Compounds, compositions and methods for treating or ameliorating nonalcoholic fatty liver disease and related diseases or disorders - Patents.com

Abstract

The present disclosure relates to the discovery that certain compounds that modulate the activity of sigma receptors can be used to treat or ameliorate non-alcoholic fatty liver disease and related diseases or disorders. In certain embodiments, the sigma receptor is sigma-1 receptor (also known as sigma 1).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 137,915, filed January 15, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] background Nonalcoholic fatty liver disease (NAFLD) describes a constellation of liver abnormalities beginning with hepatic steatosis (fatty liver). In some patients, NAFLD progresses to nonalcoholic steatohepatitis (NASH). NASH is defined histologically by hepatic steatosis with lobular inflammation, hepatocellular injury (ballooning), with or without fibrosis. NASH may then progress / lead to cirrhosis and hepatocellular carcinoma (HCC).

[0003] NAFLD is a steadily growing cause of chronic liver disease worldwide, along with the global increase in obesity and metabolic syndrome. In the United States, it is estimated that approximately 24% of the population has NAFLD, and of these people, approximately one-third have NASH. NAFLD is associated with a variety of comorbidities, including metabolic syndrome, diabetes, and cardiovascular disease (CVD). NAFLD is the fastest growing cause of HCC among liver diseases and one of the leading diseases requiring liver transplantation. Although considerable progress has been made in elucidating NAFLD pathogenesis, identifying therapeutic targets, and discovering and developing new drugs, the field still remains with significant unanswered questions and unmet clinical needs for this patient population.

[0004] The factors involved in the progression of NAFLD are not well defined. NAFLD has a highly diverse natural history reflected in different rates of progression among individuals and has heterogeneous clinical manifestations. Underlying these differences is the convergence of various environmental and social / behavioral influences, as well as metabolic comorbidities, gut microbiota, genetic, sex- and age-related / linked risk factors. Thus, the mechanisms driving NAFLD and the clinical manifestations are heterogeneous. The natural history of the disease and the factors involved in its progression are not well defined. Although several molecular pathways have been described that contribute to the development of NASH, the pathogenic drivers of NAFLD and the molecular mechanisms that result and drive the disease are not well defined, are likely to vary between patients, and are likely to be multifocal.

[0005] How to identify the approximately 20% of NAFLD patients who rapidly progress from NASH to progressive fibrosis remains elusive, and the factors underlying this rapid progression also remain unclear. There is a pressing need for predictive biomarkers of disease risk and pharmacodynamic biomarkers of drug response. However, identifying people at risk for NASH remains challenging due to a poor understanding of the natural history of NAFLD and the key pathogenic drivers and enablers / facilitators of the disease.

[0006] Currently, treatment options for NALFD remain limited, with no FDA-approved drugs to treat patients affected by this rapidly growing public health concern. There is a pressing need for new therapeutic agents and treatment strategies, as well as biomarkers of disease risk and drug response. Several agents are in clinical trials, including four different targeted agents in at least six phase 3 trials, for treating NASH with fibrosis. Of these, two recently completed phase 3 clinical trials of the ASK-1 inhibitor selonsertib failed to meet the pre-specified 48-week primary endpoint of achieving at least one histological step improvement in fibrosis without worsening of NASH.

[0007] This highlights the need for biomarker-guided therapeutic trials. Therapeutic agents and diagnostic technologies are essential to more accurately predict disease progression, develop more effective treatments, and more effectively design clinical trials. Clinical trial designs and strategies are rapidly evolving as the list of drug targets grows and our understanding of the factors underlying the heterogeneity of NAFLD improves.

[0008] There is a need in the art to identify new compounds, compositions, methods and biomarkers that enable the treatment, prevention and / or monitoring of NAFLD and related diseases. The present disclosure addresses this unmet need. Summary of the Invention

[0009] overview In certain aspects, the present specification is directed to the following non-limiting embodiments: Embodiment 1: A method for treating, ameliorating and / or preventing lipid droplet (LD) accumulation in a cell, comprising the steps of: contacting the cell with an agent that reduces and / or suppresses sigmaR1 transcription in the cell and / or reduces and / or inhibits sigma1 activity, localization and / or expression. Embodiment 2: A method of treating, ameliorating and / or preventing inflammation associated with hepatic steatosis (fatty liver) and / or steatohepatitis in a subject, comprising the steps of: administering to the subject an effective amount of an agent that reduces and / or suppresses sigma R1 transcription and / or reduces and / or inhibits sigma 1 activity and / or expression in the subject. Embodiment 3: A method of treating, ameliorating and / or preventing non-alcoholic fatty liver disease (NAFLD) in a subject, comprising the steps of: administering to the subject an effective amount of an agent that reduces and / or suppresses sigmaR1 transcription and / or reduces and / or inhibits sigma1 activity and / or expression in the subject. Embodiment 4: A method of treating, ameliorating and / or preventing non-alcoholic steatohepatitis (NASH) in a subject, comprising the steps of: administering to the subject an effective amount of an agent that reduces and / or suppresses sigmaR1 transcription and / or reduces and / or inhibits sigma1 activity and / or expression in the subject. Embodiment 5: A method for reversing, slowing or preventing progression of NAFLD to NASH in a subject suffering from NAFLD, comprising the steps of: administering to the subject an effective amount of an agent that reduces and / or suppresses sigmaR1 transcription and / or reduces and / or inhibits sigma1 activity and / or expression in the subject. Embodiment 6: A method for reversing, slowing or preventing the progression of NASH to hepatocellular carcinoma (HCC) in a subject suffering from NASH, comprising the steps of: administering to the subject an effective amount of an agent that regulates sigmaR1 transcription and / or regulates sigma1 activity and / or expression in the subject. Embodiment 7: A method for determining and / or assessing the risk of a subject suffering from NAFLD progressing to NASH, comprising the steps of: measuring the level of sigma R1 transcription and / or sigma 1 activity / expression in the subject, and comparing the measured level with the level of sigma R1 transcription and / or sigma 1 activity / expression in a control sample. Embodiment 8: The method of embodiment 7, wherein the subject at risk of developing NASH is counseled to receive an effective amount of an agent that reduces and / or suppresses sigma R1 transcription and / or reduces and / or inhibits sigma 1 activity and / or expression. Embodiment 9: The method of embodiment 7, wherein the subject at risk of developing NASH is administered an effective amount of an agent that reduces and / or suppresses sigma R1 transcription and / or reduces and / or inhibits sigma 1 activity and / or expression. Embodiment 10: A method for determining and / or assessing the risk of a subject suffering from NASH progressing to HCC, comprising the steps of: measuring the level of sigma R1 transcription and / or sigma 1 activity / expression in the subject, and comparing the measured level with the level of sigma R1 transcription and / or sigma 1 activity / expression in a control sample. Embodiment 11: The method of embodiment 10, wherein the subject at risk of progressing to HCC is counseled to receive an effective amount of an agent that modulates sigmaR1 transcription and / or modulates sigma1 activity and / or expression. Embodiment 12: The method of embodiment 10, wherein the subject at risk of progressing to HCC is counseled to receive an effective amount of an agent that modulates sigmaR1 transcription and / or modulates sigma1 activity and / or expression. Embodiment 13: The method of any of embodiments 7 to 12, wherein the control sample is derived from at least: a subject suffering from NAFDL; a subject not suffering from NAFDL; a subject suffering from NASH; a subject not suffering from NASH; a subject suffering from HCC; a subject not suffering from HCC; a subject suffering from NAFLD known not to have progressed to NASH and / or not at risk of progressing to NASH; a subject suffering from NAFLD known to have progressed to NASH and / or to be at risk of progressing to NASH; a subject suffering from NASH known not to have progressed to HCC and / or not at risk of progressing to HCC; a subject suffering from NASH known to have progressed to HCC and / or to be at risk of progressing to HCC. Embodiment 14: The method of any of embodiments 2-6, wherein the administering step reverses, prevents, and / or ameliorates at least one of the following: steatosis, inflammation, hepatocellular ballooning, fibrosis, hepatic stellate cell (HSC) activation, increased serum liver enzymes, insulin resistance / glucose tolerance, and HCC lesion formation. Embodiment 15: The method of any of embodiments 1 to 6, 8 to 9, and 11 to 14, wherein said agent comprises a CRISPR system against sigma R1, and / or a nucleic acid against sigma R1 selected from an siRNA, shRNA, microRNA, or an antisense polynucleotide. Embodiment 16: The method of embodiment 15, wherein a viral vector expressing the nucleic acid is administered to the subject. Embodiment 17: The method of embodiment 16, wherein the nucleic acid is expressed in the liver of the subject. Embodiment 18: The method of any of embodiments 1-6, 8-9, and 11-14, wherein the agent is: Formula (I): TIFF2024504632000001.tif25128, wherein Ring A is a monocyclic or bicyclic aryl ring or a monocyclic or bicyclic heteroaryl ring, wherein the aryl or heteroaryl ring is selected from the group consisting of 0 to 4 R 1 may be substituted with a group; R 1 Each of the above is -C1 to C6 alkyl, -C1 to C6 fluoroalkyl, -C1 to C6 heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR 3 , -SR 3 , -S(=O)R 3 , -S(=O)2R 3 , -NHS(=O)2R 3 , -C(=O)R 3 , -OC(=O)R 3 , -CO2R 3 , -OCO2R 3 , -CH(R 3 )2, -N(R 3 )2, -C(=O)N(R 3 )2, -OC(=O)N(R 3 )2, -NHC(=O)NH(R 3 ), -NHC(=O)R 3 , -NHC(=O)OR 3 , -C(OH)(R 3 )2, and -C(NH2)(R 3 )2 independently selected from the group consisting of; R 2each is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 heteroalkyl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), where the alkyl, heteroalkyl, or cycloalkyl group is selected from 0 to 5 R 1 or may be substituted with X 3 and R 2 together, 0 to 2 R 1 forming a (C3-C7) heterocycloalkyl group which may be substituted by a group; R 3 each is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 heteroalkyl, aryl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), where the alkyl, heteroalkyl, aryl, or cycloalkyl group is selected from 0 to 5 R 1 may be substituted with a group; X 1 is -CH2-, -S-, -O- or -(NR 2 )-and; X 2 is =CH2, =S, =O or =NR 2 and X 3 is -S-, -O-, or -NR 2 - a compound which is Formula (II): R A -R B (II) A compound of the formula: R A teeth, TIFF2024504632000002.tif20128; X 4 is selected from the group consisting of methoxy, F, Cl, Br, and I; and R B teeth, A compound selected from the group consisting of TIFF2024504632000003.tif116134; Formula (III): TIFF2024504632000004.tif20128, wherein R 1 and R 2 Each of the above is -C1 to C6 alkyl, -C1 to C6 fluoroalkyl, -C1 to C6 heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR 5 , -SR 5 , -S(=O)R 5 , -S(=O)2R 5 , -NHS(=O)2R 5 , -C(=O)R 5 , -OC(=O)R 5 , -CO2R 5 , -OCO2R 5 , -CH(R 5 )2, -N(R 5 )2, -C(=O)N(R 5 )2, -OC(=O)N(R 5 )2, -NHC(=O)NH(R 5 ), -NHC(=O)R 5 , -NHC(=O)OR 5 , -C(OH)(R 5 )2, and -C(NH2)(R 5 )2 independently selected from the group consisting of; R 3 is selected from the group consisting of -C1-C6 alkyl, -C1-C6 fluoroalkyl, -C1-C6 alkoxy, F, Cl, Br, and I; R 4 is selected from the group consisting of -C1-C6 alkyl, -C1-C6 alkoxy, F, Cl, Br, and I; R 5 each is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 heteroalkyl, aryl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), where the alkyl, heteroalkyl, aryl, or cycloalkyl group may be optionally substituted; X is selected from the group consisting of CH2, C=O, or O; n is an integer from 1 to 3; x is an integer from 0 to 4; and wherein y is an integer from 0 to 4; a salt, solvate, or N-oxide thereof; and any combination thereof. Embodiment 19: The method of embodiment 18, wherein the compound is selected from the group consisting of: 1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A); 1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (compound B); 1-(n-propyl)-3-(4-iodophenyl)guanidine (compound C); 1-(n-propyl)-3-(4-methoxyphenyl)guanidine (compound D); 1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (compound F); 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G, also referred to herein as "CT-110" or "CT-189"); Salts, solvates or N-oxides thereof, and any combination thereof. Embodiment 20: The method of embodiment 18, wherein the compound is selected from the group consisting of: 1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (compound E); 1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine (compound H); Salts, solvates or N-oxides thereof, and any combination thereof. Embodiment 21: The method of any of embodiments 18-20, wherein the compound is administered as a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier. Embodiment 22: The method of any of embodiments 18-21, wherein the subject is further administered at least one additional agent that treats, prevents, and / or ameliorates one of NAFDL, NASH, and / or HCC. Embodiment 23: The method of any of embodiments 18-22, wherein the compound is administered by a route including oral, nasal, rectal, vaginal, parenteral, buccal, sublingual, or topical. Embodiment 24: The method of any of embodiments 18 to 23, wherein the subject is a mammal. Embodiment 26: The method of embodiment 24, wherein the mammal is a human. [Brief description of the drawings]

