.0-8.0.

[0152] The pharmaceutical preparations of the present invention can be formulated and administered by any method that allows the active ingredient to contact the site of action of the drug within the body for the treatment of various disease states. They can be administered by any conventional method that can be used in combination with drugs, as a single therapeutic active ingredient or in a combination of multiple therapeutic active ingredients. They can be administered alone, but are usually administered together with a drug carrier selected based on the chosen route of administration and standard pharmaceutical practice.

[0153] Generally, water, suitable oils, saline solutions, aqueous solutions of glucose and related sugar solutions, and glycols (such as propylene glycol or polyethylene glycol) are suitable carriers for parenteral solutions. Solutions for parenteral administration contain the active ingredient, suitable stabilizers, and (if necessary) buffering agents. Antioxidants such as sodium bisulfate, sodium sulfite, or ascorbic acid (alone or in combination) are suitable stabilizers. Citric acid and its salts, and sodium ethylenediaminetetraacetate (EDTA) are also used. Furthermore, parenteral solutions may contain preservatives such as benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. Suitable drug carriers are described in Remington's Pharmaceutical Sciences (the standard reference in this field).

[0154] The active ingredients of this invention can be formulated into suspensions in pharmaceutically acceptable compositions suitable for mammals, particularly humans. Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDPs) or similar compounds described in U.S. Patent Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and 4,406,890. Other useful adjuvants include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate, and dimethyl dioctadecyl ammonium bromide (DDA), Freund's adjuvant, and IL-12. Other components may include polyoxypropylene-polyoxyethylene block polymers (Pluronic®), nonionic surfactants, and metabolizable oils such as squalene (U.S. Patent No. 4,606,918).

[0155] In addition, standard pharmaceutical methods can be used to control the duration of action. These are well known in the art and include controlled-release formulations, which may include suitable macromolecules such as polymers, polyesters, polyamino acids, polyethylene, pyrrolidone, ethylene-vinyl acetate, methylcellulose, carboxymethylcellulose, or protamine sulfate. The concentration of the macromolecules and the manner of incorporation can be adjusted to control release. Furthermore, the pharmaceutical agent can be incorporated into particles of polymeric materials, such as polyesters, polyamino acids, hydrogels, poly(lactic acid), or ethylene-vinyl acetate copolymers. Besides being incorporated, these agents can also be used to capture compounds in microcapsules.

[0156] Therefore, the pharmaceutical compositions of the present invention can be delivered to various sites within the mammalian body via various routes to achieve specific effects (see, for example, Rosenfeld et al., 1991; Rosenfeld et al., 1991a; Jaffe et al., ibid.; Berkner, ibid.). Those skilled in the art will recognize that while more than one route of administration may be used, a particular route may provide a more direct and effective response than another. Local or systemic delivery can be accomplished by applying or infusing the formulation into body cavities, inhaling or blowing it into an aerosol, administering it orally or via parenteral administration, including intramuscular, intravenous, peritoneal, subcutaneous, intradermal, and local administration.

[0157] The active ingredient of the present invention can be provided in unit dosage forms, wherein each dose unit (e.g., a teaspoon, tablet, solution, or suppository) contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. As used herein, the term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for human and mammalian subjects, each unit containing a predetermined amount of the composition of the present invention, alone or in combination with other active agents, calculated to be sufficient to produce the intended effect, and used in conjunction with a pharmaceutically acceptable diluent, carrier, or medium where appropriate. The specifications of the unit dosage forms of the present invention depend on the specific effect to be achieved and the specific pharmacodynamics associated with the pharmaceutical composition in a particular host.

[0158] The methods and uses described herein are not all-encompassing, and further methods and uses suitable for specific applications will be readily apparent to those skilled in the art. Furthermore, the effective amount of the composition can be further approximated by analogy with known compounds that produce the desired effect.

[0159] The present invention further includes several kits related to fibrosis. First provided are diagnostic, prognostic, and predictive treatment kits, which typically contain one or more components known to be effective for the diagnosis or monitoring of fibrosis or fibrosis-related diseases or conditions, and further contain at least one first-enolase inhibitor, preferably POMHEX or a derivative thereof. These kits may include, for example, pre-labeled antibodies in fully conjugated form for the detection of markers or biomarkers of fibrosis, preferably accompanied by instructions for use. Such kits preferably further include control agents, such as appropriately aliquoted biological compositions (whether labeled or unlabeled), for the preparation of a standard curve for the detection assay. Diagnostic, prognostic, and predictive treatment kits may further contain at least one or more other drugs, biologics, or therapeutic interventions suitable for the treatment of fibrosis.

[0160] The following is a combination therapy kit, which typically contains at least a first therapeutic agent or antifibrotic agent, wherein the at least first therapeutic agent or antifibrotic agent is an enolase inhibitor, preferably HMW-ENO enolase, most preferably POMHEX or a derivative thereof, and at least a second therapeutic agent or antifibrotic agent, wherein the at least second therapeutic agent or antifibrotic agent is at least a second different antifibrotic agent, i.e. at least one or more other drugs, biologics, or therapeutic interventions suitable for treating fibrosis.

[0161] Generally, the kit will contain the components in at least one suitable container (or container device). This container typically comprises at least one vial, test tube, flask, bottle, syringe, or other container or container device into which the desired reagent is placed and preferably appropriately aliquoted. The kit will also typically include a device for tightly sealing the individual vials or other similar items for transport, such as, for example, injection-molded or blow-molded plastic containers, into which the desired vials and other equipment are placed and retained. The kit preferably also includes written or electronic instructions for use, for example in quantitative, preclinical, clinical, and / or veterinary implementations, including those for combination therapy.

[0162] The kit components may be contained in an aqueous medium or in lyophilized form. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. The solvent may also be provided in a separate container within the kit. Any therapeutic component is preferably in a pharmaceutically acceptable formulation or prepared as is. The kit may also contain a device for administering the therapeutic agent to animals or patients, such as one or more needles or syringes, or eye drops, pipettes, or other similar devices from which the formulation can be injected into animals or patients or applied to the affected area of ​​the body.

