RAC1 inhibition for treating liver failure

Abstract

A method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure "ALF" consisting of drug-induced ALF, immune-induced ALF, and infectious-induced ALF; and the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is ID-142, or a pharmaceutically acceptable salt thereof.

Description

[0001] Attorney Docket No. 148839.604511.PC

[0002] RAC1 INHIBITION FOR TREATING LIVER FAILURE CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This patent application claims the benefit of and the priority to U.S. Provisional Application No. 63 / 747,698, filed on January 21, 2025, and International Application No. PCT / IB2025 / 050614, filed on January 21, 2025, the disclosures of both of which are incorporated herein by reference in their entireties.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates to the treatment or prevention of liver diseases such as acute liver failure (ALF) and acute-on-chronic liver failure (ACLF). In particular, the present disclosure relates to the treatment or prevention of liver diseases, involving RAC1 inhibition.

[0006] BACKGROUND

[0007] Acute liver failure (ALF) and acute-on-chronic liver failure (ACLF) are life threatening diseases characterized by deterioration of liver function. ALF and ACLF may lead to multi -organ dysfunction and are associated with high mortality rates.

[0008] N-acetylcysteine (NAC) is the only treatment used clinically to ameliorate ALF. However, it is only partially useful and limited to cases of ALF induced by acetaminophen (APAP) overdose. There are no currently available successful treatments that specifically target ALF and ACLF. In this scenario, liver transplant remains the sole definitive treatment for the most severe cases of ALF and ACLF, and despite advancements, the prognosis remains unfavorable. Consequently, there is an urgent need for the development of novel therapeutic options to improve outcomes for these conditions.Attorney Docket No. 148839.604511.PC RAC1 (RAS-related C3 botulinum toxin substrate 1) is a member of the subfamily of RHO GTPases, which at the same time belongs to the RAS superfamily of GTP-binding proteins. The switch between the inactive GDP-bound and the active GTP-bound state of RAC1 is strictly regulated by a collection of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), whilst its function is modulated by a great variety of downstream effectors.

[0009] It is herein surprisingly shown that inhibition of RAC 1 would be useful for the treatment of ALF and ACLF.

[0010] SUMMARY

[0011] Embodiments of the present disclosure provide a method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF). Embodiments of the present disclosure provide a method of prolonging the survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor. Embodiments of the present disclosure provide a method of increasing the survival rate, or the likelihood of survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor.Attorney Docket No. 148839.604511.PC In some embodiments, the RAC1 inhibitor is selected from the group consisting of 1D-142, 1G-55, a pharmaceutically acceptable salt thereof, and any combination of the foregoing.

[0012] In some embodiments, the RAC1 inhibitor is the compound ID-142:

[0013]

[0014] or a pharmaceutically acceptable salt thereof. In some embodiments, the RAC1 inhibitor is ID- 142. In some embodiments, the RAC1 inhibitor is a pharmaceutically acceptable salt of ID- 142.

[0015] Embodiments of the present disclosure provide a method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF), wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof. Embodiments of the present disclosure provide a method of prolonging the survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof. Embodiments of the present disclosure provide a method of increasing the survival rate, or the likelihood of survival of a subject suffering from a liver disease selected from theAttorney Docket No. 148839.604511.PC group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, the RAC1 inhibitor is the compound 1G-55:

[0017]

[0018] 5

[0019] or a pharmaceutically acceptable salt thereof. In some embodiments, the RAC1 inhibitor is 1G-55. In some embodiments, the RAC1 inhibitor is a pharmaceutically acceptable salt of 1G-55.

[0020] Embodiments of the present disclosure provide a method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF), wherein the RAC1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof. Embodiments of the present disclosure provide a method of prolonging the survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof.Attorney Docket No. 148839.604511.PC Embodiments of the present disclosure provide a method of increasing the survival rate, or the likelihood of survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC 1 inhibitor, wherein the RAC 1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof.

[0021] Embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of a RAC 1 inhibitor and a pharmaceutically acceptable excipient, for use in a method as disclosed herein. Embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of ID- 142, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, for use in a method as disclosed herein. Embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of 1G-55, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, for use in a method as disclosed herein.

[0022] Embodiments of the present disclosure provide a therapeutically effective amount of a RAC1 inhibitor for use in a method as disclosed herein. Embodiments of the present disclosure provide a therapeutically effective amount of ID- 142, or a pharmaceutically acceptable salt thereof, for use in a method as disclosed herein. Embodiments of the present disclosure provide a therapeutically effective amount of 1G-55, or a pharmaceutically acceptable salt thereof, for use in a method as disclosed herein.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A represents a Volcano plot showing differentially expressed genes (DEGs) in livers from patients with hepatitis B virus (HBV)-related ALF (n = 4) compared to liversAttorney Docket No. 148839.604511.PC from healthy donors (n = 10, public microarray dataset GSE38941) and a Gene Ontology (GO) and pathway analysis of upregulated genes. Selected genes from each panel are annotated.

[0025] Figure IB represents HBV-ALF patient clusters obtained after a principal component analysis (PCA) and k-means cluster analysis. Cluster 1 is shown on the left hand side and Cluster 2 is shown on the right hand side of this plot. Each data point represents an individual sample. The two displayed components explain 75% of total point variability. Figure IB also represents a Volcano plot showing the differential expression in Log2 fold change of genes between Cluster 1 and Cluster 2, and a GO and pathway analysis of upregulated genes.

[0026] In Figure 1A and Figure IB, the Volcano plots highlight the upregulated (right hand side, Log2(FC) > 0.6, FDR <0.05) and downregulated (left hand side, Log2(FC) < -0.6, FDR <0.05) genes.

[0027] Figure 1C represents a Venn diagram of DEGs in HBV-ALF patients and genes that correlate with RAC1 expression. GO and pathway analysis was performed on genes downregulated and upregulated in patients that correlate negatively and positively to RAC1 respectively.

[0028] In Figures 1A-C, the number of genes (right axis, column bars) and q-value FDR by Benjamini and Hochberg’s procedure (B&H, left axis, dots) are shown. DEGs, differentially expressed genes; GO, gene ontology; FDR, false discovery rate.

[0029] Figure 2A represents a Volcano plot showing DEGs in circulating monocytes derived from patients with APAP -related ALF (n = 17) compared to healthy donors (n = 5, public microarray dataset GSE80751) and GO and pathway analysis of upregulated genes.Attorney Docket No. 148839.604511.PC Figure 2B represents a Volcano plot showing DEGs in livers from mice with APAP-related ALF (n = 4) compared to healthy controls (n =4, public microarray dataset GSE111828) and GO and pathway analysis of upregulated genes.

[0030] Figure 2C represents a Venn diagram of DEGs in monocytes and genes that correlate with RAC1 expression. GO and pathway analysis was performed on genes upregulated in monocytes that correlate positively to RAC 1.

[0031] In Figures 2A-C, Volcano plots highlight the upregulated (right hand side, Log2(FC) > 0.6, FDR <0.05) and downregulated (left hand side, Log2(FC) < -0.6, FDR <0.05) genes. Bar charts show number of genes (right axis, column bars) and q-value FDR by Benjamini and Hochberg’s procedure (B&H, left axis, dots). DEGs, differentially expressed genes; GO, gene ontology; FDR, false discovery rate.

[0032] Figure 3 shows the reduction in liver injury in mice with RAC1 inhibition using ID-142 in ALF models. Liver injury was assessed by (A) determining serum ALT / AST, and (B) histopathological analysis of haematoxylin and eosin-stained sections (H&E) (magnification: xlO). Representative microphotographs are shown. *p<0.05 and ** p<0.01 vs control (ttest).

[0033] Figure 4 shows the reduction of reactive oxygen species production in hepatocytes with RAC1 inhibition using ID-142. (A) Hepatocytes isolated from mice with ConA-induced ALF (n = 2 / group, dot plots are representative of one of two independent experiments). (B) HepG2 cells and (C) primary hepatocytes isolated from healthy mice were incubated during 4 hours with TAA or APAP in the presence or not of ID-142. In Figure 4B, bars represent the average +SEM of triplicates of one of two experiments. **p<0.01 vs control (ttest).Attorney Docket No. 148839.604511.PC Figure 5A shows IL- 1 p and TNFa blood serum levels measured by Elisa in mice 6h after ALF induction by ConA injection and treated with control (n = 6) or ID-142 (10 mg / kg, n = 6).

[0034] Figure 5B shows neutrophil (LY6G+), CD4 T cell (CD3+CD4+) and CD8 T cell (CD3+CD8+) composition determined by flow cytometry analysis.

[0035] Figure 5C shows IL- 1 and TNFa mRNA levels measured by qPCR.

[0036] In Figures 5B and 5C, non-parenchymal cell fraction was isolated from mice 4 hours after the induction of ALF by ConA inoculation (treated or not with ID- 142) by collagenase type I digestion followed by Histodenz gradient.

[0037] Figure 5D represents cell viability dose response curves for Jurkat cells treated with ID 142 and ConA over 3 days evaluated by standard MTT assay. Dose response curves were plotted using a non-linear regression model and are representative of three independent assays, each containing four replicates. Error is shown as SEM. Bar graphs show IC50 values of each independent assay.

[0038] Figure 5E represents immune infiltrate composition in liver estimated using MIXTURE in the GSE38941 cohort (HBV-related ALF patients n = 4, healthy donors n = 10). Bar plots on the left show the absolute cell abundance of CD8 T cells (CD8), activated memory CD4 T cells (CD4), gamma-delta T cells (y5), plasma cells (PC), resting dendritic cells (DC) and resting natural killer (NK) on liver samples. Graph on the right represents GO and pathway analysis of genes that correlate positively with liver levels of each immune population. Number of genes (right axis, column bars) and q-value FDR by Benjamini and Hochberg’s procedure (B&H, left axis, dots) are shown. DEGs, differentially expressed genes; GO, gene ontology; FDR, false discovery rate. *p<0.05, **p<0.01 and ***p<0.001 (Mann-Whitney).Attorney Docket No. 148839.604511.PC Figure 6A represents mRNA levels of IL- 10, ARG1 and iNOS in LPS-activated J774 macrophages treated with control or ID-142 (10 pM). Bars represent average + SEM. Data represents one of two independent assays, each containing 3 replicates. *p<0.05, **p<0.01 and ***p<0.001 (ANOVA).

[0039] Figure 6B shows mRNA levels of IL- 10, ARG1 and iNOS. Data represents the average of two independent assays, each containing 3 replicates.

[0040] Figure 6C shows NF-KB immunofluorescence in HepG2 cells when incubated with conditioned media (CM) of naive or LPS- stimulated J774 treated or not with ID-142 (10 pM). Data represents one of two experiments.

[0041] Figure 6D shows the results of an in vitro migration assay of J774 cells treated with control or ID- 142 (10 pM) for 6 hours towards CM derived from ConA treated mice livers. Data represents one of three independent assays.

[0042] Figure 6E represents cell viability dose response curves for J774 cells treated with ID 142 and ConA or LPS over 3 days evaluated by standard MTT assay. Dose response curves were plotted using a non-linear regression model. Data represents one of three independent assays, each containing four replicates (paired t test). Error is shown as SEM. Bar graphs show IC50 values of each independent assay.

[0043] In Figures 6B, 6C, and 6D, results are expressed as average ±SEM. *p<0.05, ** p<0.01 and ***p<0.001 vs control (ttest).

[0044] Figure 7A shows the result of a differential expression analysis. RNA-seq was performed on liver samples from ID- 142 (n = 3) or control (n = 3) treated mice sacrificed 3 hours after APAP or ConA administration. Bar charts display GO and pathway analysis of genes downregulated by ID- 142 treatment.Attorney Docket No. 148839.604511.PC Figure 7B represents a Venn diagram of DEGs modulated by ID-142 treatment and genes dysregulated in HBV-ALF patients. GO and pathway analysis was performed on genes that are downregulated by ID- 142 treatment and are upregulated in patients.

[0045] In Figures 7A and 7B, the number of genes (right axis, column bars) and Q-value FDR (B&H, left axis, dots) are shown.

[0046] Figure 7C represents a schematic representation of the PDE model. 2x2x2 mm3 explants from healthy human livers (Healthy PDEs, n = 5) or livers from patients with liver failure (LF PDEs, n = 6) were treated overnight with ID- 142 (10 pM).

[0047] Figure 7D shows transaminase levels determined in culture medium for Healthy PDEs (t test).

[0048] Figure 7E shows a reduction in ALT levels in CM (one-tailed ratio paired t test) for treatment of LF PDEs with ID- 142.

[0049] Figure 7F shows gene expression for cytokine profile in tissues assessed by qPCR for treatment of LF PDEs with ID-142 (t test).

[0050] In Figures 7D-7F, bars represent mean ± SEM. ns: not significant, *p<0.05 and ***p<0.001 vs control.

[0051] Figures 8A and 8C represent a GO and pathways analysis of (Figure 8A) Small GTPase signaling genes and of (Figure 8C) genes downregulated in HBV-ALF patients. DEGs were identified in livers from patients with HBV- (related ALF n = 4) compared to healthy donors (n = 10, public microarray dataset GSE38941). Bar charts show number of genes (right axis, column bars) and q-value FDR by Benjamini and Hochberg’s procedure (B&H, left axis, dots).Attorney Docket No. 148839.604511.PC Figure 8B shows mRNA expression in HBV-ALF patients of genes described to be upregulated after RAC1 activation. DEGs, differentially expressed genes; GO, gene ontology; FDR, false discovery rate.

[0052] Figure 9 represents a GO and pathways analysis of (A) downregulated genes and (B) Small GTPase signaling genes from APAP-ALF human PBMCs. DEGs were identified in circulating monocytes derived from patients with APAP-related ALF (n = 17) compared to healthy donors (n = 5, public microarray dataset GSE80751). Bar charts show number of genes (right axis, column bars) and q-value FDR by Benjamini and Hochberg’s procedure (B&H, left axis, dots). DEGs, differentially expressed genes; GO, gene ontology; FDR, false discovery rate.