[0010] For the purpose of illustrating the disclosure, certain aspects of the disclosure are depicted in the drawings. However, the disclosure is not limited to the precise arrangements and instrumentalities of the aspects depicted in the drawings.

[0011] [Figure 1A] Figures 1A-1B illustrate SigmaR1 transcript expression patterns in healthy human liver and non-alcoholic fatty liver (NAFL) liver according to some embodiments. Figure 1A: SigmaR1 mRNA transcript expression levels in 27 organs (data from Fagerberg, Mol. Cell. Proteomics 2014; PMID: 24309898). Figure 1B: SigmaR1 mRNA transcript expression levels in healthy human liver and non-alcoholic fatty liver (NAFL) liver. The horizontal line on the scatter plot represents the median Z-score. [Figure 1B] See legend to Figure 1A. [Figure 2-1]Figures 2A-2E demonstrate that some embodiments protect SigmaR1 knockout (KO) mice from diet-induced hepatic steatosis. Figure 2A: Depicts steatosis grades in wild-type (WT) and SigmaR1 KO mice fed normal diet (ND) or high-fat diet (HFD). Area (% area) of liver sections composed of lipids in wild-type and SigmaR1 KO mice on normal and high-fat diets. Figure 2B: Hematoxylin and Eosin (H&E) stained images of livers from wild type (WT) and Sigma R1 KO (σ1(- / -)) mice fed a ND v HFD. Images taken at 14x magnification, scale bar = 200 um. Figure 2C: Calculated liver steatosis grades from the mice in Figure 2A. Figure 2D: Representative photographs of livers from WT and Sigma R1 KO (σ1(- / -)) mice fed a high-fat diet. Figure 2E: Oil Red-O stained images of livers from WT and Sigma R1 KO mice fed a high-fat diet. [Figure 2-2] See description of Figure 2-1. [Figure 3-1] Figures 3A-3C demonstrate that a high-fat diet induced whitening of brown adipose tissue (BAT) by several embodiments. Lipid content in BAT of wild-type (WT) and SigmaR1 KO mice fed a high-fat diet (HFD) for 8 weeks. Figure 3A: Images of representative fields from H&E stained BAT of WT and SigmaR1 KO male mice after 8 weeks of HFD are shown. Images taken at 14x, scale bar = 200 um. Figure 3B: Lipid area of ​​H&E stained sections, 3 fields per animal, n=4 animals per group are depicted. Quantification was performed using ImageJ. Statistical analysis used: One-way ANOVA Tukey's post hoc test, statistical significance of pairwise comparisons is shown. Figure 3C: Average lipid droplet (LD) size is depicted in microns squared (μM2). Quantification was performed using Aperio software. Statistical analysis used: One-way ANOVA Tukey's post hoc test, statistical significance of pairwise comparisons is shown. [Figure 3-2] See description of Figure 3-1. [Figure 4]4A-4B illustrate the finding that, according to some embodiments, sigma 1 (σ1) knockdown by introduction of sigma R1-targeting shRNA with lentiviral vectors abrogates lipid droplet (LD) accumulation. FIG. 4A: Confocal micrographs of LNCaP cells treated with dihydrotestosterone (DHT) for 72 hours (72h) to induce LD formation and accumulation. A non-specific control shRNA was compared to a sigma R1 transcript-selective shRNA in a lentiviral vector. σ1 shR #4 = MISSION / SigmaAldrich clone TRCN0000061010; σ1 shR #5 = MISSION / SigmaAldrich clone TRCN0000061008. The white box indicates the area shown in magnification immediately below. Note the near complete elimination of LDs. FIG. 4B: Immunoblot confirming sigma 1 knockdown by lentiviral sigma 1 (σ1) shRNA. LDs are stained red with HCS-LipidTox fluorescent staining. DAPI stained nuclei in blue. [Diagram 5] Some embodiments illustrate the finding that the pharmacological sigma-1 modulator / inhibitor 1-(4-iodophenyl)-3-(2-adamantyl)guanidine (IPAG) eliminates lipid droplets (LDs). Confocal micrographs of LNCaP cells treated with 1 nM dihydrotestosterone (DHT) for 72 hours (72h) to induce LD formation and accumulation. In the far right panel, LNCaP cells were treated with the prototypic sigma-1 selective small molecule compound IPAG (10 uM) for the last 16 hours of the 72-h DHT treatment period. LDs are stained red with HCS-LipidTox fluorescent stain. DAPI stained nuclei in blue. [Figure 6]6A-6C demonstrate that, according to some embodiments, a drug-like sigma 1 inhibitor (CT-110, also referred to as "CT-189" and "Compound G") prevents diet-induced hepatic steatosis. FIG. 6A: illustrates certain aspects of the study design. As shown in FIG. 6A, mice fed a high-fat diet (HFD) were co-treated orally (PO) with either vehicle or the novel drug-like small molecule sigma 1 inhibitor CT-110, 3 days per week. FIG. 6B: shows H&E stained images of the liver of the mouse in FIG. 6A. FIG. 6C: shows images of brown adipose tissue (BAT) from high-fat diet (HFD) mice treated with either vehicle or CT-110 (also referred to as "CT-189" and "Compound G"). Representative images from H&E stained BAT of mice after 8 weeks of HFD. Images taken at 14x, scale bar = 200 um. [Figure 7] Some embodiments illustrate the finding that sigma 1 inhibition by CT-189 suppresses the levels of key fibrosis-associated proinflammatory cytokines in HT-29 (human colorectal adenocarcinoma cell line).Fibrosis-associated proinflammatory cytokines include soluble IL-6, TNFα and IL-17, which are contributing factors in the progression of NAFLD to NASH with fibrosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description The study described herein ("the study"), in one aspect, discloses that the expression of sigma 1 receptor is significantly increased in the liver of people with non-alcoholic fatty liver (NAFL). Using sigmaR1 knockout mice as a non-limiting example, the study found that the elimination of sigma 1 expression can substantially reduce the level of hepatic steatosis and brown adipose tissue whitening induced by high-fat diet (HFD). Using shRNA as a non-limiting example, the study found that reducing the expression level of sigma 1 can reduce the formation and accumulation of DHT-induced lipid droplets in cells. Using the small molecule sigma 1 inhibitor IPAG (1-(4-iodophenyl)-3-(2-adamantyl)guanidine) as a non-limiting example, the study found that pharmaceutical inhibition of sigma 1 receptor can reduce the formation and accumulation of DHT-induced lipid droplets in cells. Using the small molecule sigma 1 inhibitor CT-110 (also referred to herein as "CT-189" or "compound G") as a non-limiting example, this study found that pharmacological inhibition of the sigma 1 receptor in mice can reduce hepatic steatosis and brown adipose tissue whitening induced by a high-fat diet. Additionally, this study found that pharmacological inhibition of the sigma 1 receptor can reduce the expression levels of pro-inflammatory cytokines associated with fibrosis, such as hepatic fibrosis that often follows non-alcoholic fatty liver disease.