[0163] The kit preferably has different containers for each desired component or agent, particularly for diagnostic, prognostic, or predictive testing and diagnostic components. However, for combination therapy, the kit may contain a single container with two or more premixed fibrosis treatment agents; this may be a molar equivalent combination, or one component may be in greater quantity than another. Nevertheless, even combination therapy kits preferably contain at least two different containers, the first containing at least one enolase inhibitor, preferably POMHEX or a derivative thereof, and at least the second containing one or more other drugs, biologics, or therapeutic interventions suitable for treating fibrosis.

[0164] Experimental Examples

[0165] The invention has been described in further detail with reference to the following experimental embodiments. These embodiments are for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the invention should not be construed as being limited to the following embodiments, but should be construed as encompassing any and all variations that become apparent from the teachings provided herein.

[0166] Without further description, it is believed that those skilled in the art can make and utilize the invention and practice the claimed methods and uses using the foregoing description and the following illustrative embodiments. Therefore, the following working examples specifically point to preferred embodiments of the invention, but should not be construed as limiting the remainder of this disclosure in any way.

[0167] Example 1: Enolase inhibitor ENOBLOCK does not reduce fibrosis.

[0168] Fibrosis is a connective tissue disease characterized by the excessive accumulation of extracellular matrix (ECM) components such as fibronectin and collagen in organs or tissues, and the loss of ECM homeostasis (Thannickal et al., 2014, The Journal of Clinical Investigation. 124:4673-4677; Wynn & Ramalingam, 2012, Nature Medicine). . 18:1028-1040). Fibrosis is the result of dysregulation of tissue repair responses triggered by various factors. During normal wound healing, resident fibroblasts differentiate into myofibroblasts and secrete inflammatory mediators. This leads to the temporary accumulation of ECM components in a positive feedback loop, promoting the restoration of normal tissue architecture and maintaining tissue homeostasis (Henderson et al., 2020, Nature). . 587:555-566; Cheng et al., 2022, Stem Cell Research & Therapy. 13:492). However, when the wound healing process remains uncontrolled, it leads to the dynamic accumulation of ECM components, resulting in tissue damage and eventual organ failure (Thannickal et al., 2014 The Journal of Clinical Investigation. 124:4673-4677).

[0169] The most studied experimental fibrosis inducer is transforming growth factor-β (TGF-β1). Other known fibrosis inducers include PDGF, IL6, CTGF, and IGF2 (Li et al., 2022, Int J Biol Sci. 18:5405-5414; Zhu et al., 2022, Biomolecules).. 12:1622; Waldrep et al., 2023, International Journal of Molecular Sciences . 24:11234; Feghali et al., 1992, J Rheumatol . 19:1207-1211; Effendi & Nagano, 2022, International Journal of Molecular Sciences. 23:6064; Paolini et al., 2022, International Journal of Molecular Sciences. 23:3904; Garrett et al., 2019, PLOS ONE. 14:e0225422). Although the role of SMAD and non-SMAD signaling in TGF-β1-mediated fibrosis has become a focus of extensive research, the role of glycolytic reprogramming in pulmonary fibrosis is still poorly understood. 6-phosphofructo-2-kinase (PFKFB3), pyruvate dehydrogenase kinase (PDK), lactate dehydrogenase (LDH), hexokinase 2 (HK2), and glucose transporter 1 (GLUT1) are enzymes that mediate the role of TGF-β1 in pulmonary fibrosis (Li et al., 2022, Frontiers in Pharmacology). 13:854544; Xie et al., 2015, American Journal of Respiratory and Critical Care Medicine . 192: 1462-1474; Yin et al., 2019, ScienceSignaling . 12:eaax4067).

[0170] Enolase (ENO) is an important metalloenzyme that catalyzes the conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) during glycolysis. Enolases participate in various non-catalytic functions within cells, and are thus referred to as having 'part-time' functions (Sharma et al., 2021, JCI Insight. 6:e146000; Huang et al., 2022, Molecular Therapy – Oncolytics. 24:288-298). Altered ENO expression has been reported in various diseases, including cancer, diabetes, and rheumatoid arthritis (Huang et al., 2022, Molecular Therapy – Oncolytics). 24:288-298; Cancemi et al., 2019, International Journal of Molecular Sciences. 20:3952; Didiasova et al., 2019, Frontiers in Cell and Developmental Biology. 7:61; Almaguel et al., 2021, Frontiers in Genetics. 11:614726; Qiao et al., 2021, Int J BiolSci. 17:3981-3992). There are three ENO isotypes in mammalian tissues, each with different tissue-specific functions: ENO1 is universally expressed in almost all tissues, ENO2 is a neuron-specific isotype, and ENO3 is mainly expressed in muscle (Didiasova et al., 2019, Frontiers in Cell and Developmental Biology. 7:61).

[0171] It has been demonstrated that overexpression of ENO1 in primary fibroblasts increases the expression of pro-fibrotic genes and downregulates the expression of matrix metalloproteinases (MMP-1 and MMP-3) that degrade the matrix (Sharma et al., 2021, JCI Insight. 6:e146000). The effect of ENO1 is independent of TGF-β1, unlike the effects of other TGF-β-dependent glycolytic enzymes. The pro-fibrotic effects of ENO1 have also been observed in vivo in mouse lungs and in vitro in organ-cultured human tissues (Sharma et al., 2021, JCI Insight. 6:e146000).

[0172] This embodiment describes an initial effort to identify the potential use of small molecule ENO inhibitors as fibrosis inhibitors, focusing on ENOBLOCK, a non-substrate analog ENO inhibitor. ENOBLOCK directly binds to and modulates the non-glycolytic "side job" function of ENO. ENOBLOCK has shown activity in cancer treatment models (US Patent No. 9,364,480). ENOBLOCK has a molecular weight of 594.62 g / mol, and its structure is as follows:

[0173] The study of ENOBLOCK in TGF- 1. Effects of stimulation on primary human lung fibroblasts. In the materials and methods used in these experiments, 2.0 x 10⁻⁶ cells were used. 5Human lung fibroblasts were seeded in complete DMEM (DMEM, 10% FBS, 1X Ab / Am) in 6-well tissue culture plates. After 24 hours, cells were subjected to overnight serum starvation. The next day, cells were treated with 1.68 µMENOBLOCK or an equal volume of DMSO for 1 hour, followed by treatment with 10 ng / ml human recombinant TGF-β. 1 or TGF- Cells were stimulated with a medium control (VC) of 1. After 72 hours of stimulation, cells and conditioned medium were harvested for protein analysis.