[0053] Figure 10 represents a GO and pathways analysis of (A) downregulated genes and (B) Small GTPase signaling genes from APAP-ALF mice liver. DEGs were identified in livers from mice with APAP-related ALF (n = 4) compared to healthy controls (n =4, public microarray dataset GSE111828). Bar charts show number of genes (right axis, column bars) and q-value FDR by Benjamini and Hochberg’s procedure (B&H, left axis, dots). DEGs, differentially expressed genes; GO, gene ontology; FDR, false discovery rate.

[0054] Figure HA represents a GO and pathways analysis of genes upregulated in circulating monocytes of APAP-ALF patients that died or underwent liver transplantation (n = 6) compared to those who spontaneously survived (n =6). Bar charts show number of genes (right axis, column bars) and q-value FDR by Benjamini and Hochberg’s procedure (B&H, left axis, dots). GO, gene ontology; FDR, false discovery rate.

[0055] Figure 11B shows the mRNA expression levels of genes from the “Rho GTPase Effectors” term in the group of bad prognosis.Attorney Docket No. 148839.604511.PC Figure 12 shows the effect of RAC1 inhibition using ID-142 on the LPS / D-Gal model of ALF. Liver injury was assessed by (A) determining serum ALT / AST, and (B) histopathological analysis of haematoxylin and eosin-stained sections (H&E) (magnification: x 10). Representative microphotographs are shown. *p<0.05 vs control (t test).

[0056] Figure 13 represents liver neutrophil and macrophage infiltration in HBV-ALF patients. Immune infiltrate composition estimated using MIXTURE in the GSE38941 cohort. Bar plots show the absolute cell abundance neutrophils and Ml and M2 macrophages on liver samples, ns: not significant, *p<0.05 and ***p<0.001 (Mann-Whitney).

[0057] Figure 14 represents cell viability dose response curves for THP-1 cells treated with 1D-142 and ConA or LPS over three days evaluated by standard MTT assay. Plots were made using a non-linear regression model. Each independent assay contains four replicates. Bar graphs show average ±SEM of IC50 values. *p<0.05 vs control (ttest).

[0058] Figure 15 represents a differential gene expression analysis in APAP and ConA murine models. RNA-seq was performed on liver samples from ID-142 (n = 3) or control (n = 3) treated mice sacrificed 3 hours after APAP (A) or ConA (B) administration. Volcano plots show DEGs in treated mice compared to control group (Log2(FC) > 0.6, FDR <0.05).

[0059] Figure 16 represents individual cytokine profile for LF patients. The expression of TNFa, IL-ip, IL-6, and IL- 10 was measured by qPCR in liver explants derived from six individual LF patients treated overnight with ID- 142 [10 pM] . Data are presented as fold change (2A-AACt) relative to control for each patient. Bars represent mean ± SEM of triplicates. *p < 0.05, **p < 0.01, ***p < 0.001 (ttest).Attorney Docket No. 148839.604511.PC Figure 17 shows the reduction of liver injury with RAC1 inhibition using ID-142 in an ACLF model. Chronic liver diseases was developed by 10 weeks of thioacetamide (TAA) intraperitoneal administration (200 mg / kg dose, 3 doses at week). Then, mice were fasted for 16h and injected with LPS plus APAP to induce acute on chronic liver failure. The mice were treated with a single dose of control or ID-142 1 h after liver injury induction. Liver injury was assessed by determining serum ALT / AST. *p<0.05 vs control (ttest).

[0060] Figure 18A represents a scheme of acute liver failure mouse models used in the experiments involving treatment with 1G-55. The mice were inoculated with acetaminophen (APAP) or concanavalin A (ConA) and treated with 1G-55 at 60 and 30 minutes, respectively. The mice were euthanized 6 hours after induction of ALF, and ALT and AST levels were measured.

[0061] Figure 18B represents serum ALT and AST levels in mice with ALF treated with 1G-55. *p>0.05 vs. control (t-test).

[0062] Figure 19A represents a scheme of acute-on-chronic liver failure mouse models used in the experiments involving treatment with 1G-55. Chronic liver injury was induced by intraperitoneal administration of thioacetamide (TAA) for 10 weeks (doses of 200 mg / kg, 3 doses per week). Then, the mice were subjected to fasting for 16 hours and were injected with LPS and APAP to induce acute liver injury. The mice were treated with a single dose of control or 1G-55 one hour after induction of liver injury.

[0063] Figure 19B represents the assessment of liver injury determining the serum ALT levels. ***p<0.001 vs. mice treated with control prior to acute injury and **p<0.01 vs. mice treated with control after injury.

[0064] Figure 20 shows the effects of 1G-55 in explants derived from patients (PDEs). 8 mm3explants of healthy human livers (healthy PDEs) or acute liver failure patient livers (ALFAttorney Docket No. 148839.604511.PC PDEs) were treated overnight with 1G-55 (10 pM). Transaminase levels were determined in the culture medium. (A) The absence of increased ALT release in healthy PDEs after treatment with 1G-55 suggests a lack of evident hepatotoxicity. (B) Treatment with 1G-55 enhances ALT release induced by incubation with ConA in healthy PDEs. (C) Treatment with 1G-55 reduces ALT release in ALF PDEs. The bars represent average ±SEM.

[0065] Figure 21 shows the effects of RAC1 inhibition using ID-142 at 3 h post-induction. Mice fasted for 16 h were injected with APAP (600 mg / kg, i.p.) or ConA (30 mg / kg, i.v.) to induce ALF. A single dose of control or ID-142 (10 mg / kg, i.p.) was administered at 3 h post-induction. Treatment effects were evaluated by survival in the APAP model (Mantel-Cox test) and by ALT / AST levels at 6 h in ConA-treated mice. *p <0.05 and ** p <0.01 vs. control (t test). ALF, acute liver failure; ALT, alanine aminotransferase; APAP, acetaminophen; AST, aspartate aminotransferase; ConA, concanavalin A.

[0066] Figure 22 shows the effects of ID- 142 in explants derived from patients (PDEs). 8 mm3(2x2x2 mm3) explants of healthy human livers (healthy PDEs) were treated overnight with control, ConA, or a combination of ConA and ID- 142. ALT levels were determined in the culture medium. The bars represent average ±SEM. *p <0.05 (paired ANOVA).

[0067] DETAILED DESCRIPTION

[0068] The following sections elaborate on the specific features of this disclosure. Some definitions for terms used in the disclosure are provided, which should be understood within the context of the overall disclosure. Unless otherwise indicated, all technical terms used in this disclosure have the same meaning as commonly understood by a person ordinarily skilled in the art.Attorney Docket No. 148839.604511.PC All patents, patent applications, and non-patent publications cited herein are specifically incorporated by reference in their entireties. In case of conflict, the present disclosure, including its specific definitions, will control.

[0069] Broadly speaking, the present disclosure is related to the modulation of the RAC1 pathway, for example, using a RAC1 inhibitor, for treating or preventing acute liver failure (ALF) or acute -on-chronic liver failure (ACLF).

[0070] As used herein, the term “RAC1” has its general meaning in the art and refers to RAS-related C3 botulinum toxin substrate 1, which is a member of the subfamily of RHO GTPases, which at the same time belongs to the RAS superfamily of GTP-binding proteins. The term “RAC1” as used herein refers to RAC1 from eukaryotes, preferably mammals, more preferably from humans. The naturally occurring human RAC1 gene has a nucleotide sequence as shown in Genbank Accession number NM_006908.5 and the naturally occurring human RAC1 protein has an amino acid sequence of 192 amino acids as shown in GenBank database under accession number NP_008839.2. Also encompassed by the term “RAC1” is a RAC1 protein encoded by a nucleotide sequence that has 50%, 60%, 70%, 80%, 90% or 95% or more identity over its entire nucleotide sequence with the human RAC 1 gene . Also encompassed by the term “RAC 1 ” is a RAC 1 protein that has 50%, 60%, 70%, 80%, 90% or 95% or more identity over its entire amino acid sequence with the human RAC1 protein. As used herein, the term “RAC1” further refers to the human RAC1 protein but also to the orthologs of other species.

[0071] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.Attorney Docket No. 148839.604511.PC According to the present disclosure, unless otherwise indicated, it is to be understood that, when a list is presented, each individual element of that list, and every combination of that list, is a separate embodiment.

[0072] As used herein, the term “about” or “approximately” includes and describes the value or parameter per se. For example, “about x” includes and describes “x” per se. In some embodiments, the term “about” when used to modify a value refers to variations within ± 10% of said value.

[0073] The term “excipient” as used herein refers to a substance incorporated into a formulation or composition alongside an active agent to provide a specific benefit. This term encompasses both inert materials and functional additives that enhance the properties of the composition. Illustrative examples of excipients include, but are not restricted to, polymers, glidants, carbohydrates, lubricants, salts, buffering agents, lipids, fillers, disintegrants, binders, surfactants, high surface area substrates, flavoring agents, carriers, matrix components, and diluents.

[0074] The phrase “pharmaceutically acceptable” as used herein pertains to molecular entities and mixtures that are generally anticipated to be free of adverse, allergic, or unintended reactions upon administration to animals or humans. Regarding human use, such preparations must comply with sterility, pyrogenicity, and overall safety and purity criteria as mandated by FDA regulations.

[0075] The term “pharmaceutical composition” as used herein refers to a formulation or composition consisting of at least one active compound combined with one or more pharmaceutically acceptable excipients.

[0076] In the present disclosure, all methods and techniques are generally performed, unless otherwise mentioned, according to conventional methods well known in the art.Attorney Docket No. 148839.604511.PC As used herein, the terms “subject," “patient,” or “individual,” are used interchangeably and refers to a mammal, preferably a human.

[0077] The terms "treating," "treat," or "treatment" are used interchangeably and are recognized in the art and include both prophylactic and therapeutic treatments. As used herein, "prophylactic treatment", “preventing”, or “prevention” refers to treatments that take place prior to the clinical manifestation of an unwanted condition (e.g., a disease or other unwanted state) to protect the subject against its development or against the development of symptoms, or to limit or prevent the recurrence of the disease or its symptoms in subjects previously affected or suspected of developing the disease. "Therapeutic treatment" refers to administration after the manifestation of an unwanted condition to diminish, ameliorate, stabilize, or inhibit the worsening of the condition, its severity, duration, or associated side effects. Accordingly, "treating" encompasses accomplishing one or more of the following: reducing the severity or duration of a disease, inhibiting the progression of symptoms, and preventing the onset or recurrence of a disease or its characteristic symptoms in a subject. As used herein, "treating," "treat," or "treatment" include prolonging survival of the subject when compared to expected survival if the subject were not receiving treatment. As used herein, "treating," "treat," or "treatment" further include increasing the survival rate, or the likelihood of survival, of a subject as compared to the survival rate, or the likelihood of survival, of a subject not receiving treatment. Prolonging the survival of a subject and / or increasing the survival rate, or the likelihood of survival, of a subject in the context of ALF and / or ACLF is a significant feature as it may increase the likelihood of the subject to receive liver transplant.

[0078] The terms "therapeutically effective amount" and "effective amount" are used interchangeably herein and refer to an amount effective, at dosages and for periods of time necessary, to achieve a desired beneficial or therapeutic result. A therapeuticallyAttorney Docket No. 148839.604511.PC effective amount may vary according to factors such as the state of disease, severity of the disorder, previous treatments, and general health of the individual, as well as the ability of a therapeutic or a combination of therapeutics to elicit a desired response. Such an amount can be administered in one or more administrations, applications, or dosages, and can include a single treatment or a series of treatments (e.g., from one or more times per day to one or more times per week). An "effective amount" encompasses a "combination dose" administered in conjunction with another drug product, and may be the same as or different from a "prophylactically effective amount," which is an amount necessary for preventing a disease symptoms or protect against onset of disease or symptoms.

[0079] “Administering” or “administration of’ a RAC1 inhibitor, or a pharmaceutically acceptable salt thereof, to a subject can be carried out using one of a variety of methods known to a person skilled in the art. Appropriate methods and ways of administering an agent, compound, substance or molecule to a subject may also depend, for instance, on the features of the subject and the chemical and biological properties of the agent, compound, substance or molecule (e.g., solubility, digestibility, bioavailability, stability and toxicity). “Administering” or “administration of’ a RAC1 inhibitor, or a pharmaceutically acceptable salt thereof, to a subject may also include a combined treatment of the RAC1 inhibitor with one or more additional therapeutic agents. The one or more additional therapeutic agent may be another RAC1 inhibitor or any other therapeutic agent that can be administered to treat the liver disease, such as ALF and ACLF, or the cause or symptoms of the liver disease . In some embodiments, the combined treatment may be a combination therapy where the RAC1 inhibitor is administered concurrently with the one or more additional therapeutic agents, or where the RAC1Attorney Docket No. 148839.604511.PC inhibitor is administered at different dosage regimen as compared to the one or more additional therapeutic agents.

[0080] Embodiments of the present disclosure provide a method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF). Embodiments of the present disclosure provide a method of prolonging the survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor. Embodiments of the present disclosure provide a method of increasing the survival rate, or the likelihood of survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor.

[0081] In the context of the present disclosure, a "RAC1 inhibitor" is understood as an agent, such as a compound, substance, or molecule, or a treatment that decreases or blocks the activity of RAC 1, as well as any agent, compound, substance or molecule that is capable of reducing, preventing or blocking the expression of the gene encoding RAC1 (e.g. at transcriptional, post-transcriptional, translational and / or post-translational level), or that is capable of preventing the protein encoded by said gene from performing its function. Multiple Rac-1 inhibitors are well known in the art. In addition, a novel family of guanidine alpha-aminoacyl amide derivative compounds as RAC1 inhibitors is disclosedAttorney Docket No. 148839.604511.PC in the priority application PCT / IB2025 / 050614, incorporated herein by reference in its entirety.