[0013] The present disclosure, in one aspect, relates to the discovery that pharmacological and / or genetic (i.e., gene therapy) modulation of sigma 1 receptor (also known as sigma 1) in the liver is useful for treating and / or preventing nonalcoholic fatty liver disease (NAFLD) and / or preventing and / or ameliorating the progression of fatty liver to nonalcoholic steatohepatitis with fibrosis and then to hepatocellular carcinoma (HCC). Further, according to the present disclosure, sigma 1 can act as a useful diagnostic, prognostic, and / or pharmacodynamic biomarker in the progression of any such condition and / or disease.

[0014] The present disclosure establishes a physiological role for sigma-1 as a necessary factor in the progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) and establishes sigma-1 as a novel target and biomarker in the progression of NAFLD to NASH.

[0015] Sigma 1 (gene name SIGMAR1, also known as sigma-1 receptor, sigma 1R, and several other names; see PMIDs: 28744586, 28871306) is a novel ligand-gated, intracellular integral membrane protein that is abundant in the secretory pathway of most cells. Sigma receptors are distinct from classical opioid receptors. Sigma 1 is highly conserved among mammals (>80% amino acid identity), but does not share significant homology with any traditional receptor family or other mammalian proteins. Cloned sigma 1 is a 26 kilodalton integral membrane protein found primarily in the ER and can translocate to the plasma membrane, other organelles, and endoplasmic reticulum membrane microdomains. Sigma 1 itself has no known intrinsic signaling or enzymatic activity, but rather allosterically regulates the intracellular signaling and activity of its associated proteins. Thus, sigma 1 appears to be a component of the cellular support machinery. Sigma-1 may be involved in ligand-induced cell death through lysosomal destabilization and oxidative stress.

[0016] Sigma 1 physically associates with several oncogenic driver proteins, and Sigma 1 functions through protein-protein interactions (PPIs) to regulate their stability, trafficking and degradation in a pharmacologically controllable manner. Sigma 1 also regulates lipid and protein homeostasis at multiple levels, playing a key role in the increased lipid and protein synthesis, processing and quality control associated with tumor growth. Targeting Sigma 1 therefore allows for selective regulation of multiple nodes via one drug target. A consequence of the secretory pathway regulatory properties of Sigma 1 regulators is that they can be used to elicit immunomodulatory and anti-inflammatory effects. Sigma 1 regulates lipid metabolism, and intact Sigma 1 is required for prostate cancer cells to exploit lipid droplet accumulation and metabolism for disease progression and malignancy.

[0017] Importantly, sigma1 is a multifunctional drug target that is differentially involved in pathophysiological and physiological conditions. The data provided herein show that sigmaR1 / sigma1 plays an important role in NAFLD. Although clearly high levels of sigmaR1 transcripts are observed in the liver, the role of sigmaR1 / sigma1 in liver physiology and pathophysiology remains undefined. SigmaR1 transcript levels are elevated in NAFL and fibrotic NASH livers compared with healthy livers, and interestingly, they are sharply decreased in cirrhotic livers (Figures 1A-1B). This indicates that the role of sigma1 changes as NAFLD progresses from NASH to HCC. High-fat diet (HFD) does not induce hepatic steatosis in sigmaR1 KO mice (Figures 2A-2C), suggesting that sigma1 is required for the development of NAFL. Furthermore, sigma-1 inhibition suppresses the levels of important fibrosis-associated pro-inflammatory cytokines, including soluble IL-6, TNFα, and IL-17, which are clear contributors to the progression of NAFLD to NASH.

[0018] In certain embodiments, sigma1 is required for high fat Western diet (HFWD)-induced NAFL and the progression to NASH and HCC.Therefore, sigma1 inhibition can be hepatoprotective, and can prevent or delay the progression of NAFLD to NASH by preventing inflammation associated with hepatic steatosis and steatohepatitis, and the progression to cirrhosis and HCC.

[0019] In certain embodiments, the progression of NAFL to NASH and HCC in sigmaR1 knockout (KO) mice fed a high-fat Western diet (HFWD) is characterized compared to wild-type (WT) mice. In this study, a complete sigmaR1 knockout (KO) C57BL / 6 mouse model fed HFWD+CCl4 (Tsuchida, et al., 2018, J. Hepatol. 69:385-395) is used to evaluate key biomarkers of disease progression including steatosis, inflammation, hepatocyte ballooning, fibrosis, hepatic stellate cell (HSC) activation, serum liver enzymes, insulin resistance / glucose tolerance, and / or HCC lesion formation. The status of sigmaR1 transcript and sigma1 protein levels, as well as liver immunostaining patterns at all stages of disease progression are monitored in WT C57BL / 6 mice.

[0020] In certain embodiments, the impact of sigma 1 targeted drug therapy in preventing and regressing steatosis and fibrosis is evaluated.In other embodiments, small molecule sigma 1 regulators phenotype the main aspects of the hepatoprotective effect of sigmaR1 KO mice.Then, it can be determined at which stage of disease progression sigma 1 drug therapy can be effective.It can be determined whether sigma 1 drug therapy can be used to prevent steatosis, inflammation and / or fibrosis.This helps to prove that sigma 1 drug therapy is protective and can prevent hepatic steatosis and fibrosis, and / or promote and accelerate recovery after the onset of hepatic steatosis and after the onset of fibrosis.

[0021] The present disclosure includes any compound contemplated within the disclosure, as well as compositions comprising same, wherein the compositions may optionally further comprise at least one additional therapeutic agent and / or at least one pharma- ceutically acceptable carrier.

[0022] In certain embodiments, the compounds contemplated within the present disclosure have improved drug-like properties than compounds known in the art to bind and modulate sigma receptors.In another embodiment, the compounds contemplated within the present disclosure do not cross the blood-brain barrier.In yet another embodiment, the compounds contemplated within the present disclosure cross the blood-brain barrier.

[0023] Compounds contemplated within the present disclosure may be characterized by pharmacological, cellular, biochemical, in vivo, pharmacokinetic, or pharmacodynamic properties. Selected examples of property studies include, but are not limited to, sigma 1-ligand binding properties, signal transduction pathway analysis and / or characterization, proteomic analysis of sigma 1 protein associations in response to sigma ligand treatment, tumor, brain response, and toxicity.

[0024] The entire disclosures of WO 2014 / 015157 and US 2015 / 0166472 are incorporated herein by reference in their entireties.

[0025] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described.

[0026] As used herein, each of the following terms has the meaning associated with it in this section.

[0027] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0028] As used herein, "about" when referring to a measurable value such as an amount, duration, and the like, is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, where such variations are appropriate to perform the disclosed methods.

[0029] The term "abnormal," when used in the context of an organism, tissue, cell, or component thereof, refers to an organism, tissue, cell, or component thereof that differs in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from an organism, tissue, cell, or component thereof that exhibits the "normal" (expected) respective characteristic. A characteristic that is normal or expected in one cell or tissue type may be abnormal in a different cell or tissue type.

[0030] A "disease" is a state of an animal's health where the animal is unable to maintain homeostasis and where, if the disease is not ameliorated, the animal's health will continue to deteriorate.

[0031] In contrast, an animal "disorder" is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health state is less desirable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further deterioration of the animal's health state.