[0174] The results focused on testing the efficiency of ENOBLOCK by analyzing ECM expression at the translational level. ENOBLOCK was found to reduce cell lysate (Figure A, ...). Figure 1 B) and fibroblast conditioned medium ( Figure 2 A, Figure 2 In B), the expression of known fibrosis markers COL1α1 and FN protein was not significantly affected. The expression of known matrix-degrading metalloproteinases MMP-1 and MMP-3 was observed in cell lysates (…). Figure 1 C Figure 1 D) and conditioned medium ( Figure 2 C Figure 2 In D), ENOBLOCK did not significantly upregulate the expression of ENO monomer and HMW-ENO. Furthermore, ENOBLOCK did not significantly downregulate the expression of ENO monomer and HMW-ENO. Figure 1 E, Figure 1 F).

[0175] In summary, these data suggest that ENOBLOCK has no significant effect on the fibrotic phenotype in primary human lung fibroblasts stimulated with TGF-β1.

[0176] Example 2: The enolase inhibitor POMHEX reduced fibrosis in vitro and in vivo.

[0177] As described in Example 1, ENO1 is known to have pro-fibrotic effects (Sharma et al., 2021, JCI Insight. 6:e146000). This example first demonstrates that ENO1 triggers fibrosis by regulating the expression of Yes-related protein (YAP1), a known transcriptional regulator of Hippo signaling. YAP1, along with its transcriptional coactivator (TAZ) with a PDZ-binding motif, is a key activator of TGF-β1-stimulated fibrosis. YAP1 senses tissue stiffness caused by ECM deposition in fibrotic tissue and localizes to the nucleus, triggering fibrosis in the nucleus through a feedforward loop mechanism (Enzo et al., 2015, Embo j. 34:1349-1370; He et al., 2022, JCI Insight. 7:e146243).

[0178] Next, this example describes ongoing activities that led to the identification of a small-molecule ENO inhibitor capable of successfully inhibiting fibrosis. Although ENOBLOCK lacks activity as shown in Example 1, ongoing studies have demonstrated that a different ENO inhibitor, POMHEX, can effectively reduce fibrosis. At low nanomolar concentrations, POMHEX has shown efficacy against ENO1-depleted cells in vitro and can eliminate ENO1-deficient xenograft tumors in vivo (Lin et al., 2020, Nature Metabolism. 2:1413-1426). Given that POMHEX was designed to be specific to the neuron-specific isotype ENO2 rather than ENO1 (Lin et al., 2020, Nature Metabolism. 2:1413-1426), while it is the ubiquitous ENO1 that exerts a pro-fibrotic effect, choosing to test POMHEX was counterintuitive. Nevertheless, the current experiment is the first to demonstrate that POMHEX targets enolase, alleviates fibrosis in primary lung fibroblasts, reverses fibrosis in lung fibroblasts isolated from patients with SSc and IPF, and reduces / reverses fibrosis in a bleomycin-treated mouse model of lung fibrosis, thus identifying POMHEX as a therapeutic agent for fibrosis.

[0179] The materials and methods used in the experiment are now described.

[0180] Human primary lung fibroblasts

[0181] Lung tissue was obtained from transplanted lungs of patients with SSc and IPF who received lung transplants, as well as from normal controls whose lungs were not used for transplantation, as previously reported (Hsu et al., 2011, Arthritis Rheum. 63:783-794). All tissues were obtained in accordance with approved protocols and with written informed consent obtained. Human primary lung fibroblasts were cultured from lung tissue, as previously described (Pilewski et al., 2005, Am J Pathol). 166:399-407). In short, approximately 3-5 mm 2Tissue sections were minced and cultured and maintained as fibroblasts in Dulbecco modified Eagle medium (DMEM) (Corning Incorporated Life Sciences, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA), penicillin, streptomycin, and an antifungal agent (Invitrogen, Carlsbad, CA, USA). All cells were used between passages 3 and 8.

[0182] Western blot analysis

[0183] As previously described, cell lysates, subcellular fractions, and conditioned media from fibroblast cultures were analyzed by immunoblotting (Cottin & Brown, 2019, Respiratory Research). 20:13). Cell lysates were prepared by scraping cells in 2X SDS-PAGE buffer, boiling, and storing at -80°C. Conditioned medium for SDS-PAGE was prepared by mixing with 6X SDS-PAGE buffer and treating as described above. To prepare subcellular fractions, fibroblasts were trypsinized, and the cell pellet was harvested. Cytoplasmic, nuclear, and chromatin fractions were extracted using a subcellular fractionation kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. A mixture of protease inhibitors and a phosphatase inhibitor (sodium orthovanadate (Thermo Fisher Scientific, Waltham, MA, USA)) were added to the buffer. 6X SDS-PAGE buffer was added, the sample was boiled, and stored at -80°C. The antibodies used were anti-human ENO1 antibody, Tata-binding protein (TBP) antibody, anti-human smooth muscle actin (α-SMA) antibody (Abcam, Waltham, MA, USA), anti-human type I collagen α-I (COL1α1) (CloudClone, Wuhan, Hubei, China), and type II collagen α-II (COL1α2) antibody, anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, anti-human fibronectin (FN) antibody (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA), anti-human PAI-1 antibody (Proteintech, Rosemont, IL, USA), anti-human V5 antibody (Sigma, St. Louis, MO, USA), human YAP-1 antibody, anti-human CTGF antibody (Millipore Sigma, Burlington, MA, USA), human YAP-1 antibody, TWIST-1 antibody, INHBA antibody, and thromboretin-1 (THBS-1) antibody (Novus Biologics, Centennial, Primary antibodies included histone H3 antibody (Cellsignaling, Danvers, MA, USA), mouse IgG1 isotype control antibody, and citrullinated enolase antibody (Cayman chemicals, Ann Arbor, MI, USA). Horseradish peroxidase-conjugated antibody was used as a secondary antibody. Chemiluminescence was used to detect the signal. Proteins in the conditioned medium were normalized using Ponceau S S staining.