[0082] In certain embodiments, a “RAC1 inhibitor” encompasses activity inhibitors. As used herein, an activity inhibitor of RAC1 or “RAC1 activity inhibitor” is a RAC1 inhibitor which interferes with or prevents the activity of RAC 1. An activity inhibitor may also be a RAC1 inhibitor which competes with a naturally occurring activator of RAC1 for interaction with the activation site on RAC 1. Alternatively, the activity inhibitor may bind to RAC1 at a site distinct from the activation binding site, for example, causing a conformational change in RAC 1. Alternatively, an activity inhibitor may interfere with a component upstream or downstream of RAC1 but which interferes with the activity of RAC1.

[0083] In certain embodiments, a RAC 1 activity inhibitor may interfere with the ability of RAC 1 to interact with a natural binder or ligand, such as a guanine nucleotide exchange factor (GEF). Non-limiting examples of GEFs are include T-cell lymphoma invasion and metastasis 1 (TIAM1), Vav guanine nucleotide exchange factor 1 (VAV1), Dedicator of cytokinesis 2 (D0CK2), and Rho guanine nucleotide exchange factor 6 (ARHGEF6), Guanine nucleotide exchange factor Hl (GEF-H1), but the skilled person would understand that there are many GEFs that interact with RACE Therefore, in certain embodiments, a “RAC1 inhibitor” encompasses “RAC 1 -GEF interaction inhibitors”, such as a “RAC1-TIAM1 interaction inhibitors”. Non-limiting examples of RAC1-TIAM1 interaction inhibitors are 1G-55 (see PCT / IB2025 / 050614), ID- 142 (see WO2013053726), or the compounds disclosed in RUFFONI, Alessandro, et al. MedChemComm, 2019, vol. 10, no 2, p. 310-314.

[0084] In certain embodiments, a “RAC1 inhibitor” encompasses an agent selected from the group consisting of anti-RACl antibodies (such as those described in Lawlor, M.A. et al.Attorney Docket No. 148839.604511.PC EMBO J. 2002 July 15; 21(14): 3728-3738; Collins B. J. etal EMBO J. 2003 August 15; 22(16): 4202-4211; Kharebava G. J. Neurosci. 2008 28:11409-11420; Nakamura K. et al. J. Biol. Chem 2008 283:17702-17711), microRNAs (such as those disclosed in Venugopal SK, et al. Am J Physiol Gastrointest Liver Physiol 298:G101- G106, 2010), iRNA (such as those disclosed in Monypenny J. et al., Mol. Cell. Biol. 2009 May 29(10):2730-2747), antisense oligonucleotides (such as those disclosed in Dorseuil O. et al 1992. J. Biol. Chem. Vol. 267 No. 29 15 pp. 20540-20542 1992; Jumey WM et al.

[0085] 2002 The Journal of Neuroscience, July 15, 22(14):6019- 6028), peptides (such as W56 orthose disclosed in Gao et al. 2001. J.Biol.Chem. vol. 27647530-47541), gene editors, small molecule compounds, or a combination thereof. In some embodiments, the RAC1 inhibitor is a small molecule compound. Non-limiting examples of small molecule compounds that are RAC1 inhibitors are NSC23760 and NSC 23766 and derivatives thereof (WO 2007 / 016539; Gao et al, 2004, PNAS; 101 :7618-7623), EHT 1864 and derivatives thereof (WO 2004 / 076445; Shutes et al, 2007, J Biol Chem; 282:35666-35678), GGTI-298 (N-[[4-(2-(R)-Amino-3-mercaptopropyl)amino]-2-naphthylbenzoyl] leucine methyl ester) and derivatives thereof (Sirangelo I. et al. J. Cell. Physiol. 221:412-423, 2009), lovastatine and derivatives thereof (Paintlia AS. et al. Mol Pharmacol. 2008 May; 73(5): 1381-1393), berberine and derivative thereof, azatioprine and derivatives thereof (Poppe D et al. 2006. J. Immunol. 176: 640-651), EHop-016 (MONTALVO-ORTIZ, Brenda L., et al. Journal of Biological Chemistry, 2012, vol. 287, no 16, p. 13228-13238), MBQ-167 (HUMPHRIES-BICKLEY, Tessa, et al., 2017, vol.

[0086] 16, no 5, p. 805-818), GYS32661 (AYAD, Nagi; JANGDE, Nitish. Neuro-Oncology, 2024, vol. 26, no Suppl 4, p. 0.; Molecular Cancer Therapeutics (2019), 18(5), 957-968), C20 (US2016317516), N4-(9-ethyl-9H-carbazol-3-yl)-N2-(3-morpholin-4-yl-propyl)-pyrimidine-2,4-diamine (U.S. Patent Application Publication No. 2013 / 0172552).Attorney Docket No. 148839.604511.PC Further non-limiting examples of small molecule compounds that are RAC1 inhibitors are disclosed in U.S. Patent Application Publication No. 2024 / 0067613, U.S. Patent Application Publication No. 2025 / 0346604, WO2010119050, Ferri et al. (J Med Chem 2009; 52(14):4087-90), Hernandez et al. (P R Health & / J2010; 29(4):348- 356). Further non-limiting examples of small molecule compounds that are RAC1 inhibitors are the compounds with the following chemical structures:

[0087]

[0088] Vlaar et al. Bioorganic & Medicinal Chemistry 26 (2018) 884-890),

[0089]

[0090] RUFFONI, Alessandro, et al. MedChemComm, 2019, vol. 10, no 2, p. 310-314.),Attorney Docket No. 148839.604511.PC

[0091]

[0092] (CARDAMA, Georgina A., et al. OncoTargets and therapy, 2014, p. 2021-2033.), and

[0093]

[0094] CARD AMA, Georgina A., et al. OncoTargets and therapy, 2014, p. 2021-2033).

[0095] Non-limiting examples of small molecule compounds that are RAC1 inhibitors are also disclosed in WO2013053726. For instance, the following compounds are disclosed in WO2013053726:

[0096] N -pyrimidin-2-yl-N '- [2-(trifluoromethyl)phenyl] guanidine ( 1 ) ;

[0097] N-(4-ethyl-6-methylpyrimidin-2-yl)-N'-[2-(trifluorornethyl)phenyl]guanidine (2);

[0098] N-(4-methyl-6-propylpyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (3); N-(4-isopropyl-6-methylpyrimidin-2-yl)-N'-[2-(trifluorornethyl)phenyl]guanidine (4); N-(4-butyl-6-methylpyrimidin-2-yl)-N'-[2-(trifluorornethyl)phenyl]guanidine (5);

[0099] N-(4-tert-butyl-6-methylpyrimidin-2-yl)-N'-[2-(trifluorornethyl)phenyl]guanidine (6); N-(4,6-diaminopyrimidin-2-yl)-N'-[2-(trifluorornethyl)phenyl]guanidine (7);Atorney Docket No. 148839.604511.PC N-(4,6-dichloropyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (8);

[0100] N-(4,6-difluoropyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (9);

[0101] N-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]-N'-[2-(trifluoromethyl)phenyl]guanidine (10);

[0102] N-(4-cyano-6-methylpyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (11), N-(5-methylpyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (12);

[0103] N-(4-chloro-6-methylpyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (13), N-(4-fluoro-6-methylpyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (14), N-(4-fluoropyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (15);

[0104] N-(5-fluoropyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (16);

[0105] N-[4,6-bis(trifluoromethyl)pyrimidin-2-yl]-N'-[2-(trifluoromethyl)phenyl]guanidine (17);

[0106] N-(4,6-dicyanopyrimidin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (18);

[0107] N-pyridin-2-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (19);

[0108] N-pyridin-3-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (20);

[0109] N-pyridin-4-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (21);

[0110] N-pyrimidin-4-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (22);

[0111] N-pyrimidin-5-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (23);

[0112] N-(4,6-dimethylpyridin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (24);

[0113] N-(3,5-dimethylphenyl)-N'-[2-(trifluoromethyl)phenyl]guanidine (25);Attorney Docket No. 148839.604511.PC N-(2,6-dimethylpyridin-4-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (26);

[0114] N -phenyl-N'- [2-(trifluoromethyl)phenyl]guanidine (27) ;

[0115] 2-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dimethylbenzenesulfonamide (28);

[0116] 2-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-diethylbenzenesulfonamide (29);

[0117] 2-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dipropylbenzenesulfonamide (30);

[0118] 2-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dibutylbenzene sulfonamide (31);

[0119] 3 - [(imino { [2-(trifluoromethyl)phenyl] amino } methyl)amino] -N,N -dimethylbenzenesulfonamide (32);

[0120] 3 - [(imino { [2-(trifluoromethyl)phenyl] amino } methyl)amino] -N,N -diethylbenzenesulfonamide (33);

[0121] 3 - [(imino { [2-(trifluoromethyl)phenyl] amino } methyl)amino] -N,N -dipropylbenzenesulfonamide (34);

[0122] 3 - [(imino { [2-(trifluoromethyl)phenyl] amino } methyl)amino] -N,N -dibutylbenzene sulfonamide (35);

[0123] 4-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dimethylbenzenesulfonamide (36);

[0124] 4-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-diethylbenzenesulfonamide (37);Atorney Docket No. 148839.604511.PC 4-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dipropylbenzenesulfonamide (38);

[0125] 4-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dibutylbenzene sulfonamide (39);

[0126] N-(2-nitrophenyl)-N'-[2-(trifluoromethyl)phenyl]guanidine (40);

[0127] N -(3 -nitrophenyl)-N'- [2-(trifluoromethyl)phenyl]guanidine (41);

[0128] N-(4-nitrophenyl)-N'-[2-(trifluoromethyl)phenyl]guanidine (42);

[0129] N-2-thienyl-N'-[2-(trifluoromethyl)phenyl]guanidine (43);

[0130] N-3-thienyl-N'-[2-(trifluoromethyl)phenyl]guanidine (44);

[0131] N-lH-pyrrol-2-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (45);

[0132] N-lH-pyrrol-3-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (46);

[0133] N-2-furyl-N'-[2-(trifluoromethyl)phenyl]guanidine (47);

[0134] N-3-furyl-N'-[2-(trifluoromethyl)phenyl]guanidine (48);

[0135] N-l,3-oxazol-2-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (49);

[0136] N-l,3-thiazol-2-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (50);

[0137] N-lH-imidazol-2-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (51);

[0138] N -isoxazol-5 -yl-N [2-(trifluoromethyl)phenyl] guanidine (52);

[0139] N-lH-benzimidazol-2-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (53);

[0140] N-(3,4-dimethylisoxazol-5-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (54);

[0141] N-(2-aminophenyl)-N'-(4,6-dimethylpyrimidin-2-yl)guanidine (55);Attorney Docket No. 148839.604511.PC N-(4,6-dimethylpyrimidin-2-yl)-N'-(3-ethylphenyl)guanidine (56);

[0142] l-(4-(4-amino-2-methylquinolin-7-ylamino)pyrimidin-2-yl)-3-(2-(trifluoromethyl)phenyl)guanidine (57);

[0143] N-(4-amino-2-methylquinolin-7-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (58); N-quinolin-7-yl-N'-[2-(trifluoromethyl)phenyl]guanidine (59);

[0144] N-methyl-N'-[2-(trifluoromethyl)phenyl]guanidine (60);

[0145] N-ethyl-N'-[2-(trifluoromethyl)phenyl]guanidine (61);

[0146] N-propyl-N'-[2-(trifluoromethyl)phenyl]guanidine (62);

[0147] N-butyl-N'-[2-(trifluoromethyl)phenyl]guanidine (63);

[0148] N-(2-methylphenyl)-N'-[2-(trifluoromethyl)phenyl]guanidine (64); and

[0149] N-[4,6-bis(methyl)pyrimidin-2-yl]-N'-[2-(trifluoromethyl)phenyl]guanidine (65).

[0150] In some embodiments, the RAC1 inhibitor is a small molecule compound disclosed as compound number 24 in WO2013053726, which is also referred to herein as “ID-142”. Non-limiting examples of small molecule compounds that are RAC1 inhibitors are also included in PCT / IB2025 / 050614, which encompass a novel family of guanidine alphaaminoacyl amide derivative compounds as RAC1 inhibitors. In some embodiments, the RAC1 inhibitor encompasses the following compounds that are included in PCT / IB2025 / 050614:Atorney Docket No. 148839.604511.PC

[0151]

[0152] Attorney Docket No. 148839.604511.PC

[0153]

[0154] In some embodiments, the RAC1 inhibitor is a small molecule compound disclosed as compound 1G-55 in PCT / IB2025 / 050614 and is also referred to herein as “1G-55”. Pharmaceutically acceptable salts, solvates, hydrates, deuterated derivatives, tautomers, enantiomers, stereoisomers, or other isomers of the RAC1 inhibitors mentioned above are also encompassed by some embodiments of the present disclosure.

[0155] As used herein, the term “pharmaceutically acceptable salt” refers to salts of any of the compounds described herein that within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals. Pharmaceutically acceptable salts are well known for a person skilled in the art. Non-limiting examples of pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66: 1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases.Attorney Docket No. 148839.604511.PC In some embodiments, the RAC1 inhibitor is selected from the group consisting of 1D-142, 1G-55, a pharmaceutically acceptable salt thereof, and any combination of the foregoing.

[0156] In some embodiments, the RAC1 inhibitor is the compound N-(4,6-dimethylpyridin-2-yl)-N'-[2-(trifluoromethyl)phenyl]guanidine (also referred in this disclosure as “1D-142”), which has the following chemical structure:

[0157]

[0158] or a pharmaceutically acceptable salt thereof. In some embodiments, the RAC1 inhibitor is ID- 142. In some embodiments, the RAC1 inhibitor is a pharmaceutically acceptable salt of ID- 142.

[0159] Embodiments of the present disclosure provide a method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF), wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof. Embodiments of the present disclosure provide a method of prolonging the survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof.Attorney Docket No. 148839.604511.PC Embodiments of the present disclosure provide a method of increasing the survival rate, or the likelihood of survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof.

[0160] In some embodiments, the RAC1 inhibitor is the compound N-(l-(2,5-dichlorophenyl)-2-oxo-2-(p-tolylamino)ethyl)-3 -(3 -(3 ,5 -dimethylphenyl)guanidino)-N-methylbenzamide (also referred in this disclosure as “1G-55”), which has the following chemical structure:

[0161]

[0162] or a pharmaceutically acceptable salt thereof. In some embodiments, the RAC1 inhibitor is 1G-55. In some embodiments, the RAC1 inhibitor is a pharmaceutically acceptable salt of 1G-55.