[0032] A disease or disorder is "alleviated" or "ameliorated" if the severity of the symptoms of the disease or disorder, the frequency with which such symptoms are experienced by the patient, or both, are reduced.

[0033] As used herein, the term "sigma" refers to the sigma 1 receptor (sigma 1), the sigma 2 receptor (sigma 2), any splice variants thereof, or any isoforms thereof. The canonical form of sigma 1 has the amino acid sequence of SEQ ID NO: 1. It should be noted that amino acids 2-8 target the protein to endoplasmic reticulum-associated lipid droplets, amino acids 99-106 are important for ligand binding, and amino acids 177-223 correspond to the C-terminal hydrophobic region. TIFF2024504632000005.tif64130

[0034] As used herein, a "sigma receptor modulator" is a compound that binds to a sigma receptor and modulates the activity or biological function of the receptor compared to the activity or biological function of the receptor in the absence of the modulator. In certain embodiments, a modulator can activate the receptor and thus cause a biological response that is enhanced over the baseline activity of the unbound receptor. In certain embodiments, a modulator may not fully activate the receptor and thus cause a biological response that is less in magnitude compared to that of a full modulator. In certain embodiments, a modulator may bind to the receptor but not activate it, thereby causing receptor blockage and inhibiting the binding of other modulators. Such a modulator does not attenuate the baseline intracellular response in the absence of the modulator. In certain embodiments, a modulator may function as a putative antagonist, agonist, or inverse agonist that reduces the activity of the receptor by inhibiting its constitutive activity.

[0035] As used herein, the term "sigma 1i" refers to a sigma receptor modulator. "sigma 1i" = sigma 1 inhibitor. "sigma 1a" = sigma 1 activator. sigma 1 modulator = any compound that binds / has affinity to sigma 1 and induces activity.

[0036] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal, or cells thereof, amenable to the methods described herein, whether in vitro or in situ. In non-limiting embodiments, the patient, subject, or individual is a human.

[0037] A "therapeutic" treatment is a treatment administered to a subject who exhibits symptoms of a pathology for the purpose of attenuating or eliminating those symptoms.

[0038] The term "treatment" or "treating" as used herein is defined as the application or administration of a therapeutic agent, i.e., a compound of the present disclosure (alone or in combination with another pharmaceutical agent), to a patient having a condition contemplated herein and / or symptoms of a condition contemplated herein, or to a tissue or cell line isolated from the patient (e.g., for diagnosis or ex vivo administration), for the purpose of curing, relieving, palliating, alleviating, altering, correcting, ameliorating, improving, or affecting the condition contemplated herein and / or symptoms of the condition contemplated herein. Such treatments may be specifically adapted or modified based on knowledge gained from the field of pharmacogenomics.

[0039] The term "CRISPR / Cas" or "clustered regularly interspaced short palindromic repeats" or "CRISPR" refers to DNA loci that contain short repeats of base sequences preceded by short spacer DNA segments derived from previous exposure to viruses or plasmids. Bacteria and archaea have evolved adaptive immune defenses called CRISPR / CRISPR-associated (Cas) systems that use short RNAs to direct the degradation of foreign nucleic acids. In bacteria, the CRISPR system provides adaptive immunity against invading foreign DNA through RNA-guided DNA cleavage.

[0040] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound useful within the scope of the present disclosure and a pharma- ceutical acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds to patients or subjects. In the art, there are multiple techniques for administering compounds, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary and topical administration.

[0041] As used herein, the terms "effective amount," "pharmacologically effective amount," and "therapeutically effective amount" refer to a non-toxic but sufficient amount of an agent to provide a desired biological result. The result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. The appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0042] An "effective amount" of a delivery vehicle is an amount sufficient to effectively bind or deliver a compound.

[0043] As used herein, the term "efficacy" refers to the maximum effect (E) achieved within the range of an assay. max )

[0044] As used herein, the term "haloperidol" refers to 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]-1-(4-fluorophenyl)-butan-1-one, or a salt or solvate thereof.

[0045] As used herein, the term "pharmacologically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0046] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, involved in carrying or transporting a compound useful within the scope of the present disclosure within or to a patient so that it may perform its intended function. Typically, such a construct is carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound, useful within the scope of the present disclosure, not harmful to the patient, and acceptable. Some examples of materials which can serve as pharma- ceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic, compatible substances used in pharmaceutical preparations. As used herein, "pharmaceutically acceptable carriers" also include any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like, that are compatible with the activity of the compounds useful within the scope of the present disclosure and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. "Pharmaceutically acceptable carriers" may further include pharmaceutically acceptable salts of the compounds useful within the scope of the present disclosure.Other additional ingredients that may be included in pharmaceutical compositions used in the practice of the present disclosure are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.

[0047] As used herein, the phrase "pharmaceutical acceptable salt" refers to the salt of administered compound prepared from pharmaceutical acceptable non-toxic acid, including inorganic acid, organic acid, solvate, hydrate, or clathrate thereof.The examples of such inorganic acid are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, hexafluorophosphoric acid, citric acid, gluconic acid, benzoic acid, propionic acid, butyric acid, sulfosalicylic acid, maleic acid, lauric acid, malic acid, fumaric acid, succinic acid, tartaric acid, amsonic acid, pamoic acid, p-toluenesulfonic acid, and mesylic acid. Suitable organic acids may be selected, for example, from the aliphatic, aromatic, carboxylic, and sulfonic classes of organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, camphorsulfonic acid, citric acid, fumaric acid, gluconic acid, isethionic acid, lactic acid, malic acid, mucic acid, tartaric acid, paratoluenesulfonic acid, glycolic acid, glucuronic acid, maleic acid, furoic acid, glutamic acid, benzoic acid, anthranilic acid, salicylic acid, phenylacetic acid, mandelic acid, embonic (pamoic) acid, methanesulfonic acid, ethanesulfonic acid, pantothenic acid, benzenesulfonic acid (besylate), stearic acid, sulfanilic acid, alginic acid, galacturonic acid, etc. Additionally, pharmaceutically acceptable salts include, by way of non-limiting example, alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium or calcium), and ammonium salts.

[0048] As used herein, the term "efficacy" refers to the dose required to produce half of the maximal response (ED 50 )

[0049] As used herein, the term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, an alkyl group having the designated number of carbon atoms (i.e., C 1~6 means 1 to 6 carbon atoms) and includes linear or branched chain hydrocarbons, including linear, branched, or cyclic substituents. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. Most preferred are (C1-C6) alkyl, especially ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl, and cyclopropylmethyl.

[0050] As used herein, the term "substituted alkyl" refers to an alkyl as defined above substituted with one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH2, -N(CH3)2, -C(=O)OH, trifluoromethyl, -C≡N, -C(=O)O(C1-C4)alkyl, -C(=O)NH2, -SON2NH2, -C(=NH)NH2, and -NO2, preferably selected from halogen, -OH, alkoxy, -NH2, trifluoromethyl, -N(CH3)2, and -C(=O)OH, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyl include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl, and 3-chloropropyl.

[0051] As used herein, the term "heteroalkyl", by itself or in combination with another term, means, unless otherwise specified, a stable straight or branched chain alkyl group consisting of the indicated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen atom may be optionally quaternized. The heteroatom may be located at any position of the heteroalkyl group, including between the remainder of the heteroalkyl group and the fragment to which it is attached, as well as the bond to the most terminal carbon atom of the heteroalkyl group. Examples include -OCH2CH2CH3, -CH2CH2CH2OH, -CH2CH2NHCH3, -CH2SCH2CH3, and -CH2CH2S(=O)CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2NHOCH3 or -CH2CH2SSCH3.

[0052] As used herein, the term "alkoxy", used alone or in combination with other terms, unless otherwise specified, refers to an alkyl group having the specified number of carbon atoms, as defined above, linked to the remainder of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and higher homologs and isomers. Preferred are (C1-C3)alkoxy, especially ethoxy and methoxy.

[0053] The terms "halo" or "halogen," as used herein, alone or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably fluorine, chlorine, or bromine, and more preferably fluorine or chlorine.

[0054] As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic, non-aromatic group, in which the atoms forming the ring (i.e., the skeletal atoms) are each carbon atoms. In certain embodiments, the cycloalkyl group is saturated or partially unsaturated. In another embodiment, the cycloalkyl group is fused to an aromatic ring. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include the following moieties: Examples include, but are not limited to, TIFF2024504632000006.tif46133.

[0055] Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyls include adamantane and norbornane. The term cycloalkyl includes "unsaturated non-aromatic carbocyclyl" or "non-aromatic unsaturated carbocyclyl" groups, both of which refer to a non-aromatic carbocycle, as defined herein, containing at least one carbon-carbon double bond or one carbon-carbon triple bond.

[0056] As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a heteroalicyclic group containing 1 to 4 ring heteroatoms selected from O, S, and N, respectively. In certain embodiments, each heterocycloalkyl group has 4 to 10 atoms in its ring system, provided that the ring of the group does not contain two adjacent O or S atoms. In another embodiment, the heterocycloalkyl group is fused to an aromatic ring. In certain embodiments, the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The heterocyclic ring system may be attached at any heteroatom or carbon atom that provides a stable structure, unless otherwise specified. The heterocyclic ring may be aromatic or non-aromatic in nature. In certain embodiments, the heterocyclic ring is a heteroaryl.