[0184] Quantitative PCR

[0185] Total RNA was extracted from mouse lung tissue and human fibroblasts using TRIZOL™ lysis reagent (phenol and guanidine isothiocyanate solution, Life Technologies, CA, USA). Reverse transcription was performed using SuperScript™ IV (reverse transcriptase, Invitrogen, CA, USA). TaqMan was used. ® Real-time PCR systems (quantitative PCR systems, LifeTechnologies) are manufactured according to the manufacturer's protocol and are available in TaqMan. ® In the gene expression assay, step one involved measuring gene mRNA expression levels using a real-time PCR system (quantitative PCR system, Life Technologies, CA, USA) via quantitative PCR. The expression of each gene was measured relative to... B2M or B2m The ratio of gene expression. Used for amplifying coding genes. Col1A1 (Hs00164004_m1) Col1A2 (Hs00164099_m1) FN (Hs00365052_m1) ACTA2 (Hs00426835_g1) B2M (Hs00187842_m1) Fn (Mm01256744_m1) Col1a1 (Mm00801666_g1) Col1a2 (Mm00483888_m1) Acta2 (Mm00725412_s1) Eno (Mm01619597_g1) B2m The specific primers and probes for the gene (Mm00437762_m1) were purchased from Life Technologies.

[0186] Immunoprecipitation assay

[0187] Total 1 × 10 6 Primary human lung fibroblasts were cultured on 10 cm culture dishes and treated with TGF-β1 or VC. After 72 hours, nuclear fractions of the fibroblasts were extracted using a subcellular protein fractionation kit according to the manufacturer's protocol. The nuclear fractions were incubated overnight at 4°C on a rotating body with citrullinated ENO antibody or mouse isotype control. The next day, the complex was pulled down with agarose G beads (Thermoscientific) at 4°C for 2 hours. The immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting.

[0188] In vitro POMHEX treatment

[0189] As previously described, with some modifications, actively growing primary human lung fibroblasts and fibroblasts from SSc patients were stimulated (Nguyen et al., 2018, Frontiers in Endocrinology. 9:601). In short, 2.0 x 10 5 Primary fibroblasts were seeded in complete DMEM in 6-well tissue culture plates. After 48 hours, the cells were serum starved in DMEM for 1 hour, followed by stimulation with POMHEX or an equal volume of DMSO. Normal lung fibroblasts were stimulated with (0.001 to 5 μM) POMHEX or DMSO for 1 hour, followed by stimulation with 10 ng / ml human recombinant TGF-β1 or PBS. SSc fibroblasts were treated with 5 and 10 μM POMHEX or DMSO. Cells and conditioned medium were harvested 48 hours (for RNA) or 72 hours (for protein) after stimulation. The structure of POMHEX is shown below:

[0190] Small interfering RNA (siRNA) transfection

[0191] 24 hours before siRNA transfection, primary human lung fibroblasts were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in six-well plates. Dharmacon™ ON-TARGETplus™ (modified siRNA for reducing off-target effects) ENO siRNA, YAP-1 siRNA, and control siRNA (siCTRL) were used (Lafayette, CO, USA). Transfection was performed according to the manufacturer's recommendations using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) and Opti-MEM I low-serum medium (Life Technologies). In brief, fibroblasts were transfected with 30 nmol of ENO, YAP-1, or siCTRL-specific siRNA for 24 hours in antibiotic- and antifungal-free DMEM medium supplemented with 10% FBS. The medium was then replaced with serum-free DMEM. Two hours later, 10 ng / ml TGF-β1 or 3 µg rENO was added to the wells transfected with siCTRL / siENO or siCTRL / siYAP-1, respectively. Fibroblasts were harvested after 72 hours.

[0192] rENO processing

[0193] As described above, normal lung fibroblasts were silenced with siCTRL or siYAP-1. Twenty-four hours later, fibroblasts were stimulated with rENO (Abcam). Cells were serum starved one hour before rENO treatment. 4 μggrENO protein was added to each well. Tris buffer (20 mM Tris-HCl, pH 7.5, 1 mM MgSO4) was used as a medium control. Fibroblasts were harvested 72 hours after siRNA transfection and protein extraction was performed.

[0194] Transfection with ENO expression plasmid

[0195] The ENO gene was subcloned into the mammalian plasmid OG1082 (Oxford Genetics) and expressed as a V5-tagged protein as previously described (Sharma et al., 2021, JCI Insight. 6:e146000). Transfection was performed using the X-tremeGENE™ transfection reagent (MilliporeSigma) according to the manufacturer's instructions. Briefly, human lung fibroblasts were transfected at 1.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well Corning plates. 24 hours after seeding, the medium was replaced with antibiotic-free medium, and fibroblasts were transfected with 1 µg of control empty vector (EV) or ENO-expression plasmid. Cells were harvested 72 hours after transfection for Western blotting.

[0196] L-lactic acid ester / salt quantification

[0197] L-lactate / salt was quantified in conditioned medium from SiENO or SiCTRL-transfected and VC or TGF-β1-stimulated human lung fibroblasts using a glycolytic cell-based assay kit (Cayman Chemical) following the manufacturer’s instructions.

[0198] In vivo POMHEX treatment

[0199] Pulmonary fibrosis was induced in mice according to an approved protocol, as previously described (Yamaguchi et al., 2012, Sci Transl Med. 4:136ra171). Briefly, bleomycin (1.2 mU / g body weight) was administered orally in 50 µl PBS to 6–8 week old male CB57BL / 6J mice (The Jackson Laboratory, Bar Harbor, ME, USA). PBS alone was administered as a mediator control. POMHEX (10 mg / kg mouse) or DMSO was administered orally every three days, starting 24 hours after bleomycin administration, for a total of four doses. Mice were sacrificed 14 days after treatment, and lung tissue was collected. The four treatment groups were PBS / PBS, PBS / DMSO, bleomycin / DMSO, and PBS / POMHEX.

[0200] For the treatment studies, mice were administered POMHEX or DMSO orally every three days after 7 days of bleomycin treatment (a total of four doses). Lungs were harvested on day 21 after bleomycin treatment. The three groups of mice treated were PBS / DMSO, bleomycin / DMSO, and PBS / POMHEX.