[0163] Embodiments of the present disclosure provide a method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF), wherein the RAC1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof.Attorney Docket No. 148839.604511.PC Embodiments of the present disclosure provide a method of prolonging the survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof. Embodiments of the present disclosure provide a method of increasing the survival rate, or the likelihood of survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC 1 inhibitor, wherein the RAC 1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof.

[0164] Embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of a RAC 1 inhibitor and a pharmaceutically acceptable excipient, for use in a method as disclosed herein. Embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the group consisting of ID- 142, 1G-55, a pharmaceutically acceptable salt thereof, or a combination of the foregoing, and a pharmaceutically acceptable excipient, for use in a method as disclosed herein. Embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of ID- 142, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, for use in a method as disclosed herein. Embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of 1G-55, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, for use in a method as disclosed herein.Attorney Docket No. 148839.604511.PC Embodiments of the present disclosure provide a therapeutically effective amount of a RAC1 inhibitor for use in a method as disclosed herein. Embodiments of the present disclosure provide a therapeutically effective amount of a compound selected from the group consisting of ID- 142, 1G-55, a pharmaceutically acceptable salt thereof, or a combination of the foregoing, for use in a method as disclosed herein. Embodiments of the present disclosure provide a therapeutically effective amount of ID- 142, or a pharmaceutically acceptable salt thereof, for use in a method as disclosed herein. Embodiments of the present disclosure provide a therapeutically effective amount of 1G-55, or a pharmaceutically acceptable salt thereof, for use in a method as disclosed herein. Embodiments of the present disclosure provide the use of a RAC1 inhibitor for preparing or manufacturing of a medicament for use in a method as disclosed herein. Embodiments of the present disclosure provide the use of a compound selected from the group consisting of ID- 142, 1G-55, a pharmaceutically acceptable salt thereof, or a combination of the foregoing, for preparing or manufacturing a medicament for use in a method as disclosed herein. Embodiments of the present disclosure provide the use of ID-142, or a pharmaceutically acceptable salt thereof, for preparing or manufacturing a medicament for use in a method as disclosed herein. Embodiments of the present disclosure provide the use of 1G-55, or a pharmaceutically acceptable salt thereof, for preparing or manufacturing a medicament for use in a method as disclosed herein.

[0165] In the methods and uses according to some embodiments of the present disclosure, the liver disease is selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF).

[0166] In some embodiments, the liver disease is ALF. As used herein, “acute liver failure” and “ALF” are used interchangeable and are defined as a liver disease characterized by a rapid reduction or loss of liver function. The reduction in liver function may occur a few days,Attorney Docket No. 148839.604511.PC a few weeks, or a few months after exposure to a causative factor or from the onset of a disease or condition. As used herein, the term “acute liver failure” or “ALF” includes, but is not limited to, the conditions referred to by the terms hyperacute liver failure (characterized as failure of the liver within one week), acute liver failure (characterized as the failure of the liver within 8-28 days), subacute liver failure (characterized as the failure of the liver within 4-12 weeks), and fulminant hepatic failure (FHF), which is characterized by a severe impairment of hepatic functions in the absence of pre-existing chronic liver disease. As used herein, ALF further includes all grades of the disease and any rapidly developing liver failure, regardless of the cause. ALF may be caused by several agents, diseases or conditions, such as exposure to drugs (such as acetaminophen, antibiotics, nonsteroidal anti-inflammatory drugs or anticonvulsants), toxins (such as mushroom toxins or carbon tetrachloride), some herbal supplements, autoinunune diseases and conditions (such as autoimmune-mediated hepatitis or transplant rejection), infectious diseases or sepsis, viral infections (such as hepatitis A, hepatitis B, hepatitis E, Epstein-Barr, cytomegalovirus, herpes simplex), metabolic diseases, pregnancy, vascular diseases (such as Budd-Chiari syndrome), cancer, or heat stroke. Other agents, diseases or conditions that have been reported to cause ALF will be known to a person skilled in the art. Non-limiting examples of ALF categorized by the cause include drug-induced ALF, immune-induced ALF, ALF induced by immune-mediated hepatitis, autoimmune-induced ALF, ALF induced by transplant rejection, toxin-induced ALF, alcohol-induced ALF, infectious-induced ALF, sepsis-induced ALF, bacterial-induced ALF, viral-induced ALF, heat-stroke induced ALF, hypoxic hepatitis or pregnancy-induced ALF, ALF induced by an inflammatory process, and any combination thereof. For example, in case of drug-induced ALF, acetaminophen (APAP) overdose is the most common causeAttorney Docket No. 148839.604511.PC of this type of ALF. However, there are multiple drugs that have been reported to lead to less common cases of ALF, which will be known for the person skilled in the art.

[0167] In some embodiments, the ALF is selected from the group consisting of drug-induced ALF, toxin-induced ALF, alcohol-induced ALF, immune-induced ALF, ALF induced by immune-mediated hepatitis, autoimmune -induced ALF, ALF induced by transplant rejection, infectious-induced ALF, sepsis-induced ALF, bacterial-induced ALF, viral-induced ALF, heat-stroke induced ALF, hypoxic hepatitis or pregnancy-induced ALF, ALF induced by an inflammatory process, and any combination thereof. In some embodiments, the ALF is selected from the group consisting of drug-induced ALF, immune-induced ALF, and infectious-induced ALF. In some embodiments, the ALF is drug-induced ALF, for example, the ALF may be acetaminophen (APAP)-induced ALF. Several insults or agents may lead to the development of ALF. In some embodiments, the RAC1 inhibitor is administered within a therapeutically effective time window after the insult or agent has been received by the subject. For example, the RAC1 inhibitor may be administered within about 48 hours; within about 36 hours; within about 24 hours; within about 22 hours; within about 20 hours; within about 18 hours; within about 16 hours; within about 14 hours; within about 12 hours; within about 6 hours; within about 3 hours; within about 1 hour; within about 30 minutes; or immediately after the insult or agent has been received by the subject.

[0168] In some embodiments, the subject to be treated has received an overdose of acetaminophen (APAP). APAP overdose is generally defined as the intake of APAP in an amount that exceeds the recommended maximum daily dose, resulting in a serum concentration that poses a risk of hepatotoxicity. The intake may be accidental or intentional and may be auto administered or not. In the context of this disclosure, APAP overdose encompasses acute intake (such as a single ingestion of about 150 mg / kg orAttorney Docket No. 148839.604511.PC more, which could be about. 7.5g to about 10g or more in adults) within a 24-hour period. In the context of this disclosure, APAP overdose also encompasses acute intake (such as a single ingestion of about 150 mg / kg or more, which could be about. 7.5g to about 10g or more in adults) within a 24-hour period. APAP overdose may also encompass mild overdose (such as the intake of about. 3.5g to about 7.5g or more in adults). In the context of this disclosure, APAP overdose also encompasses chronic / supratherapeutic intake (such as repeated intake of doses exceeding therapeutic recommendations, for example, a mild overdose, repeatedly).

[0169] In some embodiments, the RAC1 inhibitor is administered within a therapeutically effective time window after the overdose of APAP. For example, the RAC1 inhibitor may be administered within about 48 hours; within about 36 hours; within about 24 hours; within about 22 hours; within about 20 hours; within about 18 hours; within about 16 hours; within about 14 hours; within about 12 hours; within about 6 hours; within about 3 hours; within about 1 hour; within about 30 minutes; or immediately after the overdose of APAP has been received.

[0170] In some embodiments, the liver disease is ACLF. As used herein, acute-on-chronic liver failure" or "ACLF" refers to a liver disease characterized by an acute episode of liver failure in a subject with underlying chronic liver disease (with or without previously diagnosed cirrhosis), which is generally further complicated by one or more extrahepatic organ failures. For purposes of this disclosure, "ACLF" is defined broadly to encompass any diagnostic criteria recognized in the art, including but not limited to the EASL-CLIF (European Association for the Study of the Liver-Chronic Liver Failure) criteria, the APASL (Asian Pacific Association for the Study of the Liver) criteria, the NACSELD (North American Consortium for the Study of End-Stage Liver Disease) criteria, among others. As used herein, ACLF further includes all grades of the disease (e.g., ACLF GradeAttorney Docket No. 148839.604511.PC 1, 2, or 3) and any acute decompensation of chronic liver disease involving, for example, systemic inflammation and / or multi-organ dysfunction, regardless of the precipitating event (e.g., alcoholic hepatitis, infection, or idiopathic causes).

[0171] In some embodiments, the present disclosure provides methods of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF). In some embodiments, a subject may be determined to be in need of treatment based on one or more clinical, biochemical, functional, or prognostic parameters or markers, such as biomarkers, associated with a liver disease and / or with a risk of developing a liver disease, such as ALF and ACLF. The person skilled in the art would understand that there are multiple clinical, biochemical, functional, or prognostic parameters or markers associated with ALF and ACLF. In some embodiments, the subject is determined to be in need of treatment based on one or more clinical, biochemical, functional, or prognostic parameters or markers associated with liver injury. Non-limiting examples of parameters or markers associated with liver injury in ALF and / or ACLF may include clinical, biochemical, functional, and prognostic indicators of hepatic injury or dysfunction, including but not limited to elevations in serum aminotransferases, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST); increased lactate dehydrogenase (LDH); abnormalities in bilirubin metabolism, including increased total bilirubin and direct (conjugated) bilirubin; impairment of hepatic synthetic capacity, as evidenced by prolonged prothrombin time (PT), increased international normalized ratio (INR), reduced activity of coagulation factors, including factor V and / or factor VII, reduced serum albumin levels, reduced fibrinogen levels, and diminished serum cholinesterase activity; metabolic and biochemical derangements associated with hepaticAttorney Docket No. 148839.604511.PC failure, including hyperammonemia, hypoglycemia, lactic acidosis, and altered lactate clearance; and additional markers of hepatic injury, cholestasis, or functional reserve, including changes in alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), alpha-fetoprotein (AFP), and other parameters reflective of hepatocellular injury, impaired hepatic metabolism, or loss of hepatic functional capacity. Non-limiting examples of clinical parameters associated with ALF and / or ACLF may include the hepatic encephalopathy; coagulopathy or coagulopathy-related bleeding; jaundice; the presence, new onset, or worsening of ascites, each of which may be indicative of severe liver injury and may correlate with underlying biochemical or functional markers of liver injury. Notwithstanding the above, a subject may be determined to be in need of treatment without any symptom or diagnosis of liver injury. For instance, a subject that has an increased risk of developing ALF and / or ACLF may be determined as being in need of treatment.

[0172] In some embodiments, the RAC 1 inhibitor may be administered immediately or at any timepoint after the diagnosis of the liver disease. In some embodiments, the RAC1 inhibitor may be administered before the diagnosis if the liver disease, for example, when a subject is at risk of developing a liver disease, such as ALF or ACLF. In some embodiments, the RAC 1 inhibitor is administered immediately or at any timepoint after the onset of the liver disease. In some embodiments, the RAC1 inhibitor is administered before the onset of the liver disease. The onset of the liver disease may be determined by detecting any sign of liver injury such as one or more clinical, biochemical, functional, or prognostic parameters or markers, such as biomarkers, associated with a liver disease and / or with a risk of developing a liver disease, such as ALF and ACLF. As used herein, in the context of liver diseases, a “late treatment” or a treatment at a “later stage” is a treatment that begins after liver injury has been detected, and an “early treatment” or aAttorney Docket No. 148839.604511.PC treatment at an “early stage” is a treatment that begins before liver injury has been detected.

[0173] In some embodiments, the administration of the RAC1 inhibitor may reduce liver injury. Liver injury may be determined by multiple ways, including but not limited to serum alanine aminotransferase (AST) levels, serum aspartate aminotransferase (ALT levels), liver tissue damage, liver cell death, among other parameters known to the person skilled in the art.

[0174] In some embodiments, the RAC1 inhibitor may reduce liver injury, liver tissue damage, liver inflammation, immune cell infdtration, release of proinflammatory cytokines (such as IL-ip and TNFa), expression of pro-inflammatory genes, ROS formation, liver oxidative damage, liver oxidative stress, liver cell death, ALT release, AST release, or any combination of the foregoing. In some embodiments, the administration of the RAC1 inhibitor reduces the levels of serum alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST). The reduction of serum levels of ALT and / or AST may be relative to the respective levels prior to the administration of the RAC 1 inhibitor, or to an expected or reference value, or to a control subject that has not received the RAC1 inhibitor. As used herein, the term “reduces,” or other forms of the word, such as “reduce”, “reducing,” “reduction,” and “reduced” refers to a significant decrease, attenuation, mitigation, or suppression, of a measurable parameter, which may include, without limitation, a reduction relative to an untreated control, a baseline level, a reference level, an expected level, or a pre-treatment value. Such reduction may be evidenced by a decrease in absolute value, a relative or percentage decrease, a decrease in the rate of generation, production, accumulation, or progression, or a delay in onset or development of the measured parameter. The terms further encompass a reduction in peak levels, a shift in a distribution toward lower values, a decrease in severity or frequency,Attorney Docket No. 148839.604511.PC or a slowing of pathological processes, as determined by any qualitative or quantitative method known in the art.

[0175] In some embodiments, the subject to be treated is a mammal, including but not limited to, humans, non-human primates, horses, cows, dogs, cats, sheep, pigs, goats, rats, mice, rabbits, etc. In a preferred embodiment, the subject is a human.

[0176] The following Examples serve to illustrate the embodiments of the present disclosure and must not be considered as limiting the scope thereof.