[0057] Examples of 3-membered heterocycloalkyl groups include, but are not limited to, aziridine. Examples of 4-membered heterocycloalkyl groups include, but are not limited to, azetidine and beta-lactam. Examples of 5-membered heterocycloalkyl groups include, but are not limited to, pyrrolidine, oxazolidine, and thiazolidinedione. Examples of 6-membered heterocycloalkyl groups include, but are not limited to, piperidine, morpholine, and piperazine. Other non-limiting examples of heterocycloalkyl groups are The file is TIFF2024504632000007.tif74133.

[0058] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepine, and hexamethylene oxide.

[0059] As used herein, the term "aromatic" refers to a carbocyclic or heterocyclic ring having one or more polyunsaturated rings and having aromatic character, i.e., having (4n+2) delocalized π (pi) electrons, where n is an integer.

[0060] As used herein, the term "aryl", used alone or in combination with other terms, means, unless otherwise specified, a carbocyclic aromatic system containing one or more rings (typically 1, 2 or 3 rings), where such rings may be linked together in a pendant manner, such as in biphenyl, or may be fused, such as in naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl. Preferred examples are phenyl and naphthyl, with phenyl being the most preferred.

[0061] As used herein, the term "aryl-(C1-C3)alkyl" refers to a functional group in which a 1-3 carbon alkylene chain is attached to an aryl group, e.g., -CH2CH2-phenyl. Preferred are aryl-CH2- and aryl-CH(CH3)-. The term "substituted aryl-(C1-C3)alkyl" refers to an aryl-(C1-C3)alkyl functional group in which the aryl group is substituted. Preferred is substituted aryl(CH2)-. Similarly, the term "heteroaryl-(C1-C3)alkyl" refers to a functional group in which a 1-3 carbon alkylene chain is attached to a heteroaryl group, e.g., -CH2CH2-pyridyl. Preferred is heteroaryl-(CH2)-. The term "substituted heteroaryl-(C1-C3)alkyl" refers to a heteroaryl-(C1-C3)alkyl functional group in which the heteroaryl group is substituted. Preferred is substituted heteroaryl-(CH2)-.

[0062] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. Polycyclic heteroaryls may contain one or more rings that are partially saturated. Examples include the following moieties: An example is TIFF2024504632000008.tif50138.

[0063] Examples of heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (especially 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (especially 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (especially 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-trizolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

[0064] Examples of polycyclic heterocycles and heteroaryls include indolyl (especially 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (especially 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (especially 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (especially Examples of such radicals include benzothienyl (especially 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (especially 3-, 4-, 5-, 6- and 7-benzothienyl), benzoxazolyl, benzothiazolyl (especially 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (especially 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolidinyl, and quinolidinyl.

[0065] As used herein, the term "substituted" means that an atom or group of atoms replaces hydrogen as a substituent bonded to another group. The term "substituted" further refers to any level of substitution where such substitution is permitted, i.e., mono-, di-, tri-, tetra-, or penta-substitution. The substituents are independently selected and the substitution can be at any chemically feasible position. In certain embodiments, the number of substituents varies between 1 and 4. In another embodiment, the number of substituents varies between 1 and 3. In yet another embodiment, the number of substituents varies between 1 and 2.

[0066] As used herein, the term "optionally substituted" means that the referenced group may be substituted or unsubstituted.In certain embodiments, the referenced group may be substituted with zero substituents, i.e., the referenced group is unsubstituted.In another embodiment, the referenced group may be substituted with one or more additional groups, each independently selected from the groups described herein.

[0067] In certain embodiments, a substituent is oxo, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, alkyl (including linear, branched and / or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoroalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=O)2 alkyl, -C(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl. and -C(NH)[substituted or unsubstituted alkyl]. In another embodiment, by way of example, the optional substituents are selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CH(CH3)2, -CF3, -CH2CF3, -OCH3, -OCH2CH3, -OCH(CH3)2, -OCF3, -OCH2CF3, -S(=O)2-CH3, -C(=O)NH2, -C(=O)-NHCH3, -NHC(=O)NHCH3, -C(=O)CH3, and -C(=O)OH. 1~6 Alkyl, -OH, C 1~6 In yet another embodiment, the substituents are independently selected from the group consisting of alkoxy, halo, amino, acetamido, oxo and nitro. 1~6 Alkyl, C 1~6 Independently selected from the group consisting of alkoxy, halo, acetamido, and nitro. As used herein, when a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight chain or cyclic, although straight chain is preferred.

[0068] Ranges: Throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0069] compound The compounds contemplated within this disclosure may be synthesized using techniques well known in the art of organic synthesis. The starting materials and intermediates required for the synthesis may be obtained from commercial sources or may be synthesized according to methods known to those skilled in the art.

[0070] In one aspect, compounds contemplated within the present disclosure are compounds of formula (I), or a salt, solvate, or N-oxide thereof: TIFF2024504632000009.tif27128 formula: Ring A is a monocyclic or bicyclic aryl ring or a monocyclic or bicyclic heteroaryl ring, wherein the aryl or heteroaryl ring is selected from the group consisting of 0 to 4 R 1 may be substituted with a group; R 1 Each of these is -C1-C6 alkyl, -C1-C6 fluoroalkyl, -C1-C6 heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR 3 , -SR 3 , -S(=O)R 3 , -S(=O)2R 3 , -NHS(=O)2R 3 , -C(=O)R 3 , -OC(=O)R 3 , -CO2R 3 , -OCO2R 3 , -CH(R3 )2, -N(R 3 )2, -C(=O)N(R 3 )2, -OC(=O)N(R 3 )2, -NHC(=O)NH(R 3 ), -NHC(=O)R 3 , -NHC(=O)OR 3 , -C(OH)(R 3 )2, and -C(NH2)(R 3 )2 independently selected from the group consisting of; R 2 each is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 heteroalkyl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), where the alkyl, heteroalkyl, or cycloalkyl group is selected from 0 to 5 R 1 or may be substituted with X 3 and R 2 together, 0 to 2 R 1 forming a (C3-C7) heterocycloalkyl group which may be substituted by a group; R 3 each is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 heteroalkyl, aryl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), where an alkyl, heteroalkyl, aryl, or cycloalkyl group is selected from 0 to 5 R 1 may be substituted with a group; X 1 is -CH2-, -S-, -O- or -(NR 2 )-and; X 2 is =CH2, =S, =O or =NR 2 and X 3 is -S-, -O-, or -NR 2 -It is.

[0071] In certain embodiments, ring A is selected from 0 to 4 R 1In another embodiment, ring A is an optionally substituted monocyclic aryl or heteroaryl ring. In another embodiment, ring A is unsubstituted. In yet another embodiment, ring A is phenyl or substituted phenyl.

[0072] In a preferred embodiment, X 1 and X 3 are both -NH-, and X 2 is =NH.

[0073] In another aspect, compounds contemplated within the present disclosure are compounds of formula (II) or a salt, solvate, or N-oxide thereof: R A -R B (II) During the ceremony, R A teeth TIFF2024504632000010.tif20128; X 4 is selected from the group consisting of methoxy, F, Cl, Br, and I; and R B teeth: TIFF2024504632000011.tif108128.

[0074] In another aspect, compounds contemplated within the present disclosure are compounds of formula (III), or a salt, solvate, or N-oxide thereof: TIFF2024504632000012.tif20128 formula: R 1 and R 2 Each of these is -C1-C6 alkyl, -C1-C6 fluoroalkyl, -C1-C6 heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR 5 , -SR 5 , -S(=O)R 5 , -S(=O)2R 5 , -NHS(=O)2R 5 , -C(=O)R 5 , -OC(=O)R5 , -CO2R 5 , -OCO2R 5 , -CH(R 5 )2, -N(R 5 )2, -C(=O)N(R 5 )2, -OC(=O)N(R 5 )2, -NHC(=O)NH(R 5 ), -NHC(=O)R 5 , -NHC(=O)OR 5 , -C(OH)(R 5 )2, and -C(NH2)(R 5 )2 independently selected from the group consisting of; R 3 is selected from the group consisting of -C1-C6 alkyl, -C1-C6 fluoroalkyl, -C1-C6 alkoxy, F, Cl, Br, and I; R 4 is selected from the group consisting of -C1-C6 alkyl, -C1-C6 alkoxy, F, Cl, Br, and I; R 5 each is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 heteroalkyl, aryl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), where the alkyl, heteroalkyl, aryl, or cycloalkyl group may be optionally substituted; X is selected from the group consisting of CH2, C=O, or O; n is an integer from 1 to 3; x is an integer from 0 to 4; and y is an integer from 0 to 4.

[0075] In certain embodiments, compounds contemplated within the present disclosure are selected from the group consisting of: 1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A; also known as JMS-51-58 or 51-58); 1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (compound B); 1-(n-propyl)-3-(4-iodophenyl)guanidine (compound C); 1-(n-propyl)-3-(4-methoxyphenyl)guanidine (compound D); 1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (compound E); 1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (compound F); 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G, also referred to herein as CT-189 or CT-110); 1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine (compound H); a salt, solvate, or N-oxide thereof; and any combination thereof.