[0201] Histological analysis

[0202] Mice were euthanized and their left lungs were fixed in 10% formalin. Masson's trichrome staining was performed in an AML laboratory (St. Augustine, FL). Images were taken using an Axio Observer microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany).

[0203] Hydroxyproline assay

[0204] To quantify the amount of collagen in lung tissue, hydroxyproline content was measured using some modifications as previously described (Santos et al., 2009, J Clin Invest. 119:3613-3625). Briefly, the right upper lung sample was dissolved in 1 ml of 1N HCl at 105°C for 4 hours. The sample was cooled to room temperature, and 20 µl of the lysed, clear sample was carefully transferred into a 96-well plate and dried at 65°C for 2 hours. A standard was prepared by dissolving cis-4-hydroxy-L-proline (Sigma) in 1 mM HCl. 100 µl of chloramine-T was added to each well and incubated with shaking at room temperature for 15 min. Ehrlich solution (100 µl) was added to the well and incubated at 65°C for 15 minutes. The absorbance was measured at 550 nm. The hydroxyproline content in the sample is automatically calculated from the standard curve generated on the BioTek Synergy™ plate reader (Agilent, Santa Clara, CA).

[0205] statistics

[0206] All continuous variables were expressed as mean ± standard deviation. All statistical analyses were performed using GraphPad Prism 9.00 for Windows (GraphPad Software, La Jolla, California, USA, www.graphpad.com). Paired t-tests were used for comparisons between two groups to test for statistical significance. ANOVA and subsequent Tukey multiple comparison tests were used for comparisons between three or more groups. Logarithmic transformation was applied according to tests for normality and log-normality.

[0207] The experimental results will now be described.

[0208] ENO1 upregulates YAP-1 expression.

[0209] YAP-1 is a well-known transcription factor regulated in aerobic glycolysis and is considered a key regulator of the fibrotic response. Overexpression of YAP-1 promotes collagen production in lung fibroblasts, while knockdown inhibits ECM production (He et al., 2022, JCI Insight. 7:e146243). Using in vitro, ex vivo, and in vivo assays, we demonstrated that ENO1 promotes lung fibrosis independently of TGF-β1 (Sharma et al., 2021, JCI Insight. 6:e146000). To determine the role of YAP signaling in the fibrotic response to ENO, we tested the effect of ENO on YAP-1 expression by transfecting fibroblasts with a plasmid overexpressing ENO and by stimulating fibroblasts with recombinant ENO (rENO). In cell lysates of ENO-overexpressing normal lung fibroblasts ( Figure 3 A) and in rENO-treated normal lung fibroblasts ( Figure 3 B), YAP-1 protein expression increased. Upon TGF-β1 stimulation, YAP-1 localizes to the nucleus to activate pro-fibrotic genes. Therefore, the effect of ENO silencing on the nuclear translocation of YAP-1 was tested. ENO1 in human lung fibroblasts was silenced using siRNA, followed by TGF-β1 stimulation. Fibroblasts were subcellularly graded, and ENO levels in the nuclear fraction were analyzed. Significant silencing of ENO in the nuclear fraction was observed (B). Figure 9 A). TGF-β1 significantly increased YAP-1 expression in the nuclear fraction (A). Figure 3 C), and a significant decrease in YAP-1 levels was observed in ENO-silenced and TGF-β1-stimulated fibroblasts compared to siCTRL-transfected and TGF-β1-stimulated fibroblasts. Figure 3 C). In summary, these findings suggest that ENO1 regulates YAP-1 expression and its nuclear translocation.

[0210] ENO1 increases TWIST-1 expression.

[0211] Next, the effects of ENO on Twist family BHLH transcription factor 1 (TWIST-1) were investigated. Studies have shown that YAP-1 upregulates pulmonary fibrosis by transcribedly activating TWIST-1 in the nucleus. TWIST-1 is a transcription factor, and its expression is known to trigger epithelial-to-mesenchymal transition (EMT), thereby promoting pulmonary fibrosis (Ning et al., 2018, Journal of Cellular and Molecular Medicine. 22:1383-1391; Chen et al., 2019, CellDeath & Differentiation). 26:1832-1844). Its expression was also found to be elevated in patients with idiopathic pulmonary fibrosis (IPF) (King et al., 2014, New England Journal of Medicine. 370:2083-2092). Since YAP-1 regulates TWIST-1 expression and ENO knockdown inhibits YAP-1 expression, the effect of ENO1 on TWIST-1 levels was examined. rENO1 upregulated TWIST-1 expression levels ( Figure 3 D、 Figure 9 B). TGF-β1 stimulation increased TWIST-1 levels in fibroblast staining fractions, while ENO silencing decreased TWIST-1 levels (B). Figure 3 E, Figure 9 C), which is similar to the effect observed on YAP-1.

[0212] ENO promotes fibrosis through YAP1.

[0213] To further investigate whether ENO regulates fibrosis through YAP-1, the cytoplasmic fraction and ECM protein levels in the culture medium of ENO1-silenced and TGF-β1-stimulated fibroblasts (in which YAP-1 expression was downregulated) were examined. In the cytoplasm of fibroblasts ( Figure 4 A) and conditioned medium ( Figure 4 In (B), the protein levels of type I collagen α1 chain (COL1α1), type I collagen α2 chain (COL1α2), fibronectin (FN), and plasminogen inhibitor activator-1 (PAI-1) were significantly downregulated. Furthermore, the expression of the myofibroblast marker α-smooth muscle actin (α-SMA) was also significantly downregulated. Figure 4 A). Interestingly, independent of TGF-β1, the protein expression of all these proteins was significantly reduced in ENO-silenced cells (Sharma et al., 2021, JCI Insight. 6:e146000).

[0214] Connective tissue growth factor (CTGF) (a secreted stromal cell protein) is known to activate myofibroblasts and promote ECM deposition in affected organs (Lipson et al., 2012, Fibrogenesis & Tissue Repair). . 5:S24). CTGF is a known downstream target protein of YAP-1 and is upregulated in various cancers (Zhang et al., 2018, Molecular Cancer. 17:134). Significant downregulation of CTGF expression was detected in ENO-silenced and TGF-β1-stimulated fibroblasts. Figure 4 A and Figure 10 Furthermore, glycolysis was measured in fibroblasts silenced with SiENO and stimulated with TGF-β1, and no significant changes were observed compared with control fibroblasts. Figure 13 This indicates that the fibrotic activity of ENO is independent of its glycolytic function.