[0177] EXAMPLES

[0178] The following general protocols and methods were used in the Examples unless otherwise mentioned:

[0179] Bioinformatics analysis

[0180] The microarray dataset GSE38941 ((https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE38941) was examined to explore RAC1 activation status in human ALF. The cohort consists of 4 patients with Hepatitis B virus (HBV)-Associated ALF (4-5 samples per patient) who underwent liver transplantation and 10 individual normal liver donors. Further, the role of RAC1 in circulating monocytes from patients with APAP -induced liver failure was evaluated using the microarray dataset GSE80751 (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE80751). The study contains data from peripheral monocyte samples from 5 healthy controls and 17 patients with ALF, with or without hepatic encephalopathy (n=12 and n=5, respectively). In addition, the RNA-seq dataset GSE111828 (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSEl 11828 ) of C57BL / 6J mice with hepatic injury was used to assess RAC1 activation in mice models of ALF. TheAttorney Docket No. 148839.604511.PC cohort comprises 4 healthy controls and 4 mice sacrificed 12 h after acetaminophen administration.

[0181] For microarray posterior analysis, data from multiple probes that target the same gene were combined into a gene expression index. For this, Robust Multi-array Averaging (RMA) was applied on normalized log2 expression data. Then, differentially expressed genes (DEGs) were identified using the Limma package (Log2(fold change)>|0.6|, FDR<0.05). For correlation analyses, Pearson correlation coefficients were calculated using log -transformed data. To identify HBV-ALF patient clusters, Principal Component Analysis (PCA) was applied for dimensionality reduction, followed by K-means cluster analysis. The results with FDR <0.05 were considered significant. Additionally, bulk transcriptomic data was deconvoluted applying MIXTURE to estimate the absolute quantification of immune cell types in patients with ALF. The ToppGene (ToppFun) was used to perform gene ontology and pathway analysis. All the above analyzes were performed using "R" software and graphs were generated with GraphPad prism software.

[0182] Animal models

[0183] This study was performed in 3 mouse models of ALF and a model of ACLF developed in male C57BL / 6J mice (six-to-eight weeks old), purchased from the Institute de Medicina Experimental (Consejo Nacional de Investigaciones Cientificas y Tecnicas -Academia Nacional de Medicina, Argentina). Animals were maintained at Animal Resources Facilities in accordance with institutional guidelines and conformed to requirement of the state authority for animal research conduct. All protocols dealing with animals were reviewed and approved by the Austral University Animal Studies Committee.

[0184] Randomization and control of confounding variables:Attorney Docket No. 148839.604511.PC Mice were randomly assigned to their respective experimental groups before the studies were started. Each cage used for the experiment contains an equal number of animals from both the control group and the treated group. All the procedures were performed by alternating between a control mouse and a treated mouse.

[0185] Blinding:

[0186] The experimenter was not blinded to the experimental group allocations. The inventors were aware of the group assignments, the execution of the experiment, and the data analysis.

[0187] Concanavcilin-A ALF model

[0188] Mice were fasted overnight and treated with Con-A [30 mg / kg] (Sigma-Aldrich) diluted in saline solution (0.9% NaCl) by a single tail vein injection. Treatment with ID-142 [10 mg / kg] or 1G-55 [19 mg / kg] was given intraperitoneally (i.p.) 30 min after Con-A injection, and mice were euthanized at the indicated time points.

[0189] Acetaminophen ALF model

[0190] APAP (Sigma-Aldrich) was dissolved in 70 °C warm PBS, incubated in a water bath at 60 °C for 30 min and i.p. injected at 55 °C into overnight fasted mice (600 mg / kg). One hour after APAP administration, a group of mice was administered ID- 142 [10 mg / kg], 1G-55 [19 mg / kg] or control by intraperitoneal route. All animals were euthanized at the indicated time points.

[0191] LPS-Galactosamine ALF model

[0192] D-galactosamine (D-GalN) was dissolved in saline and injected i.p. (500 mg / kg) in combination with LPS [25 pg / kg] into overnight fasted mice. ID-142 [10 mg / kg] wasAttorney Docket No. 148839.604511.PC administrated i.p. one hour after GalN injection. All animals were euthanized 6 hours after D-GalN+LPS administration.

[0193] ACLF model

[0194] Fibrosis was induced by the intraperitoneal (i.p.) administration of 0.2 mg / g of body weight of thioacetamide (TAA) (Sigma-Aldrich, Missouri, USA) 3 times per week for 10 weeks. The last dose of TAA was 0.4 mg / g of body weight, and 24 hours later APAP (600 mg / kg) and LPS (25 pg / kg) were injected by intraperitoneal route.

[0195] Detection of serum biochemical markers

[0196] At the end of in vivo experiments, mice were euthanized and blood samples collected for serum quantification of inflammatory markers. IL- 1 and TNFa levels were measured by ELISA, while alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were quantified using an Alinity instrument (Abbott).

[0197] Analysis of liver histopathology

[0198] Liver samples were fixed in 4% formalin for 48 h and embedded in paraffin to prepare 5 pm sections. Paraffin sections were deparaffinized and rehydrated, and then used for hematoxylin and eosin (H&E) staining. Stained liver specimens were evaluated by light microscopy at lOx.

[0199] Cell culture and viability assay

[0200] Murine J774 macrophages (ATCC), THP-1 human monocytes (ATCC) and Jurkat human lymphocytes (ATCC) were cultured in RPMI media, while HepG2 (ATCC) cells were maintained in DMEM media. Both culture media were supplemented with 2 pM glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS). Cell lines were confirmed to be mycoplasma-free using e-Myco kit (Boca Scientific). All cells were cultured at 37 °C in a 5% CO2 atmosphere. For cellAttorney Docket No. 148839.604511.PC viability assays, cells were plated at 500-2000 cells / well in 96 well plates and treated the next day with increasing doses of RAC 1 inhibitor ID- 142, in presence or absence of LPS [5 ng / ml] or ConA [15 pg / ml], Standard MTT assays were performed after 3 days of treatment. Dose-response curves were plotted using a non- linear regression model and IC50 values were determined from the fitted curves using GraphPad prism.

[0201] Isolation of primary hepatocytes, macrophages and nonparenchymal cells

[0202] Livers were perfused through the portal vein and digested with collagenase (Sigma-Aldrich,) for the extraction of hepatocytes and nonparenchymal cells (NPC), which include macrophages, LSECs, HSCs, lymphocytes, NK cells, DCs and neutrophils. Then, hepatocytes were isolated by a 30 min decantation followed by two steps of centrifugation (50 g for 10 min), and plated onto 6-well plates at a density of 250.000 living cells per well. Hepatocytes were grown in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) 10% FBS, with 2 mM glutamine, 100 pg / mL streptomycin, and 100 pg / mL penicillin. After 3 h, the medium was replaced with DMEM / F12 (70% DMEM + 30% Fl 2) 10% SFB. Nonparenchymal cell fraction was separated from the supernatant by a density gradient with Histodenz (Sigma- Aldrich) 30% in PBS and incubated for 40 min in a RPMI supplemented with 10% SFB to allow macrophages to adhere to the plastic. Supernatant was collected to obtain nonadherent cells (NPC), and adherent macrophages were maintained temporarily in RPMI supplemented with 10% SFB.

[0203] Measurement of intracellular ROS levels

[0204] Measurement of ROS production was performed by flow cytometry using 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA), a dye that diffuses into cells where is de-acetylated by intracellular esterases into dichlorodihydrofluorescein (DCFH). In the presence of intracellular H202 or low molecular-weight peroxides, DCFH oxidizes to the fluorescent derivate 2 ’,7 ’-dichlorofluorescein (DCF). Hence, fluorescence intensity isAttorney Docket No. 148839.604511.PC proportional to the intracellular level of ROS. For ex vivo assays, primary hepatocytes isolated as previously described were seeded in 6-well plates (250.000 cells per well) and then exposed to TAA [50 mM or 100 mM] or APAP [10 Mm or 25 mM] in the presence or absence of ID- 142 [10 pM] for 4h at 37°C. Next, cells were washed twice with PBS, detached with trypsin / EDTA and incubated for 30 min at 37°C with DCFH-DA (5 pM in PBS). For the assessment of in vivo ROS production, mice received ID-142 treatment (10 mg / kg, i.p.) or control 30 min after Con-A injection (30 mg / km, i.v.). Then, isolated hepatocytes were directly incubated with DCFH-DA (5 pM in PBS) for 30 min at 37°C. After the incubation period, extracellular residual DCFH-DA was washed away with PBS, and DCF fluorescence of cells was measured.

[0205] Flow cytometry

[0206] Liver nonparenchymal cells were isolated as previously described from mice treated with Con-A and ID- 142 or DMSO, and subsequently stained for flow cytometry. Antibodies against mCD3 (FITC; 555274), mCD4 (PeCy5; 553050), mCD8a (PeCy5; 553034) and mLy-6G (PE; 551461) were purchased from BD Biosciences. Cells were incubated with the corresponding combination of antibodies (CD3+ / CD4+ helper T cells (Th), CD3+ / CD8+ cytotoxic T lymphocytes and Ly6G+ neutrophils) for 25 min on ice in the dark, washed twice and analyzed on a BD Accuri™ C6 Plus flow cytometer. Data was analyzed with the BD Accuri C6 Software.

[0207] Quantitative real-time PCR

[0208] Total RNAs from J774, nonparenchymal cells, hepatic macrophages, and patient derived explants were extracted using TRIzol reagent (Sigma- Aldrich). cDNA was synthesized with 200 U of SuperScript II Reverse Transcriptase (Invitrogen) using random hexamers. mRNA levels were measured by qRT-PCR using SYBR Green (Invitrogen). PCR amplifications were carried out using a cycle of 95°C for 10 min and 45 cycles under theAttorney Docket No. 148839.604511.PC following parameters: 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 min. At the end of PCR reaction, the temperature was increased from 60°C to 95°C at a rate of 2°C / min, and the fluorescence was measured every 15 seconds to construct the melting curve. GAPDH and ACTB served as internal normalization controls. See Table 2 for the list of oligonucleotides utilized for qPCR. Changes in gene expression were calculated using the AAct method. A non-template control was run in every assay, and all determinations were performed as triplicates.

[0209] Table 2: oligonucleotides for quantitative real time PCR used in this study.

[0210]

[0211] NF-KB immunofluorescence staining

[0212] J774 cells were treated with ID-142 (50 pM) for 90 minutes and then incubated for 24 h with LPS [100 ng / ml]. To generate conditioned medium (CM), cell supernatant was collected and centrifuged at 2500xg for 10 min at 4 °C to remove cell debris. Next, HepG2 cells were incubated with CM for 30 min at 37 °C and fixed. NF-KB nuclear translocation was visualized by immunofluorescence staining with rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology). Secondary goat anti-rabbit FluoroLinkTM CyTM2Attorney Docket No. 148839.604511.PC antibodies were purchased from GE Healthcare Bio-Sciences. Nuclei were counterstained with DAPI.

[0213] Migration assay

[0214] In vitro migration was assessed using a 48-Transwell microchemotaxis Boyden Chamber unit (Neuroprobe). Cells (1.2 x 103 / well) were placed in the upper chamber in the presence or not of ID- 142 [10 pM], CM from Con-A treated mice livers were generated as described below and used as chemoattractant. Briefly, 6 h after ALF induction in mice as described above mice were euthanized and 2x2x2 mm3liver sections were cultured during 16 h in DMEM without SFB. Migration assay was carried out during 6h at 37°C in a 5% CO2 humidified atmosphere. Cells that migrated to the underside of the chamber were fixed with 2% formaldehyde, stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma Aldrich), and counted using fluorescent-field microscopy at 100X.

[0215] Transcriptome analysis

[0216] Animals from APAP and ConA models (n=3 / group) with representative transaminase levels were selected from each experimental group. RNA extraction from liver samples was performed using TRIzol protocol. RNA was precipitated by adding 0.1 volumes of a 3 M sodium acetate solution (NaOAc; pH 5.5) and 2 volumes of 100% ethanol. RNA-seq was performed using the Illumina NextSeq 500 platform (Macrogen). RNA-seq reads were mapped to mouse genome version GRCm39 and aligned using St. Jude Cloud RNAseq Standard v2 pipeline (https: / / github.com / stjudecloud / workflows). Differential expression was performed with DESeq2 package from R. Genes were considered differentially expressed when Log2(fold change) > |0.6| with a false discovery rate (FDR) < 0.05.Attorney Docket No. 148839.604511.PC Patient Derived-Explant assays

[0217] Liver tissue samples were obtained from healthy individuals and patients with liver failure of different etiologies at Austral University Hospital, Pilar, Buenos Aires, Argentina. The project was approved by the “Institutional Evaluation Committee” (CIE) from School of Biomedical Sciences and written informed consent was obtained from all patients. Healthy liver explants (healthy PDEs) and liver failure explants (LF PDEs) were obtained from liver tissue discarded during liver transplant procedures for patients with liver failure. Explants were cut into 2x2x2 mm3sections and treated overnight with ID-142 [10 pM], 1G-55 (10 pM), or control at 37°C in DMEM culture medium in a 5% CO2 humidified atmosphere. For the ConA treatment, explants were incubated overnight with ConA 15 pg / ml. Conditioned media and tissues samples were obtained for qPCR analysis and ALT determination.

[0218] Statistical analyses

[0219] Data are reported as arithmetic means ± SEM. Statistical analysis was performed using ‘R’ software and PRISM software (GraphPad). Statistical analyses were performed using t-test, Mann-Whitney test, ANOVA or Kruskal-Wallis according to data distribution. Data distribution was analyzed by D’Agostino-Pearson omnibus normality test. Values with associated p<0.05 and FDR <0.05 were considered as statistically significant.

[0220] Example 1: Transcriptomic analysis of both human and murine samples of acute liver failure (ALF).