[0076] In some aspects, compounds contemplated within the present disclosure include 1-(4-iodophenyl)-3-(2-adamantyl)guanidine (IPAG).

[0077] Preparation of compounds Compounds contemplated within this disclosure may be prepared according to the general schemes described herein using synthetic methods known to those skilled in the art. The following examples illustrate non-limiting aspects of the disclosure.

[0078] In non-limiting embodiments, the synthesis of unsymmetrical N,N'-disubstituted guanidines is accomplished by coupling aryl cyanamides and amines. In certain embodiments, the coupling reaction occurs at elevated temperatures ranging from 80°C to 250°C. Anilines may be converted to aryl cyanamides with cyanogen bromide in ether. Unsymmetrical N,N'-disubstituted guanidines are then generated by coupling aryl cyanamides with amines. Non-limiting examples of coupling methods include heating at reflux in acetonitrile and heating at 120°C in a microwave. TIFF2024504632000013.tif21128

[0079] In another non-limiting embodiment, unsymmetrical N,N'-disubstituted guanidines may be synthesized by coupling benzimidothioates and amines. For example, anilines may be reacted with potassium isothiocyanate to give thioureas. The thioureas may then be treated with methyl iodide in acetone heated to reflux to give the desired benzimidothioates. Unsymmetrical N,N'-disubstituted guanidines may then be produced by coupling benzimidothioates with amines. Non-limiting examples of coupling methods include heating in ethanol at reflux. TIFF2024504632000014.tif23128

[0080] The compounds of the present disclosure may have one or more stereocenters, and each stereocenter may exist independently in either R or S configuration. In certain embodiments, the compounds described herein exist in optically active or racemic forms. It should be understood that the compounds described herein include racemates, optically active forms, positional and stereoisomers, or combinations thereof, that have the therapeutically useful properties described herein. Preparation of optically active forms is accomplished in any suitable manner, including, by way of non-limiting example, resolution by recrystallization techniques of racemates, synthesis from optically active starting materials, or chromatographic separation using chiral stationary phases. In certain embodiments, a mixture of one or more isomers is used as the therapeutic compound described herein. In another embodiment, the compounds described herein include one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis, and / or separation of mixtures of enantiomers and / or diastereomers. Resolution of the compounds and their isomers can be accomplished by any means, including, but not limited to, chemical methods, enzymatic methods, fractional crystallization, distillation, and chromatography.

[0081] The methods and formulations described herein include the use of N-oxides (where appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and / or pharmaceutically acceptable salts of compounds having the structure of any of the compounds disclosed herein, as well as metabolites and active metabolites of these compounds having the same type of activity.Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates, and the like.In certain embodiments, the compounds described herein exist in solvated form with pharmaceutically acceptable solvents such as water and ethanol.In another embodiment, the compounds described herein exist in unsolvated form.

[0082] In certain embodiments, the compounds of the present disclosure may exist in tautomeric forms, and all tautomeric forms are included within the scope of the compounds presented herein.

[0083] In certain embodiments, the compounds described herein are prepared as prodrugs. "Prodrug" refers to an agent that is converted to parent drug in vivo.In certain embodiments, after in vivo administration, prodrug is chemically converted to biologically, pharmacologic or therapeutically active form of compound.In another embodiment, prodrug is enzymatically metabolized to biologically, pharmacologic or therapeutically active form of compound by one or more steps or processes.

[0084] In certain embodiments, for example, sites on the aromatic ring portion of the compounds of the present disclosure are susceptible to various metabolic reactions.By incorporating suitable substituents on the aromatic ring structure, this metabolic pathway may be reduced, minimized, or eliminated.In certain embodiments, suitable substituents for reducing or eliminating the susceptibility of the aromatic ring to metabolic reactions are, by way of example only, deuterium, halogen, or alkyl groups.

[0085] The compounds described herein also include isotopically labeled compounds in which one or more atoms are replaced with an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include: 2 H, 3 H, 11 C. 13 C. 14 C. 36 Cl, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O. 17 O. 18 O. 32 P, and 35 In certain embodiments, isotope-labeled compounds are useful in drug and / or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium provides greater metabolic stability (e.g., increased in vivo half-life or reduced dose required). In yet another embodiment, 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes, such as N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds are prepared by any suitable method or process, employing an appropriate isotopically labeled reagent in place of a non-labeled reagent that would otherwise be used.

[0086] In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0087] The compounds described herein, and other related compounds having different substituents, are synthesized using techniques and materials described herein and, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4 th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999), all of which are incorporated herein by reference for such disclosures. The general methods for the preparation of the compounds described herein are modified by the use of appropriate reagents and conditions to introduce the various moieties found in the formulas provided herein.

[0088] The compounds described herein are synthesized using any suitable means, starting from compounds available from commercial sources or prepared using the procedures described herein.

[0089] In certain embodiments, reactive functional groups, such as hydroxyl, amino, imino, thio or carboxy groups, are protected to avoid their deleterious participation in reactions. Protecting groups are used to block some or all of the reactive moieties, preventing such groups from participating in chemical reactions until the protecting group is removed. In another embodiment, each protecting group is removable by a different means. Protecting groups that are cleaved under completely different reaction conditions meet the requirements for differential removal.

[0090] In certain embodiments, the protecting groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and / or oxidative conditions. Groups such as trityl, dimethoxytrityl, acetal, and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with hydrogenolysis-removable Cbz and base-labile Fmoc groups. Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl in the presence of amines blocked with acid labile groups such as t-butyl carbamate, or carbamates that are both acid and base stable, but removable by hydrolysis.

[0091] In certain embodiments, carboxylic acid and hydroxy reactive groups are blocked with hydrolytically removable protecting groups such as benzyl groups, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc. Carboxylic acid reactive moieties are protected by conversion to simple esters exemplified herein, including conversion to alkyl esters, or are blocked with oxidatively removable protecting groups such as 2,4-dimethoxybenzyl, while coexisting amino groups are blocked with fluoride labile silyl carbamates.

[0092] Allyl blocking groups are useful in the presence of acid and base protecting groups because they are stable and subsequently removed by metal or pi-acid catalysis. For example, allyl-blocked carboxylic acids are deprotected with palladium catalysis in the presence of acid labile t-butyl carbamate or base labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, the functional group is blocked and will not react. Once released from the resin, the functional group is available to react.

[0093] Typically, the blocking / protecting groups are TIFF2024504632000015.tif75129.

[0094] Detailed descriptions of other protecting groups and applicable techniques for the creation of protecting groups and their removal are described in Greene & Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are incorporated herein by reference for such disclosures.

[0095] Nucleic acid inhibitors / regulators In certain embodiments, the sigma 1 regulator comprises an isolated nucleic acid. In other embodiments, the regulator is an RNAi molecule (such as, but not limited to, siRNA and / or shRNA and / or miRNA) or an antisense molecule that inhibits sigma 1 expression and / or activity. In still other embodiments, the nucleic acid preferably comprises a promoter / regulatory sequence that can direct the expression of the nucleic acid. Thus, the present disclosure provides expression vectors and methods for the introduction of exogenous DNA into cells with co-expression of the exogenous DNA in the cells, such as those described in, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), and elsewhere herein.

[0096] In certain embodiments, sigma 1 can be inhibited by inactivating and / or sequestering sigma 1. Thus, inhibition of sigma 1 activity can be achieved by using transdominant negative mutants.

[0097] In certain embodiments, siRNA is used to reduce levels of sigma 1. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a variety of organisms and cell types leads to the degradation of complementary mRNA. Inside the cell, a ribonuclease known as Dicer cleaves the long dsRNA into short, 21-25 nucleotide small interfering RNAs, or siRNAs. The siRNAs then assemble with protein components into RNA-induced silencing complexes (RISCs), unwinding in the process. The activated RISC then binds to the complementary transcripts through base-pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, and sequence-specific degradation of the mRNA leads to gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describe chemical modifications to siRNA that aid in intravenous systemic delivery. To optimize siRNA, it is necessary to consider the overall G / C content, the C / T content at the end, Tm and the nucleotide content of 3' overhang.See, for example, Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216.Therefore, the present disclosure also includes the method of reducing the level of sigma 1 using RNAi technology.

[0098] In certain embodiments, the present disclosure provides a vector comprising an siRNA or antisense polynucleotide. In other embodiments, the siRNA or antisense polynucleotide inhibits the expression of sigma 1. The incorporation of a desired polynucleotide into a vector and the selection of the vector are well known in the art.

[0099] In certain embodiments, the expression vector described herein encodes a short hairpin RNA (shRNA) inhibitor.shRNA inhibitors are well known in the art and are directed to target mRNA, thereby reducing the expression of the target.In certain embodiments, the encoded shRNA is expressed by cells and then processed into siRNA.For example, in some cases, cells possess natural enzymes (e.g., dicer) that cleave shRNA to form siRNA.

[0100] The siRNA, shRNA, or antisense polynucleotides can be cloned into several types of vectors, as described elsewhere herein. For expression of the siRNA or antisense polynucleotide, at least one module in each promoter functions to position the start site of RNA synthesis.