[0215] Next, the regulation of YAP-1 in fibrosis mediated by ENO was tested. YAP-1 was silenced in normal lung fibroblasts and then stimulated with rENO protein. Significant silencing of YAP-1 was observed in fibroblasts. Figure 11 As expected, rENO significantly upregulated the expression of the pro-fibrotic proteins COL1a1 and PAI-1 in cell lysates. Figure 4 C). Interestingly, rENO failed to induce the expression of COL1a1 and PAI-1 in YAP-1-silenced and rENO-treated fibroblasts. Figure 4 C). These data collectively demonstrate that ENO and YAP-1 are functionally interdependent and alter each other's roles in fibrillation.

[0216] The ENO inhibitor POMHEX attenuates fibrosis in normal lung fibroblasts.

[0217] The effects of the ENO inhibitor POMHEX on pulmonary fibrosis were investigated. Primary lung fibroblasts were treated with POMHEX prior to TGF-β1 stimulation. POMHEX significantly reduced ECM gene expression. COL1A1 , COL1A2 , FN and ACTA2 level of expression ( Figure 5 A) and the protein levels of COL1α1, COL1α2, FN, and α-SMA in cell lysates ( Figure 5 B) and the protein levels of COL1α1, COL1α2 and FN in fibroblast conditioned medium ( Figure 5 C). Changes in CTGF levels were not significant. The effect of POMHEX on MMPs was also tested. POMHEX significantly induced MMP-1 expression in conditioned medium of untreated and TGF-β1-treated fibroblasts. Figure 5 (C), but no significant effect on MMP-3 expression was observed. Overall, these results indicate that POMHEX can reduce the experimentally induced fibrotic phenotype of TGF-β1 in primary lung fibroblasts from different donors.

[0218] Notably, POMHEX was identified as a small-molecule ENO inhibitor that successfully inhibited fibrosis, a stark contrast to the lack of antifibrotic activity in the first tested inhibitor, ENOBLOCK. This is significant for the presence of FN, COL1α1, and HMW-ENO in cell lysates. Figure 18 A and Figure 18 B comparison) and FN, COL1α1 and MMP-1 in conditioned medium for POMHEX- and ENOBLOCK-treated fibroblasts (B comparison) and (B comparison) Figure 18 C and Figure 18 (D Comparison) provides data showing the different effects of POMHEX compared to ENOBLOCK.

[0219] POMHEX attenuates the fibrotic response of SSc lung fibroblasts.

[0220] The ability of POMHEX to reverse fibrosis was analyzed by testing its effects on primary fibroblasts derived from lung tissue of patients with systemic sclerosis (SSc), a prototypical fibrotic disease, who had undergone lung transplantation and suffered from severe lung disease. POMHEX efficiently downregulated the levels of fibroblast lysates in fibroblasts. COL1A1 , COL1A2 , FN and ACTA2 mRNA levels ( Figure 6 A) and the corresponding protein levels ( Figure 6 B). CTGF expression was also significantly downregulated in the lysate ( Figure 6 B). Furthermore, in fibroblast conditioned medium, in SSc fibroblasts treated with POMHEX, FN and COL1α2 were significantly downregulated and MMP-1 was significantly induced ( Figure 6 C). Although there was significant downregulation of COL1α1 in the total lysate, a higher concentration of POMHEX (10 µM) was required to significantly reduce COL1α1 secretion levels. Figure 6 D). These data suggest that POMHEX can reverse the fibrotic phenotype of fibroblasts derived from SSc patients with severe end-stage pulmonary fibrosis.

[0221] POMHEX attenuates the fibrotic response of IPF lung fibroblasts.

[0222] Based on the results from late-stage SSc fibroblasts, the effects of POMHEX on fibroblasts derived from the lungs of IPF patients were tested. POMHEX efficiently downregulated the levels of COL1A1, COL1A2, FN, and ACTA2 mRNA. Figure 14 A). Protein levels of COL1α2, FN, and α-SMA were downregulated in fibroblast lysates. Figure 14B), and the secretion level of FN in the conditioned medium was significantly lower ( Figure 14 C). Similar to SSc fibroblasts, the expression of secreted MMP-1 was significantly increased in conditioned medium. Figure 14 C). In contrast to SSc, POMHEX did not up- or down-regulate COL1α1 at the protein level in lysates and conditioned media. Interestingly, in IPF fibroblasts treated with POMHEX, the mRNA level of COL1A1 was significantly lower ( Figure 14 A).

[0223] POMHEX regulates YAP-1 expression.

[0224] ENO regulates YAP-1 expression. Since POMHEX is an ENO inhibitor, its effect on YAP-1 was examined. In POMHEX-treated normal ( Figure 7 A), SSc lung fibroblasts ( Figure 7 B) and IPF lung fibroblasts ( Figure 15 In this study, YAP-1 expression was significantly attenuated. POMHEX also significantly reduced cytoplasmic expression in normal and SSc lung fibroblasts. Figure 7 C) and nuclear grade ( Figure 7 The level of YAP-1 protein in D) indicates that its mechanism of action is at least in part through downregulation of YAP-1.

[0225] TGF-β1 triggers ENO core localization.