[0221] The potential activation of RAC 1 pathway was explored in patients with ALF to evaluate its role in mediating reactive oxygen species (ROS) production and inflammation. To address this, the GSE38941 microarray dataset consisting of transcriptomic data of liver samples from patients with hepatitis B virus-related ALF (HBV-ALF) was analyzed. First, a Gene Ontology (GO) and pathway analysis was performed on genes that areAttorney Docket No. 148839.604511.PC overexpressed in HVB-ALF compared to healthy livers, which showed alterations in inflammatory processes, blood coagulation, and response to oxidative stress observed in the disease. In addition, this analysis also revealed an enrichment of genes involved in the Rho GTPase signaling pathways (Figure 1A). Moreover, GO analysis of “Small GTPase mediated signaling transduction” genes confirmed an enrichment in genes directly associated with RAC1 pathway (Figure 8A). These included several RAC1 guanine nucleotide exchange factors (GEFs) such as Tiaml, Vavl, Dock2, and Arhgef6, as well as numerous RAC1 effectors including PAK1, Wasf2, IQGAP1, among others. On the other hand, a similar analysis on genes that are downregulated in livers from HVB-ALF patients shows terms related with physiological liver process (Figure 8C). Then, to evaluate whether the activation of RAC1 pathway was associated with the extent of patient’s liver injury, a dimensionality reduction of gene expression data followed by a GO analysis was performed. Said analysis showed the segregation of HBV-ALF patients into two distinct clusters (Figure IB). Cluster 1 (patients #31 and #241) not only overexpresses terms related with liver injury, such as “Cytokine production”, “Leukocyte mediated cytotoxicity”, “Apoptotic signaling pathway” and “Blood coagulation”, but also genes associated with RAC1 when compared to patients in cluster 2 (patients #32 and #219) (Figure IB).

[0222] In order to identify biological processes altered in ALF potentially associated with RAC1 activation, an overlap analysis between previously identified DEGs and genes whose expression correlates with RAC1 mRNA levels was performed. GO analysis of genes that are negatively correlated with RAC1 and downregulated in HBV-ALF patients revealed an enrichment in GO categories associated with liver physiological metabolic processes (Figure 1C). This suggests that RAC1 activation could contribute to liver function damage, as revealed by dysregulation of processes such as detoxification, secretion,Attorney Docket No. 148839.604511.PC proteolysis and lipid metabolism. On the other hand, enrichment analysis of genes that show a positive correlation with RAC1 and that are upregulated in HBV-ALF patients indicated the alteration of biological processes closely associated to the pathophysiology of ALF, such as immune system activation, cell migration, NFkB and MAPK signaling and apoptosis. This subset of genes also yielded enrichment in terms associated with Rho GTPase pathways, such as “RAC1 GTPase cycle”. Meanwhile, terms related to oxidative stress appear in both subsets of genes. The antioxidant genes SOD1, CAT, PRDX4, MSRA, GCLC, and MGST1 are downregulated in patients and exhibit a negative correlation with RAC1. Together, these results suggest a link between RAC1 activation and inflammation and ROS accumulation in the context of ALF.

[0223] During ALF, the release of soluble mediators induces phenotypic and functional alterations in circulating monocytes, consequently prompting their recruitment to the liver. Upon migration to the liver, monocytes undergo differentiation into macrophages, a process pivotal in both the initiation and resolution phases of hepatocellular damage. In order to evaluate whether circulating monocytes derived from ALF patients exhibited an increased activation of RAC 1 signaling pathway, a DEG analysis was conducted on the GSE80751 transcriptomic database comprising peripheral monocytes from patients who developed ALF as consequence of paracetamol overdose (APAP-ALF patients). GO and pathway analysis of upregulated genes revealed overrepresentation of terms related to RHO GTPase signaling pathways (Figure 2A), which in turn are enriched in RAC1 GTPase cycle-associated genes (Figure 9A). Moreover, genes from this subset that are also positively correlated with RAC 1 expression are enriched in immune-related terms, such as “Myeloid cell activation involved in immune response”, “Leukocyte degranulation”, “Chemokine signaling pathway”, “TNF-alpha signaling pathway”, and “NIK / NF-kappaB signaling”, as well as in terms associated with Rho GTPase pathwaysAttorney Docket No. 148839.604511.PC (Figure 2C). This suggests that RAC1 activation may potentially intensify monocyte activation and infiltration into the injured liver. On the other hand, GO analysis of downregulated genes revealed an enrichment in terms that may indicate a phenotype of monocyte exhaustion or dysregulation (Figure 9B). Consistently, it was observed that genes from the RHO GTPase signaling, previously found to be enriched in ALF monocytes (Figure 2A) are overexpressed in patients who either died or underwent liver transplantation, compared to those who recovered spontaneously (Figure 11). Then, further analyses were performed to evaluate whether mouse models of ALF could successfully replicate the activation of RAC1 signaling pathways observed in patients. Analysis of transcriptomic data from C57BL / 6J mice with acetaminophen-induced hepatic injury (APAP-ALF mice) from GSE111828 dataset consistently demonstrated a significant enrichment in Rho GTPase-mediated signaling pathways among genes that are upregulated in APAP-ALF mice (Figure 2B). Specifically, GO analysis of genes from those terms revealed an enrichment in genes related to RAC1 pathway (Figure 10A). Conversely, as seen in the livers from HVB-ALF patients, analysis of downregulated genes suggests an impaired hepatic function (Figure 10A). Together, these findings support the notion that RAC 1 activation is indeed a relevant feature in ALF in both human patients and murine models, and thus underscore the potential of targeting RAC1 as a therapeutic approach in the management of ALF.

[0224] Example 2: RAC1 inhibition using ID-142 reduces liver injury in mouse models of ALF.

[0225] To evaluate the effect of RAC 1 inhibition on the evolution of ALF in mice, the compound 1 D- 142, an inhibitor of the interaction between RAC 1 and its GEFs, was used across three different murine models of ALF. ALF was induced with acetaminophen (APAP) or Concanavalin A (ConA) to cause oxidative liver damage and acute immune-mediatedAttorney Docket No. 148839.604511.PC hepatitis, respectively, and using a D-GalN / LPS-induced model of ALF, which combines direct liver injury with immune-mediated injury.

[0226] Parts of the results are shown in Figure 3. For the experiment of Figure 3, the following protocol was used. After fasting for 16 h, mice received either APAP (600 mg / kg. i.p.) or ConA (30 mg / kg i.v.) to induce ALF. Mice were treated with a single dose of control or ID-142 (10 mg / kg). ID-142 was administered 1 h (APAP model) or 30 min (ConA model) after liver injury induction. Mice were euthanized after 3 h (APAP model: n = 8 per group; ConA model: n = 11 per group) or 6 h (APAP model: control n = 21, ID- 142 n = 19; ConA model: control n = 9, ID-142 n = 10).

[0227] The results are also partially shown in Figure 12. For the experiment of Figure 12, the following protocol was used. After overnight fasting, mice received an intraperitoneal administration of D-GalN (500 mg / kg i.p) together with LPS (25 pg / kg) to induce ALF. Mice were treated with a single dose of control (n= 4) or ID- 142 (10 mg / kg, n = 4) one hour after liver injury induction and euthanized after 6 h.

[0228] Administration of ID- 142 resulted in a notable reduction in the severity of liver injury across all tested models. This was evidenced by a significant reduction in serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Figure 3A and 12A). Additionally, treated mice exhibited a remarkable preservation of liver structure, characterized by visibly reduced areas of focal hepatocyte death primarily confined to centrilobular regions (Figure 3B and 12B). This contrasts with the widespread necrosis observed in the untreated group. Moreover, treatment with ID- 142 alleviated inflammatory cell infiltration and congestion in the liver.Attorney Docket No. 148839.604511.PC Example 3: RAC1 inhibition using ID-142 reduces ROS formation and oxidative damage in hepatocytes during ALF.

[0229] Oxidative stress has been associated with both the onset and progression of ALF. Prior reports indicate that RAC1 is involved in the production of reactive oxygen species (ROS). In consequence, the inventors posed the hypothesis that RAC1 inhibition, for example, using ID-142, could reduce ROS formation and oxidative damage during ALF. To test this hypothesis, ROS levels were assessed in hepatocytes isolated from mice with ConA-induced ALF, both untreated and treated with ID-142.

[0230] In particular, ID- 142 (10 mg / kg or 10 pM for in vivo and in vitro, respectively) effect on ROS production was measured by DCFH-DA staining followed by flow cytometry analysis. The results are shown in Figure 4. Remarkably, hepatocytes from treated mice exhibited significantly lower levels of ROS intracellular accumulation compared to control (27.69% vs. 49.58% DCF positive cells) (Figure 4A). This suggests that the RAC1 inhibition after administration of ID-142 strongly mitigates oxidative damage in hepatocytes during ALF. These results were confirmed in HepG2 cells. DCFH-DA staining revealed that ROS intracellular formation dramatically increased in APAP- and thioacetamide (TAA)-treated cells but was reduced when co-incubated with ID- 142 (Figure 4B). Even more, primary mouse hepatocytes were used to validate ID-142 the antioxidant effect observed in HepG2 cells (Figure 4C). Taken together, these findings collectively demonstrate that RAC1 inhibition using ID-142 exerts a direct protective effect on hepatocytes by reducing ROS formation and oxidative damage.

[0231] Example 4: Local and systemic inflammatory response is reduced by RAC1 inhibition using ID- 142 in ALF.Attorney Docket No. 148839.604511.PC To evaluate the immunomodulatory properties of RAC 1 inhibition within the context of ConA-induced ALF, the inflammatory profde in mice was characterized. Remarkably, RAC1 inhibition after treatment with ID- 142 resulted in a significant reduction of the inflammatory response evidenced by a reduction in circulating IL-ip and TNFa proinflammatory cytokines in ConA-ALF mice (Figure 5A). The immune cell infiltrate (LY6G+ neutrophils, CD4 T and CD8 T cells) and mRNA levels of IL- 1 p and TNFa was further examined in the non-parenchymal hepatic fraction (Figures 5B-5C). ConA induces a pronounced recruitment of the three populations (6) that was impaired by 1D-142 treatment (Figure 5B). Additionally, ID- 142 also induced a significant reduction in the expression levels of IL- i and TNFa in non-parenchymal liver cells (Figure 5C). Deconvolution of publicly available datasets were evaluated using MIXTURE to estimate the abundance of liver immune infiltration in HBV-ALF patients. These patients showed an increase in activated memory CD4 and CD8 T cells, gamma-delta T cells (y5), plasma cells (PCs), resting natural killer cells (NK) and resting dendritic cells (DCs) when compared to healthy donors (Figure 5E), as well as in macrophages (Figure 13). A similar trend was observed for neutrophils, although it did not reach statistical significance (Figure 13). Next, genes correlated positively with the levels of these populations followed by a GO and pathway analysis were identified (Figure 5E), and an enrichment in signaling mediated by RHO GTPases (and specifically by RAC1 in CD8 and PCs related genes) was observed, suggesting that RAC1 may be involved in the hepatic inflammatory response developed in HBV-ALF patients. This result reinforces the potential of RAC1 inhibition (for example, using ID-142) for ALF therapy.

[0232] Example 5: Effect of RAC 1 inhibition using ID-142 on macrophages.

[0233] The enrichment in gene ontology (GO) terms associated with Rho GTPase signaling pathways observed in human monocytes suggests the potential impact of RAC 1 inhibitionAttorney Docket No. 148839.604511.PC on the functional profile of hepatic macrophages, thereby influencing their role in the progression of the disease. To test this hypothesis, in vitro experiments were conducted using naive or LPS-activated J774 macrophages. As shown in Figure 6A, treatment with ID- 142 induced a phenotypic switch towards an anti-inflammatory and tissue-protective profile, as evidenced by the reduction of Ml and M2 markers (IL-10, ARG1, iNOS). These findings were further validated by experiments conducted on isolated hepatic macrophages from mice with ConA-induced ALF that were treated with ID- 142. In particular, mice were challenged with ConA (30 mg / kg, i.v.) and treated with control (n = 3) or ID-142 (10 mg / kg, n = 3) one hour after injection. After 3 h, livers were perfused in situ, hepatic macrophages (hMo) were isolated and pooled. The results are shown in Figure 6B.

[0234] Within hepatocytes, NF-KB plays a dual role, mediating both inflammatory and anti-apoptotic responses. In unstimulated conditions, NF-KB remains inactive in the cytoplasm of hepatocytes, where it interacts with IKB inhibitors and different pathogen-associated molecular patterns (PAMPs), such as LPS and proinflammatory cytokines, can trigger their activation and nuclear translocation. This defensive mechanism shields hepatocytes from the detrimental impact of inflammatory mediators like TNFa and IL-ip while ensuring a balanced immune response. In the context of ALF, a significant proportion of these cytokines are released by activated macrophages during the initiation of inflammation. Therefore, the effect of RAC1 inhibition by ID-142 on the functional profile of J774 was evaluated by evaluating the impact of their supernatant on the activation of NF-KB in HepG2 hepatocytes. The results demonstrate that NF-KB nuclear internalization is strongly induced after incubation with the supernatant of LPS-activated J774, but this effect is significantly abrogated when J744 cells are pre-incubated with a RAC1 inhibitor such as ID- 142 (Figure 6C). This suggests that RAC1 inhibition mayAttorney Docket No. 148839.604511.PC modify the cytokine secretion profile in macrophages, preventing the generation of inflammatory mediators that could induce the activation of NF-KB signaling pathway in hepatocytes.

[0235] Furthermore, the effects of RAC1 inhibition by ID-142 on cellular processes related to macrophage expansion and recruitment was explored. ID-142 treatment not only significantly reduced J774 cell migration (Figure 6D) but also exhibited selective effects on cell survival of ConA or LPS-activated J774 (Figure 6E) and THP-1 (Figure 14) cells compared to control cells. Together, these data suggests that RAC1 inhibition by ID-142 treatment in vivo might reduce the hepatic macrophage pool by attenuating monocyte infiltration and reducing the viability of activated macrophages. These findings provide evidence that pharmacological inhibition of RAC1 leads to a significant reduction in immune cell recruitment and induces a shift in macrophages towards an antiinflammatory and pro-regenerative phenotype.

[0236] Example 6: RAC1 inhibition using ID-142 in mice normalizes transcriptional programs found dysregulated in patients with HBV-associated ALF.