[0101] To evaluate the expression of siRNA, shRNA or antisense polynucleotide, the expression vector introduced into the cell can also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected with the viral vector.In certain embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure.Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences that allow expression in the host cell.Useful selectable markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin resistance.

[0102] Therefore, in another aspect, the present disclosure relates to a vector comprising the nucleotide sequence of the present disclosure or the construct of the present disclosure. The choice of vector will depend on the host cell into which the vector is subsequently introduced. In certain embodiments, the vector of the present disclosure is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors and mammalian cell vectors. Systems based on prokaryotic and / or eukaryotic vectors can be employed to produce polynucleotides, or their cognate polypeptides, with the present disclosure. Many of such systems are commercially available and widely available.

[0103] Furthermore, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector comprises an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers. (See, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193.)

[0104] The viral vector can be any suitable adeno-associated virus (AAV), such as the AAV1-AAV8 family of adeno-associated viruses. In some embodiments, the viral vector is a viral vector capable of infecting humans. The desired nucleic acid sequence can be inserted between the inverted terminal repeats (ITRs) in the AAV. In various embodiments, the viral vector is AAV8. The promoter can be a thyroxine-binding globulin (TBG) promoter. In various embodiments, the promoter is a human promoter sequence that allows for expression of the desired nucleic acid in the liver. The AAV can be a recombinant AAV, in which the capsid is derived from one AAV serotype and the ITRs are derived from another AAV serotype. In various embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8 capsids. In various embodiments, the ITR in AAV is at least one ITR selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8 ITR.In various embodiments, the present disclosure contemplates an AAV8 viral vector (recombinant or non-recombinant) that comprises a desired nucleic acid expression sequence and at least one promoter sequence that causes the systemic expression of the desired nucleic acid to increase when administered to a subject.In some embodiments, the viral vector is a recombinant or non-recombinant AAV2 or AAV5 that comprises any of the desired nucleic acid expression sequences described herein.

[0105] By way of example, the vector into which the nucleic acid sequence is introduced can be a plasmid that may or may not integrate into the genome of the host cell when introduced into the cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequences of the present disclosure or the genetic constructs of the present disclosure can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0106] The vector may be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0107] In certain embodiments, the recombinant expression vector may also include a nucleic acid molecule encoding a peptide or peptidomimetic inhibitor of the present disclosure, as described elsewhere herein.

[0108] A promoter may be one that is naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages may be gained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. Similarly, a recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., those that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically producing promoter and enhancer nucleic acid sequences, nucleic acid amplification techniques, including recombinant cloning and / or PCR™, may be used in conjunction with the compositions disclosed herein to produce the sequences (U.S. Pat. Nos. 4,683,202 and 5,928,906).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, and the like, may similarly be utilized.

[0109] It will be important to utilize a promoter and / or enhancer that efficiently directs the expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use a combination of promoters, enhancers, and cell types for protein expression. The promoter utilized may be constitutive, tissue-specific, inducible, and / or useful under suitable conditions to direct high-level expression of the introduced DNA segment, which is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0110] Recombinant expression vector may contain a selectable marker gene that facilitates the selection of transformed or transfected host cells.Suitable selectable marker genes are G418 and hygromycin, which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or genes that code proteins such as immunoglobulins or parts thereof, such as the Fc part of immunoglobulins, preferably IgG.Selectable markers may be introduced into a vector separate from the nucleic acid of interest.

[0111] After generation of a siRNA polynucleotide, one of skill in the art will appreciate that the siRNA polynucleotide has certain properties that can be modified to improve the siRNA as a therapeutic compound. Therefore, siRNA polynucleotides may be further designed to resist degradation by modification to include phosphorothioate or other linkages, methylphosphonates, sulfones, sulfates, ketyl, phosphorodithioates, phosphoramidates, phosphate esters, and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett. 26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117). (1989)).

[0112] Any polynucleotide may be further modified to enhance stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends; the use of phosphorothioate or 2' O-methyl linkages rather than phosphodiester linkages in the backbone; and / or the inclusion of acetyl, methyl, thio and other modified forms of adenine, cytidine, guanine, thymine and uridine, as well as non-traditional bases such as inosine, queosine and wybutosine.

[0113] In certain embodiments, antisense nucleic acid sequences expressed by plasmid vectors are used to inhibit expression of sigma 1 protein. Antisense expression vectors are used to transfect mammalian cells or the mammal itself, thereby causing a reduction in the endogenous expression of sigma 1.

[0114] Antisense molecules and their use for inhibiting gene expression are well known in the art (see, for example, Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press).Antisense nucleic acid, as that term is defined elsewhere herein, is a DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40).Intracellularly, antisense nucleic acid hybridizes with corresponding mRNA, forming a double-stranded molecule and inhibiting the translation of genes.

[0115] The use of antisense methods to inhibit the translation of genes is known in the art and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules may be provided to cells via gene expression using DNA encoding the antisense molecule, as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

[0116] Alternatively, the antisense molecules of the present disclosure may be made synthetically and then provided to cells. Antisense oligomers of about 10 to about 30 nucleotides, more preferably about 15 nucleotides, are preferred as they are easily synthesized and introduced into target cells. Synthetic antisense molecules contemplated by the present disclosure include oligonucleotide derivatives known in the art that have improved biological activity compared to unmodified oligonucleotides (see U.S. Pat. No. 5,023,243).

[0117] In certain embodiments, ribozymes are used to inhibit the expression of sigma 1 protein. Ribozymes useful for inhibiting the expression of target molecules can be designed, for example, by incorporating a target sequence complementary to the mRNA sequence encoding sigma 1 into the basic ribozyme structure. Ribozymes targeting sigma 1 can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, Calif.) or genetically expressed from DNA that encodes them.

[0118] CRISPR / Cas9 The CRISPR / Cas9 system is a simple and efficient system for inducing targeted gene modifications. Target recognition by the Cas9 protein requires a conserved dinucleotide containing a "seed" sequence within the guide RNA (gRNA) and a protospacer adjacent motif (PAM) sequence upstream of the gRNA binding region. The CRISPR / Cas9 system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein and two or more gRNAs, making the system uniquely suitable for multiple gene editing or synergistic activation of target genes.

[0119] The Cas9 protein and guide RNA form a complex that identifies and cleaves the target sequence. Cas9 is composed of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC. The RecI domain binds to the guide RNA, and the Bridge helix binds to the target DNA. The HNH and RuvC domains are nuclease domains. The guide RNA is engineered to have a 5' end that is complementary to the target DNA sequence. Binding of the guide RNA to the Cas9 protein causes a conformational change that activates the protein. Once activated, Cas9 searches for the target DNA by binding to a sequence that matches its protospacer adjacent motif (PAM) sequence. The PAM is a two- or three-nucleotide base sequence within one nucleotide downstream of the region that is complementary to the guide RNA. In one non-limiting example, the PAM sequence is 5'-NGG-3'. When the Cas9 protein finds its target sequence with the appropriate PAM, it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. The RuvC and HNH nuclease domains then cleave the target DNA starting at the third nucleotide base upstream of the PAM.

[0120] One non-limiting example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in US Patent Application Publication No. US20140068797. CRISPRi utilizes an RNA-guided Cas9 endonuclease to introduce DNA double-strand breaks, inducing permanent gene disruption that triggers an error-prone repair pathway that results in frameshift mutations. Catalytically dead Cas9 lacks endonuclease activity. When co-expressed with a guide RNA, it generates a DNA recognition complex that specifically interferes with transcription elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently silences the expression of target genes.

[0121] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific to a target gene and a Cas endonuclease are introduced into a cell to form a complex that allows the Cas endonuclease to introduce double-strand breaks into the target gene. In certain embodiments, the CRISPR / Cas system comprises an expression vector, such as, but not limited to, the pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces the expression of Cas9 endonuclease. Other endonucleases may be used, including, but not limited to, T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleases known in the art, and any combination thereof.

[0122] In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector comprises an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, such as doxycycline). However, it should be understood that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, such as an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.

[0123] In certain embodiments, guide RNA and Cas9 can be delivered to cells as a ribonucleoprotein (RNP) complex. RNPs are composed of purified Cas9 protein complexed with gRNA and are well known in the art to be efficiently delivered to multiple types of cells, including but not limited to stem cells and immune cells (Addgene, Cambridge, MA, Mirus Bio LLC, Madison, WI).

[0124] Guide RNA is specific to the genome region of interest and targets the region with Cas endonuclease-induced double-strand break.The target sequence of guide RNA sequence can be in the locus of gene or in the non-coding region of genome.In certain embodiments, guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

[0125] Guide RNAs (gRNAs), also referred to as "short guide RNAs" or "sgRNAs", provide both targeting specificity and scaffolding / binding ability to Cas9 nuclease. gRNAs can be synthetic RNAs composed of targeting and scaffolding sequences derived from endogenous bacterial crRNAs and tracrRNAs. gRNAs are used to target Cas9 to specific genomic loci in genome engineering experiments. Guide RNAs can be designed using standard tools well known in the art.