[0226] ENO has been primarily studied as a cytoplasmic enzyme in glycolysis. However, ENO can translocate to different cellular compartments and perform non-glycolytic functions (Terrier et al., 2007, Autoimmunity Reviews. 6:176-182; Pancholi, 2001, Cellular and Molecular Life Sciences CMLS. 58:902-920; Plow & Das, 2009, Blood. 113:5371-5372). Previous data reported TGF-β1-independent non-glycolytic functions of ENO. Since TGF-β1 is known to regulate the expression of certain glycolytic enzymes, the effect of TGF-β1 on ENO expression or function was tested. In primary lung fibroblasts, TGF-β1 had no effect on the expression of monomeric ENO1 (45 kDa). However, TGF-β1 stimulation resulted in the detection of a higher molecular weight band (HMW-ENO) (70 kDa) in the cell lysates of TGF-β1-stimulated fibroblasts, which may be a dimer of ENO1. Figure 7G). Furthermore, HMW-ENO was detected only in the nuclear fraction of TGF-β1-stimulated fibroblasts. Figure 7 E), indicating that TGF-β1 triggers nuclear localization of ENO. The levels of HMW-ENO were also examined in unstimulated healthy fibroblasts and fibroblasts derived from fibrotic lungs of patients with SSc and IPF. Fibroblasts derived from fibrotic lungs had significantly higher levels of HMW-ENO (E) compared to healthy controls. Figure 7 F, Figure 13 This further points to the presence of HMW-ENO in fibrosis.

[0227] Next, using citrullinated ENO antibody, HMO-ENO was reduced from the nuclear fraction of normal lung fibroblasts treated with TGF-β1 using immunoprecipitation. When detected with ENO antibody, citrullinated ENO antibody was observed to reduce HMW-ENO (…). Figure 16 A). This indicates that HMW-ENO is a citrullinated form of ENO triggered by TGF-β1 stimulation, which, upon translocation to the nucleus, either undergoes dimerization or forms a complex with another unrecognized protein. To confirm this post-translational modification, inhibition experiments were performed using the following citrullinated protein arginase deaminase (PAD) inhibitors: AFM32a (PAD2 inhibitor) and GSK199 (PAD4 inhibitor). In cell lysates of TGF-β1-stimulated lung fibroblasts, an inhibitory effect was observed with GSK199 (…). Figure 16 Compared to C), AFM32a ( Figure 16 B) HMW-ENO expression was significantly downregulated, indicating that ENO was citrullinated and that citrullination was PAD2-specific.

[0228] POMHEX reduces HMW-ENO.

[0229] The effect of POMHEX on HMW-ENO levels was then tested. POMHEX had no effect on the level of 45 kDa ENO monomers. Figure 7 G), but it significantly reduced the level of HMW-ENO triggered by TGF-β1 in normal lung fibroblasts. POMHEX on SSc fibroblasts ( Figure 7 H) and IPF fibroblasts ( Figure 14 A similar effect was observed on HMW-ENO levels in D). Overall, these findings suggest that TGF-β1 promotes nuclear localization of HMW-ENO and indicates that it is the HMW-ENO form of ENO (citrullinated and possibly undergoing other post-translational modifications) that mediates the fibrotic response. These findings also suggest that POMHEX reduces HMW-ENO levels, which is associated with the anti-fibrotic response of POMHEX in fibroblasts.

[0230] POMHEX reduces bleomycin-induced pulmonary fibrosis.

[0231] Results from primary lung fibroblasts and fibroblasts derived from the lungs of SSc and IPF patients showed that the ENO inhibitor POMHEX significantly downregulated ECM mRNA and protein levels. Next, the effects of POMHEX were tested in vivo in a mouse bleomycin-induced pulmonary fibrosis model. PBS or bleomycin, along with DMSO or POMHEX, was administered intratracheally to C57BL6 / J mice. Lung tissue was collected 14 days after bleomycin treatment. In mouse lungs, POMHEX administration following bleomycin significantly reduced ECM deposition (as assessed by Masson's trichrome staining). Figure 8 A), and significantly reduced hydroxyproline content ( Figure 8 C). The effect of POMHEX on the mRNA expression of the ECM gene in mouse lungs was also tested. POMHEX significantly reduced bleomycin-induced ECM gene expression. Fn、Col1a1 , Col1a2 , Fn and Acta2 The expression ( Figure 8 B). Furthermore, POMHEX significantly reduced... Eno Transcription expression levels ( Figure 8 B). These findings suggest that POMHEX improves fibrosis in vivo.

[0232] POMHEX reverses bleomycin-induced pulmonary fibrosis.

[0233] To further investigate the biological efficacy of POMHEX, its effect in reversing fibrosis in mice was evaluated. Mice were administered POMHEX or DMSO 7 days after treatment with bleomycin or PBS, and lungs were harvested 21 days after bleomycin treatment. Masson trichrome staining showed that POMHEX reduced fibrosis in the lungs, but the effect was not significant as determined by the Ashcroft score. Figure 17 A). However, POMHEX significantly reduced hydroxyproline content ( Figure 17 B). Furthermore, POMHEX significantly downregulated the mRNA expression levels of Fn, Col1a1, and Col1a2 (B). Figure 17 C). In contrast to the 14-day mouse experiment, it had no effect on Eno transcription levels. These data from the bleomycin-induced mouse fibrosis model collectively suggest that POMHEX has the potential to reverse fibrosis.

Claims

1. A composition for use in treating fibrosis in subjects for whom treatment is required, wherein, The composition contains an effective amount of POMHEX or a POMHEX derivative.

2. The composition for use according to claim 1, wherein, The composition is intended for use in the treatment of fibrosis in subjects suffering from fibrosis or fibrosis-related diseases or conditions selected from the group consisting of: pulmonary fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis (IPF), interstitial pulmonary fibrosis, familial pulmonary fibrosis, pulmonary arterial hypertension (PAH), radiation-induced pulmonary fibrosis, coal worker's pneumoconiosis, asbestosis, bleomycin-induced lung disease, sarcoidosis, silicosis, acute lung injury, fibrotic mediastinitis, and acute respiratory distress syndrome (ARDS). RDS, pulmonary fibrosis with emphysema (CPFE), asthma; cardiac fibrosis, vascular fibrosis, endocardial myocardial fibrosis (EMF), atherosclerosis, aortic valve sclerosis (AVS); skin fibrosis and wound healing disorders or conditions, hypertrophic scars, keloids, postoperative scars, systemic scleroderma, localized scleroderma, morphea, eosinophilic fasciitis, palmar aponeurosis contracture; cirrhosis, hepatitis, metabolic dysfunction-associated steatohepatitis (MASH), congenital liver fibrosis, alcoholic liver disease. Liver fibrosis induced by hepatitis C virus (HCV) or hepatitis B virus (HBV), primary sclerosing cholangitis, primary biliary cirrhosis; kidney fibrosis, fibrotic nephropathy, IgA nephropathy, transplant nephropathy, diabetic nephropathy, lupus nephritis, glomerulonephritis, focal segmental glomerulosclerosis (FSGS); ocular fibrosis, capsular fibrosis, conjunctival fibrosis, corneal fibrosis, retinal fibrosis, subretinal fibrosis, dry eye syndrome, macular edema, retinopathy, glaucoma, age-related fibrosis. AMD; fibrosis caused by neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), multiple sclerosis, or Alzheimer's disease; fibrosis caused by graft-versus-host disease (GVHD), subepithelial fibrosis, uterine fibrosis, Peronis disease, myelofibrosis, retroperitoneal fibrosis, renal systemic fibrosis, multifocal fibrosis, rheumatoid arthritis, tumor-associated fibrosis, radiation-induced fibrosis, chemotherapy-induced fibrosis, systemic sclerosis, and Sjögren's syndrome.