[0237] To characterize the molecular mechanisms underlying the protective effect of RAC1 inhibition in the APAP- and ConA-ALF models, a global gene expression profiling of bulk liver samples from treated and untreated mice was conducted by RNAseq. First, a differential expression analysis was performed to identify genes that exhibit a negative modulation in response to RAC1 inhibition by ID-142 (Figure 7A and Figure 15). In addition, the genes modulated by ID-142 in the ConA model were overlapped with the DEGs previously identified in HBV-ALF patients (Figure 7B). This revealed genes overexpressed in HBV-ALF patients that are downregulated by ID-142. Notably, GO analysis of both the downregulated genes from APAP and ConA models, and the overlapping genes between mouse and patient data revealed their involvement in the RHOAttorney Docket No. 148839.604511.PC GTPases pathway. This indicates the potential of RAC 1 inhibition, for example, by 1D-142 treatment, to modulate the RAC 1 -related transcriptional program identified in patients. Moreover, the enrichment in immune related terms such as “Inflammatory response”, “Positive regulation of cytokine production”, “Response to cytokine”, “Leukocyte activation” and “Regulation of macrophage activation” underscores the pivotal role of RAC1 in immune response and suggests that RAC1 inhibition, for example, by ID- 142 could be effective in attenuating the ongoing inflammation in ALF patients. Similarly, the enrichment in terms like “Reactive oxygen species metabolic process” and “Response to oxidative stress” provides additional insights for the protective effect of RAC 1 inhibition against oxidative stress-induced tissue damage. Furthermore, GO terms related like “Positive regulation of apoptotic process” and “Stress-activated MAPK cascade” were also enriched. This is in line with the reduction in liver injury observed in ID- 142 treated mice and suggests that this hepatoprotective activity could be translated to patients. To note, most of these processes were previously identified for the RAC 1 -positively correlated - upregulated in HBV-ALF genes (Figure 1C), highlighting the potential of these findings for future clinical translation.

[0238] Finally, patient-derived explants (PDEs) were used as a model closely resembling clinical conditions to further demonstrate the therapeutic potential of RAC 1 inhibition (Figure 7C). Explants (2x2x2 mm3) from healthy human livers (healthy PDEs) showed no signs of injury after treatment with ID-142, as indicated by unaltered ALT levels (Figure 7D). Notably, overnight treatment with ID-142 of liver explants from patients with liver failure (LF PDEs) significantly reduced ALT release compared to untreated paired samples (Figure 7E). Additionally, ID-142 significantly increased mRNA levels of IL-10, IL-1 P and IL-6, while reducing TNFa expression (Figure 7F). This cytokine patterns could indicate a complex modulation of inflammatory process toward resolution and liverAttorney Docket No. 148839.604511.PC regeneration, driven by the anti-inflammatory role of IL- 10, the regenerative properties of IL-6, and the suppression of TNF-a, which may help limit excessive tissue damage. Moreover, despite its pro-inflammatory role, Il lb signaling has been reported to be essential for normal tissue regeneration. These combined preclinical and ex vivo results highlight the therapeutic potential of RAC1 inhibition, for example, using ID-142, for clinical management of ALF.

[0239] Example 7: RAC1 inhibition using ID-142 reduces liver injury in mouse models of ACLF.

[0240] To assess the effects of ID-142 on acute-on-chronic liver failure (ACLF), a mouse model with underlying fibrosis was used. These mice were subjected to a combination of acetaminophen (APAP) and lipopolysaccharide (LPS) to induce liver injury involving both oxidative stress and inflammation, characteristic of ACLF.

[0241] Consistent with the results observed in the ALF models, treatment with ID- 142 resulted in significantly lower levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating reduced liver injury (Figure 17).

[0242] Example 8: Late treatment effects ofRACl inhibition using ID-142.

[0243] Mice fasted for 16 h were injected with APAP (600 mg / kg, i.p.) or ConA (30 mg / kg, i.v.) to induce ALF. A single dose of control or ID-142 (10 mg / kg, i.p.) was administered at 3 h post-induction. Treatment effects were evaluated by survival in the APAP model (Mantel-Cox test) and by ALT / AST levels at 6 h in ConA-treated mice.

[0244] Crucially, therapeutic benefits were also observed when a RAC1 inhibitor, such as 1D- 142, was administered 3 hours post-induction, supporting the surprising beneficial effect of a late treatment with a RAC 1 inhibitor. In the ConA model, late treatment significantlyAttorney Docket No. 148839.604511.PC lowered ALT levels (Figure 21). In the APAP model, the late treatment extended survival, as shown in Figure 21.

[0245] Example 9. Preparation of N-(l-(2,5-dichlorophenyl)-2-oxo-2-(p-tolylamino)ethyl)-3-(3-(3,5-dimethylphenyl)guanidino)-N-methylbenzamide (1G-55)

[0246] The compound N-( 1 -(2,5 -dichlorophenyl)-2-oxo-2-(p-tolylamino)ethyl)-3 -(3 -(3 ,5 -dimethylphenyl)guanidino)-N-methylbenzamide (1G-55) is a RAC1 inhibitor disclosed in International Application No. PCT / IB2025 / 050614, incorporated herein by reference in its entirety. The process for the preparation of 1G-55 is disclosed in PCT / IB2025 / 050614 and is summarized below:

[0247] All chemical compounds were acquired from commercial sources or were synthesized. The solvents used were of analytical grade or higher.

[0248] Use of anhydrous solvents and moisture-sensitive reagents: the solvents used were of analytical grade or higher. THF was distilled with sodium -benzophenone prior to use, and CH2CI2 was refluxed with P2O5, prior to distillation and storage on molecular sieves (4 A). All reactions involving moisture-sensitive solvents or reagents were carried out in dry glassware and under nitrogen or argon atmosphere.

[0249] Thin layer chromatography: silica gel F2540,2 mm thick aluminum sheets (Merck) were used. The solvent system used (ascending method) are indicated in each case. The development was carried out by: (a) exposure to ultraviolet light (X = 254 nm); (b) immersion in Mo7O24(NH4)e 0.04 M, Ce(SC>4)2 3 mM in solution H2SO4-H2O (9:1) followed by heating; (c) immersion in ninhydrin 0.1% w / v solution in ethanol followed by heating; (d) exposure to molecular iodine.

[0250] Column chromatography: it was carried out with silica gel 60 230-400 mesh (Merck). Eluting solvents are indicated in each case.Attorney Docket No. 148839.604511.PC Flash chromatography: it was carried out in a CombiFlash Teledyne Isco instrument with disposable RediSep silica gel columns. Solvents and elution conditions are indicated in each case.

[0251] High resolution liquid chromatography: it was carried out in a Waters Alliance 2695 instrument equipped with a photodiode array (PDA) detector.

[0252] Melting points: these were determined with an Electrothermal IA9000series apparatus and are not corrected.

[0253] Nuclear magnetic resonance: nuclear magnetic resonance spectra were recorded with Bruker Fourier 300 (operating at 300 MHz for 'H and 75 MHz for13C), Avance DPX 400 (operating at 400 MHz for 'H and 100 MHz for13C) spectrometers at 303 K. The following deuterated solvents were used, as indicated in each case: CDCh, CD3OD, DMSO-d6. Chemical shifts are indicated in parts per million (ppm) relative to tetramethylsilane (TMS) signal. Coupling constants are expressed in Hz. Signals are described as s (singlet), d (doublet), t (triplet), q (quartet), quint, (quintet), dd (double doublet), ddd (double double doublet), dt (double triplet), tt (triple triplet), dist. t (distorted triplet), bs (broad singlet) and m (multiplet).

[0254] In some cases, the assignment of 'H or13C magnetic resonance spectral signals was carried out using a combination of one- and two-dimensional techniques, such as heteronuclear 'H-I3C (HSQC and HMBC) and / or homonuclear (COSY-45 and ROESY1H-1H) correlation experiments.

[0255] Mass spectrometry: electronic impact (El) and eletrospray (ESI) ionization techniques were used. Low resolution El spectra were acquired in a QP2010 ultra spectrometer at 70 eV. ESI spectra were acquired in a Quattro Premier XE spectrometer. High resolution mass spectra were acquired in CIBION (CONICET) with a Xevo G2S Q-TOFAttorney Docket No. 148839.604511.PC spectrometer calibrated in the m / z 50-1200 interval with a sodium formate 0,5 mM solution prepared in 90:10 2 -propanol: water v / v. The data were corrected during acquisition using a reference compound (LockSpray).

[0256] Synthesis of 1G-55.

[0257]

[0258] Synthesis of intermediate N-(Lbutoxycarbonyl)-N'-(3.5-dimethylDhenyl)thiourea (ID-195)

[0259] To a solution of thiourea (521 mg; 6.84 mmol) in 117 mL of anhydrous THF 60% NaH (1.23 g; 30.75 mmol) was added portion-wise at 0 °C under argon atmosphere. It was warmed to room temperature and reacted for 45 min. Then it was placed in an ice bath at 0 °C and di-tert-butyl dicarbonate (3.3 mL, 14.36 mmol) was added dropwise. The reaction mixture was warmed to room temperature and reacted overnight. Then it was cooled to 0 °C and 60% NaH (349.2 mg; 8.73 mmol) was added portion-wise, and reacted for 1 h before adding trifluoroacetic anhydride (1.0 mL; 7.08 mmol) dropwise. The mixture was reacted at 0 °C for another 1 h to form the thioacylating agent and 3,5-dimethylaniline (1.23 mL; 9.87 mmol) was added dropwise. Then it was warmed to room temperature and reacted overnight. The reaction was quenched by the addition of H2O (120 mL) at 0 °C and extracted with EtOAc (3 x 100 mL). The combined organic layersAttorney Docket No. 148839.604511.PC were washed with NaCl saturated solution (30 mL), dried (Na2SC>4) and the solvent was evaporated. The residue was purified by flash chromatography (silica gel) eluted with hexane-EtOAc gradient followed by recrystallization on MeOH-EbO to provide 774 mg (40%) of ID- 195 as a white solid (17).

[0260] Rf 0.26 (95:5 hexane :EtO Ac). Mp 122 °C.1HNMR(300 MHz, CDC13): 5 11.39 (s, 1H), 8.01 (s, 1H), 7.24 (s, 2H), 6.89 (s, 1H), 2.33 (s, 6H), 1.53 (s, 9H).13C NMR (75 MHz, CDC13): 5 178.2, 152.0, 138.7, 137.6, 128.6, 122.1, 84.6, 28.1, 21.4. EM-ESI m / z 281 ([M+H]+).

[0261] Synthesis of intermediate 3-(2-(t-butoxycarbonyl)-3-(3,5-dimethylphenyl)guanidino)benzoic acid (1F-123)

[0262] To a solution of ID-195 (1.56 g; 5.56 mmol), benzyl 3 -aminobenzoate (1.70 g; 7.48 mmol) and triethylamine (0.9 mL; 6.46 mmol) in 60 mL of anhydrous CH2CI2 EDCI (2.48 g; 12.94 mmol) was added at 0 °C and under argon atmosphere. The residue was purified by flash chromatography (silica gel) eluted with hexane-EtOAc gradient. The reaction product (2.24 g; 4.73 mmol) was dissolved in 50 mL of EtOAc and treated with hydrogen at 2 bar in the presence of 10% Pd on carbon (227 mg) for 24 h at room temperature. The mixture was filtered through a Celite bed and the solvent was evaporated to provide 1.82 g of IF- 123 (100%) as a white solid.

[0263] Rf 0.50 (1:1 hexane: EtOAc). Mp 118-119 °C.3H NMR (300 MHz, CDCh): 5 10.75 (bs, 3H ), 7.77 (s, 1H), 7.72 (d, J= 7.5 Hz, 1H), 7.28 (bs, 1H), 7.20 (t, J= 7.8 Hz, 1H), 6.71 (s, 2H), 6.58 (s, 1H), 2.11 (s, 6H), 1.54 (s, 9H).13C NMR (75 MHz, CDCh): 5 171.8, 156.3, 149.3, 138.8, 138.5, 137.2, 133.4, 128.6, 126.9, 126.2, 126.1, 124.2, 121.0, 83.0, 28.2, 21.2. EM-ESI m / z 384 ([M+H]+).

[0264] General procedure for the synthesis of 1G-55 by Ugi reactionAttorney Docket No. 148839.604511.PC To a solution of IF- 123 (1,0 eq.) in MeOH, an amine (1,0 eq.) and an aldehyde (1,0 eq.), isocyanide (1,0 eq.) was added while stirring, and reacted for 2 to 7 days at room temperature. Then the solvent was evaporated and the residue was purified by chromatography (silica gel) to provide a product that was treated with 15.5% HC1 in isopropanol for 4-5 h at room temperature. Then the pH was adjusted to 8-9 by the addition of NaHCCh saturated solution at 0 °C. The mixture was extracted with CH2CI2 (x 5), the combined organic layers were dried (Na2SC>4) and the solvent was evaporated. The residue was purified by column chromatography (silica gel) to provide the desired product 1G-55.

[0265] Synthesis of N-(l-(2, 5-dichlorophenyl)-2-oxo-2-(p-tolylammo)ethyl)-3-(3-(3, 5-dimethylphenyDsuanidinoj-N-methylbenzamide (1G-55)

[0266]

[0267] 1G-55

[0268] Following the general procedure for the synthesis of 1G-55 by Ugi reaction, 2,5-dichlorobenzaldehyde (88 mg; 0.50 mmol), methylamine hydrochloride (35 mg; 0.52 mmol), 1F-123 (196 mg; 0.51 mmol), triethylamine (70 pL; 0.50 mmol) and 4-methylphenyl isocyanide (95 mg; 0.81 mmol) were reacted in 1 mb of MeOH for 6 days. The reaction was purified by column chromatography (silica gel) eluted with hexane-EtOAc to provide 67 mg of a product that was treated with HC1 in IPA (0.5 mb) andAttorney Docket No. 148839.604511.PC purified by column chromatography (silica gel) eluted with CH2Ch-MeOH-NH3 to provide 42 mg (14 %, total) of 1G-55 as a white solid.

[0269] Rf 0.25 (95:5:0.5 CH2C12-MeOH-NH3). Mp 156-157 °C.1HNMR (300 MHz, MeOD): 5 7.56 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.43 (s, 1H), 7.40 (d, J = 7.9 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 7.30 (s, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 7.4 Hz, 1H), 6.80 (maj) and 6.76 (min) (rot., s, 2H), 6.65 (s, 1H), 6.48 (maj) and 5.76 (min) (rot., s, 1H), 2.87 (min) and 2.80 (maj) (rot., s, 3H), 2.31 (maj) and 2.28 (min) (rot., s, 3H), 2.24 (s, 6H).13C NMR (75 MHz, MeOD): 5 175.4 (min) and 174.6 (maj) (rot.), 169.4 (min) and 169.2 (maj) (rot.), 152.9 (maj) and 152.6 (min) (rot.), 147.8 (min) and 147.5 (maj) (rot.), 144.0 (min) and 143.9 (maj) (rot.), 139.7 (maj) and 139.6 (min) (rot.), 137.7 (min) and 137.3 (maj) (rot.), 136.8, 136.2 (maj) and 135.9 (min) (rot.), 135.7 (min) and 135.4 (maj) (rot.), 135.2 (maj) and 135.1 (min) (rot.), 134.3, 132.8 (min) and 132.7 (maj) (rot.), 131.7 (min) and 131.5 (maj) (rot.), 131.4, 130.5, 130.4, 125.6, 125.4 (min) and 125.3 (maj) (rot.), 121.8 (maj) and 121.7 (min) (rot.), 121.6 (maj) and 121.5 (min) (rot.), 121.3, 120.8 (maj) and 118.3 (min) (rot.), 65.7 (min) and 61.4 (maj) (rot.), 36.4 (maj) and 31.9 (min) (rot.), 21.5, 21.0. EM-ESI m / z 588 ([M+H]+).

[0270] Example 10. Liver injury reduction in mouse models of ALF and ACLF after RAC1 inhibition using compound 1G-55

[0271] Prior reports indicate that RAC1 is involved in the production of ROS and in the inflammatory process. In consequence, the inventors posed the hypothesis that 1G-55 may have a therapeutic effect 1G-55 in acute liver failure (ALF) induced by oxidative damage or immune system-mediated acute hepatitis. In order to confirm this hypothesis, the protective effect of 1G-55 was assessed in two ALF models. The first ALF model was induced with acetaminophen (APAP), where ROS play a major role, and the second one with Concanavalin A (ConA) to mimic immune system-mediated acute hepatitis,Attorney Docket No. 148839.604511.PC mediated by macrophages, T cells, natural killer cells and neutrophils (Figure 18A). Notably, RAC1 inhibition after treatment with 1G-55 significantly reduced the degree of liver injury, as evaluated by AST and ALT measurements in both ALF models (Figure 18B).

[0272] In addition, evaluated was the protective effect of 1G-55 in a mouse model of acute-on-chronic liver failure (ACLF) that involved establishment of liver fibrosis by administering TAA for 10 weeks, followed by the induction of acute liver injury by the administration of acetaminophen (APAP) and lipopolysaccharide (LPS) to generate both oxidative stress and inflammation, which are the specific features of ACLF. In line with the results obtained in ALF models, RAC1 inhibition after the treatment with 1G-55 resulted in significantly lower levels of alanine aminotransferase (ALT), indicating a reduction in liver injury (Figure 19).

[0273] Example 11. Effects ofRACl inhibition using 1G-55 in liver explants from patients The effects of 1G-55 in liver explants from patients (healthy livers and acute liver failure patient livers) were evaluated (Figure 20). The in vitro incubation of liver explants with 1G-55 (10 pM) induced no changes in transaminase (ALT) levels (Figure 20A). Then, a healthy liver explant was exposed to Concanavalin A to induce liver injury. As shown in (Figure 20B), the treatment with 1G-55 reduces ALT levels. Finally, the incubation of acute liver failure patient liver explants induced a reduction in ALT released into the supernatant (Figure 20C).

[0274] Example 12. Effects ofRACl inhibition using ID-142 in liver explants derived from patients (PDEs).

[0275] 8 mm3(2x2x2 mm3) explants of healthy human livers (healthy PDEs) were treated overnight with (i) control in DMEM culture medium, (ii) ConA (15 pg / ml), or (iii) a combination of ConA (15 pg / ml) and ID- 142 (10 pM). ALT levels were determined inAttorney Docket No. 148839.604511.PC the culture medium. ALT levels were measured in the culture medium using an Alinity instrument (Abbott). Healthy liver explant was exposed to Concanavalin A to induce liver injury, as evidenced by the increase in ALT levels restoring the ALT levels observed in control (Figure 22).

[0276] Taken together, the results presented in the above Examples demonstrate that the use of RAC1 inhibitors leads to a significant reduction in liver injury across different models of liver disease (such as, ALF and ACLF) representing liver diseases of diverse etiologies, which highlight the therapeutic potential of RAC 1 inhibition.

[0277] It is of particular relevance that ID-142 (Examples 1-7) and 1G-55 (Examples 8-10) represent distinct chemical scaffolds. Despite their structural differences, both RAC1 inhibitors consistently achieve comparable biological results. Without wishing to be bound by theory, these data, suggest a functional correlation between the modulation of the RAC1 pathway and the mitigation of liver injury associated with ALF and ACLF. Accordingly, the findings provided herein indicate that inhibition of RAC 1 is the main factor behind the observed results. Consequently, these results support that other agents acting as RAC 1 inhibitors are reasonably expected to be useful for the treatment of ALF and ACLF, as the biological results are shown to be independent of the specific chemical structure of the assayed inhibitors.

[0278] Furthermore, without wishing to be bound by theory, the beneficial effects observed in the treatment of ALF and ACLF are believed to be associated with inhibition of the interaction between RAC1 and its GEFs, for example, the interaction between RAC1 and TIAM1, as both inhibitors ID- 142 and 1G-55, which act on RAC1 via the same mechanism, produced beneficial therapeutic effects in the context of ALF and ACLF.Attorney Docket No. 148839.604511.PC REFERENCES

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[0281] 3. Shi Y, Bollam SR, White SM, et al. Rael -Mediated DNA damage and inflammation promote Nf2 tumorigenesis but also limit cell-cycle progression. Developmental Cel 2016;39(4):452— 465.

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[0284] 6. Liu Y, Hao H, Hou T. Concanavalin A-induced autoimmune hepatitis model in mice: Mechanisms and future outlook. Open life sciences. 2022; 17(1):91-101.

[0285] 7. Wu Z, Han M, Chen T, Yan W, Ning Q. Acute liver failure: mechanisms of immune-mediated liver injury. Liver international : official journal of the International Association for the Study of the Liver. 2010;30(6):782-94.

[0286] 8. Liao H, Du S, Jiang T, Zheng M, Xiang Z, Yang J. UMSCs Attenuate LPS / D-GalN-induced Acute Liver Failure in Mice by Down-regulating the MyD88 / NF-kappaB Pathway. Journal of clinical and translational hepatology. 2021;9(5):690-701.Attorney Docket No. 148839.604511.PC 9. da Silveira Cruz-Machado S, Carvalho-Sousa CE, Tamura EK, Pinato L, Cecon E, Fernandes PA, et al. TLR4 and CD 14 receptors expressed in rat pineal gland trigger NFKB pathway. Journal of pineal research. 2010;49(2): 183-92.

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Claims

Attorney Docket No. 148839.604511.PC CLAIMS1. A method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF).

2. A method of prolonging the survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC 1 inhibitor.

3. A method of increasing the survival rate, or the likelihood of survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor.

4. The method according to any one of claims 1-3, wherein the RAC1 inhibitor is selected from the group consisting of ID- 142, 1G-55, a pharmaceutically acceptable salt thereof, and any combination of the foregoing.

5. The method according to any one of claims 1-4, wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof.

6. The method according to claim 5, wherein the RAC1 inhibitor is ID-142.

7. The method according to claim 5, wherein the RAC1 inhibitor is a pharmaceutically acceptable salt of ID- 142.

8. The method according to any one of claims 1-4, wherein the RAC1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof.Attorney Docket No. 148839.604511.PC 9. The method according to claim 8, wherein the RAC1 inhibitor is 1G-55.

10. The method according to claim 8, wherein the RAC1 inhibitor is a pharmaceutically acceptable salt of 1G-55.

11. The method according to any one of claims 1-10, wherein the liver disease is ACLF.

12. The method according to any one of claims 1-10, wherein the liver disease is ALF.

13. The method according to claim 12, wherein the ALF is selected from the group consisting of drug-induced ALF, toxin-induced ALF, alcohol-induced ALF, immune-induced ALF, ALF induced by immune-mediated hepatitis, autoimmune-induced ALF, ALF induced by transplant rejection, infectious-induced ALF, sepsis-induced ALF, bacterial-induced ALF, viral-induced ALF, heat-stroke induced ALF, hypoxic hepatitis or pregnancy-induced ALF, ALF induced by an inflammatory process, and any combination thereof.

14. The method according to claim 13, wherein the ALF is selected from the group consisting of drug-induced ALF, immune-induced ALF, toxin-induced ALF, and infectious-induced ALF.

15. The method according to claim 13, wherein the ALF is drug-induced ALF.

16. The method according to claim 15, wherein ALF is acetaminophen (APAP)-induced ALF.

17. The method according to claim 16, wherein the subject has received an overdose of APAP.Attorney Docket No. 148839.604511.PC 18. The method according to any one of claims 1-17, wherein the RAC1 inhibitor is administered immediately or at a predetermined timepoint after the onset of the liver disease.

19. The method according to any one of claims 1-17, wherein the RAC1 inhibitor is administered before the onset of the liver disease.

20. The method of any one of claims 1-19, wherein the administration of the RAC1 inhibitor reduces liver injury, liver tissue damage, liver inflammation, immune cell infiltration, release of proinflammatory cytokines (such as IL- 1 and TNFa), expression of pro-inflammatory genes, ROS formation, oxidative damage, liver cell death, and / or any combination of the foregoing.

21. The method of any one of claims 1-19, wherein the administration of the RAC1 inhibitor reduces liver injury.

22. The method of any one of claims 1-20, wherein the administration of the RAC1 inhibitor reduces the levels of serum alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST).

23. The method of any one of claims 1-22, wherein the subject is a mammal, such as a human.

24. A pharmaceutical composition comprising a therapeutically effective amount of a RAC 1 inhibitor and a pharmaceutically acceptable excipient, for use in a method according to any one of claims 1-23.

25. A therapeutically effective amount of a RAC1 inhibitor for use in a method according to any one of claims 1-23.Attorney Docket No. 148839.604511.PC 26. A method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof.

27. A method of prolonging the survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof.

28. A method of increasing the survival rate, or the likelihood of survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is ID- 142, or a pharmaceutically acceptable salt thereof.

29. A method of treating or preventing a liver disease in a subject in need thereof comprising administering a therapeutically effective amount of a RAC1 inhibitor to the subject, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), wherein the RAC1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof.

30. A method of prolonging the survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeuticallyAttorney Docket No. 148839.604511.PC effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof.

31. A method of increasing the survival rate, or the likelihood of survival of a subject suffering from a liver disease selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), the method comprising administering to the subject a therapeutically effective amount of a RAC1 inhibitor, wherein the RAC1 inhibitor is 1G-55, or a pharmaceutically acceptable salt thereof.

32. The method according to any one of claims 26-31, wherein the liver disease is ACLF.

33. The method according to any one of claims 26-31 , wherein the liver disease is ALF.

34. The method according to claim 33, wherein the ALF is selected from the group consisting of drug-induced ALF, toxin-induced ALF, alcohol-induced ALF, immune-induced ALF, ALF induced by immune-mediated hepatitis, autoimmune -induced ALF, ALF induced by transplant rejection, infectious-induced ALF, sepsis-induced ALF, bacterial-induced ALF, viral-induced ALF, heat-stroke induced ALF, hypoxic hepatitis or pregnancy-induced ALF, ALF induced by an inflammatory process, and any combination thereof.

35. The method according to claim 34, wherein the ALF is selected from the group consisting of drug-induced ALF, immune-induced ALF, and infectious-induced ALF.

36. The method according to claim 33, wherein the ALF is drug-induced ALF.

37. The method according to claim 36, wherein ALF is acetaminophen (APAP)-induced ALF.Attorney Docket No. 148839.604511.PC 38. The method according to claim 37, wherein the subject has received an overdose of APAP.

39. The method according to any one of claims 26-38, wherein the RAC1 inhibitor is administered immediately or at any timepoint after the onset of the liver disease, such as a late treatment.

40. The method according to any one of claims 26-38, wherein the RAC1 inhibitor is administered before the onset of the liver disease, such as an early treatment.

41. The method of any one of claims 26-40, wherein the administration of the RAC1 inhibitor reduces liver injury, liver tissue damage, liver inflammation, immune cell infiltration, release of proinflammatory cytokines (such as IL- 1 and TNFa), expression of pro-inflammatory genes, ROS formation, oxidative damage, liver cell death, and / or any combination of the foregoing.

42. The method of any one of claims 26-40, wherein the administration of the RAC1 inhibitor reduces liver injury.

43. The method of any one of claims 26-42, wherein the administration of the RAC1 inhibitor reduces the levels of serum alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST).

44. The method of any one of claims 26-43, wherein the subject is a mammal, such as a human.

45. A pharmaceutical composition comprising a therapeutically effective amount of ID- 142, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, for use in a method of treating or preventing a liver disease in aAttorney Docket No. 148839.604511.PC subject in need thereof, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute -on-chronic liver failure (ACLF).

46. A therapeutically effective amount of ID- 142, or a pharmaceutically acceptable salt thereof, for use in a method of treating or preventing a liver disease in a subject in need thereof, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF).

47. A pharmaceutical composition comprising a therapeutically effective amount of 1G-55, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, for use in a method of treating or preventing a liver disease in a subject in need thereof, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF).

48. A therapeutically effective amount of 1G-55, or a pharmaceutically acceptable salt thereof, for use in a method of treating or preventing a liver disease in a subject in need thereof, wherein the liver disease is selected from the group consisting of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF).

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