3. The composition for use according to claim 2, wherein, The composition is intended for use in treating fibrosis in subjects with idiopathic pulmonary fibrosis.

4. The composition for use according to claim 2, wherein, The composition is intended for use in treating fibrosis in subjects with hypertrophic scars or keloids.

5. The composition for use according to claim 2, wherein, The composition is intended for use in the treatment of fibrosis in subjects with systemic scleroderma.

6. The composition for use according to claim 2, wherein, The composition is intended for use in the treatment of fibrosis in subjects with Peroni disease.

7. The composition for use according to any one of claims 1 to 6, wherein, The composition contains POMHEX.

8. The composition for use according to any one of claims 1 to 7, wherein, The composition is administered together with a second agent, wherein the second agent is an anti-fibrotic agent.

9. The composition for use according to any one of claims 1 to 8, wherein, The subjects were non-primate mammals, preferably dogs.

10. The composition for use according to any one of claims 1 to 8, wherein, The subjects were human subjects.

11. A method for reducing fibrosis, comprising contacting a group of cells or tissues with a composition comprising POMHEX or a POMHEX derivative to effectively reduce fibrosis in the cells or tissues.

12. The method according to claim 11, wherein, The tissue in question is lung tissue.

13. The method according to claim 11, wherein, The tissue in question is skin tissue.

14. The method according to any one of claims 11 to 13, wherein, The composition contains POMHEX.

15. The method according to any one of claims 11 to 14, wherein, The cells or tissues are kept in vitro.

16. The method according to any one of claims 11 to 14, wherein, The cells or tissues are contained within the subject.

17. A method of treating fibrosis, comprising administering to a subject suffering from fibrosis a composition comprising POMHEX or a POMHEX derivative in an amount effective in treating fibrosis in the subject.

18. The method according to claim 17, wherein, The subjects with fibrosis had fibrosis or fibrosis-related diseases or conditions selected from the following groups: pulmonary fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis (IPF), familial pulmonary fibrosis, pulmonary arterial hypertension (PAH), radiation-induced pulmonary fibrosis, coal worker's pneumoconiosis, asbestosis, bleomycin-induced lung disease, sarcoidosis, silicosis, acute lung injury, acute respiratory distress syndrome (ARDS), pulmonary fibrosis with emphysema (CPFE), asthma; cardiac fibrosis, vascular fibrosis, endocardial myocardial fibrosis (EMF), arteriosclerosis, aortic valve sclerosis (AVS); skin fibrosis and wound healing diseases or conditions, hypertrophic scars, keloids, postoperative scars, systemic scleroderma, localized scleroderma, morphea, eosinophilic fasciitis; cirrhosis, hepatitis, metabolic dysfunction-associated steatohepatitis (MASH), congenital liver fibrosis, alcoholic liver disease, hepatitis C virus (HCV). Hepatitis B virus (HBV)-induced liver fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis; renal fibrosis, fibrotic nephropathy, IgA nephropathy, transplanted nephropathy, diabetic nephropathy, lupus nephritis, glomerulonephritis, focal segmental glomerulosclerosis (FSGS); ocular fibrosis, lenticule fibrosis, conjunctival fibrosis, corneal fibrosis, retinal fibrosis, subretinal fibrosis, dry eye syndrome, macular edema, retinopathy, glaucoma, age-related macular degeneration (AMD). AMD); fibrosis caused by neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), multiple sclerosis, or Alzheimer's disease; fibrosis caused by graft-versus-host disease (GVHD), subepithelial fibrosis, uterine fibrosis, Peronis disease, myelofibrosis, retroperitoneal fibrosis, renal systemic fibrosis, multifocal fibrosis, rheumatoid arthritis, tumor-associated fibrosis, radiation-induced fibrosis, chemotherapy-induced fibrosis, systemic sclerosis, and Sjögren's syndrome.

19. The method according to claim 18, wherein, The subject had idiopathic pulmonary fibrosis.

20. The method according to claim 18, wherein, The subjects had hypertrophic scars or keloids.

21. The method according to claim 18, wherein, The subject had systemic scleroderma.

22. The method according to claim 18, wherein, The subject had Peroni disease.

23. The method according to any one of claims 17 to 22, wherein, The composition contains POMHEX.

24. The method according to any one of claims 17 to 23, further comprising administering an effective amount of a second therapeutic agent to the subject, wherein, The second treatment agent is an antifibrotic agent.

25. The method according to any one of claims 17 to 24, wherein, The subjects were dogs.

26. The method according to any one of claims 17 to 24, wherein, The subjects were human subjects.

27. A composition comprising a first antifibrotic agent and a second distinct antifibrotic agent in a combined amount effective in treating fibrosis in a subject, wherein, The first anti-fibrotic agent is POMHEX or a POMHEX derivative.

28. A treatment kit comprising: a first antifibrotic agent and a second distinct antifibrotic agent in at least a first suitable container, wherein, The first anti-fibrotic agent is POMHEX or a POMHEX derivative.

29. The kit according to claim 28, wherein, The first and second different anti-fibrotic agents are contained in the first and second different containers.

30. Use of an effective amount of POMHEX or a POMHEX derivative in the preparation of a medicament for the treatment of fibrosis in subjects who require it.

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