[0126] In the context of the formation of a CRISPR complex, "target sequence" refers to a sequence that the guide sequence is designed to have a degree of complementarity with, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. In certain embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be in an organelle of a eukaryotic cell, such as a mitochondria or nucleus. Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (comprising a guide sequence hybridized with the target sequence and complexed with one or more Cas proteins) causes cleavage of one or both strands within or near the target sequence (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs). As with target sequences, it is believed that perfect complementarity is not required, provided it is sufficient for function.

[0127] In certain embodiments, one or more vectors are introduced into a host cell that drive the expression of one or more elements of the CRISPR system, such that the expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors that provide any components of the CRISPR system that are not included in the first vector. The CRISPR system elements that are combined in a single vector can be arranged in any suitable orientation, such as one element being located 5' to the second element ("upstream" of the second element) or 3' to the second element ("downstream" of the second element). The coding sequence of one element can be located on the same strand or on the opposite strand as the coding sequence of the second element, and can be oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of a guide sequence, a tracr mate sequence (which may be operably linked to a guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).

[0128] In certain embodiments, the CRISPR enzyme is part of a fusion protein that includes one or more heterologous protein domains (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to the CRISPR enzyme). The CRISPR enzyme fusion protein may include any additional protein sequence and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Additional domains that can form part of fusion proteins that include CRISPR enzymes are described in U.S. Patent Application Publication No. US20110059502, which is incorporated herein by reference. In certain embodiments, the tagged CRISPR enzyme is used to identify the location of the target sequence.

[0129] Conventional virus and non-virus based gene transfer methods can be used to introduce nucleic acid into mammalian and non-mammalian cells or target tissues.Such methods can be used to administer the nucleic acid encoding the components of CRISPR system to cultured cells or cells in host organisms.Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcription products of vectors described herein), naked nucleic acid, and nucleic acid complexed with delivery vehicles such as liposomes.Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery to cells (Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy 1:13-26).

[0130] In certain embodiments, the CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species.

[0131] Generally, Cas proteins contain at least one RNA recognition domain and / or RNA binding domain. The RNA recognition domain and / or RNA binding domain interact with the guide RNA. Cas proteins can also contain nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, and other domains. Cas proteins can be modified to increase nucleic acid binding affinity and / or specificity, alter enzymatic activity, and / or change another property of the protein. In certain embodiments, the Cas-like protein of the fusion protein can be derived from a wild-type Cas9 protein or a fragment thereof. In other embodiments, the Cas can be derived from a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, domains of the Cas9 protein that are not involved in RNA-guided cleavage can be omitted from the protein such that the modified Cas9 protein is smaller than the wild-type Cas9 protein. Generally, Cas9 protein comprises at least two nuclease (i.e., DNase) domains. For example, Cas9 protein can comprise RuvC-like nuclease domain and HNH-like nuclease domain. RuvC domain and HNH domain cooperate to break single strand and generate double strand break in DNA (Jinek, et al., 2012, Science, 337:816-821). In certain embodiments, Cas9-derived protein can be modified to contain only one functional nuclease domain (either RuvC-like or HNH-like nuclease domain). For example, Cas9-derived protein can be modified to delete or mutate one of the nuclease domains so that it is no longer functional (i.e., no nuclease activity).In some embodiments in which one of the nuclease domains is inactive, the Cas9 derived protein can introduce nicks into double-stranded nucleic acids (such proteins are referred to as "nickases") but do not cleave double-stranded DNA. In any of the above embodiments, any or all of the nuclease domains can be inactivated by one or more deletion, insertion, and / or substitution mutations using well-known methods such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.

[0132] In one non-limiting embodiment, the vector drives the expression of CRISPR system. The art is rich in suitable vectors that are useful in the present disclosure. The vector used is suitable for replication and optionally integration in eukaryotic cells. Typical vectors include transcription and translation terminators, initiation sequences and promoters that are useful for regulating the expression of desired nucleic acid sequences. The vector of the present disclosure can be used in standard gene delivery protocols for nucleic acids. Methods for gene delivery are known in the art (U.S. Patent Nos. 5,399,346, 5,580,859 and 5,589,466, which are incorporated herein by reference in their entirety).

[0133] Furthermore, vectors may be provided to cells in the form of viral vectors.Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4th Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012) and other virology and molecular biology manuals.Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis viruses, gamma retroviruses and lentiviruses.In general, suitable vectors include an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0134] method The present disclosure includes a method of treating, preventing and / or ameliorating lipid droplet (LD) accumulation in cells. The present disclosure further includes a method of treating, preventing and / or ameliorating fatty liver and / or inflammation associated with steatohepatitis in a subject. The present disclosure includes a method of treating, preventing and / or ameliorating non-alcoholic fatty liver disease (NAFLD) in a subject. The present disclosure also includes a method of reversing, slowing or preventing the progression of NAFLD to non-alcoholic steatohepatitis (NASH). The present disclosure also includes a method of reversing, slowing or preventing the progression of NASH to hepatocellular carcinoma (HCC). The present disclosure also includes a method of determining and / or assessing the risk of a subject suffering from NAFLD progressing to NASH. The present disclosure also includes a method of determining and / or assessing the risk of a subject suffering from NASH progressing to HCC.

[0135] In certain embodiments, the method comprises administering to a subject an effective amount of at least one compound and / or composition contemplated within the present disclosure.In other embodiments, the subject is further administered at least one additional agent that treats or prevents the disease and / or disorder contemplated herein.In yet other embodiments, the compound and at least one additional agent are co-administered to the subject.In yet other embodiments, the compound and at least one additional agent are co-formulated.

[0136] In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

[0137] Combination therapy The compounds contemplated within this disclosure are intended to be useful in combination with one or more additional compounds, which may include at least one additional antiviral agent and / or at least one additional agent that treats the compounds of this disclosure and / or one or more diseases or disorders contemplated herein.

[0138] Synergistic effects, for example, sigmoid-E max The combination of drugs may be calculated using any suitable method, such as the α-additive curve (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and median effective curve (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each of the above-mentioned formulas may be applied to experimental data to generate corresponding graphs to aid in the evaluation of drug combination effects. The corresponding graphs associated with the above-mentioned formulas are the concentration-effect curve, the isobologram curve and the combination index curve, respectively.

[0139] Administration / Dosage / Formulation Dosage schedules can affect what constitutes an effective amount. The therapeutic formulations contemplated within the present disclosure can be administered to the subject either before or after the onset of the disease and / or disorder contemplated herein. Furthermore, several divided doses and alternating doses can be administered once a day or sequentially, or the doses can be continuously infused or bolus injected. Furthermore, the doses of the therapeutic formulations contemplated within the present disclosure can be proportionally increased or decreased as indicated by the exigencies of the treatment or prevention situation.

[0140] The administration of the compositions contemplated within the present disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at a dosage and for a period of time effective to treat the disease and / or disorder contemplated herein in the patient. The effective amount of the therapeutic compound required to obtain a therapeutic effect may vary depending on factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the present disclosure to treat the disease and / or disorder contemplated herein in the patient. The administration schedule may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered once a day, or the dose may be proportionally reduced as dictated by the exigencies of the therapeutic situation. A non-limiting example of an effective dosage range of the therapeutic compound contemplated within the present disclosure is about 1 to 5,000 mg / kg body weight / day. One skilled in the art would be able to test the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0141] Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within this disclosure may be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration.

[0142] In particular, the selected dosage level will depend on a variety of factors, including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0143] A medical practitioner, e.g., a physician or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start doses of a compound contemplated within the present disclosure utilized in a pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0144] In certain embodiments, it is particularly advantageous to formulate the compound into dosage unit form for ease of administration and uniformity of dosage.As used herein, dosage unit form refers to a physically separate unit suitable as a unitary dose for the patient to be treated; each unit contains a predetermined amount of therapeutic compound calculated to produce a desired therapeutic effect together with the necessary pharmaceutical vehicle.The dosage unit form contemplated within the present disclosure is dictated by and directly depends on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the technical field of compounding / formulating such therapeutic compound for the treatment of the disease and / or disorder contemplated herein.

[0145] In certain embodiments, the compositions of the present disclosure are formulated with one or more pharma- ceutically acceptable excipients or carriers.In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a therapeutically effective amount of the compounds of the present disclosure and a pharma- ceutically acceptable carrier.

[0146] The carrier may be a solvent or dispersion medium, for example, containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent in the composition, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol. Prolonged absorption of the injectable composition may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0147] In certain embodiments, the compositions of the present disclosure are administered to a patient in a dosage range of 1 to 5 or more times per day. In another embodiment, the compositions of the present disclosure are administered to a patient in a dosage range including, but not limited to, once per day, once every 2 days, once every 3 days to once per week, and once every 2 weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the present disclosure will vary from individual to individual depending on many factors including, but not limited to, age, disease or disorder being treated, sex, general health, and other factors. Thus, the present disclosure should not be construed as being limited to any particular dosing regimen, and the exact dose and composition to be administered to any patient will be determined by the attending physician, taking into account all other factors related to the patient.

[0148] The compounds of the present disclosure for administration may range from about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 900 mg, about 90 mg to about 1000 mg, about 150 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, about 400 mg to about 500 mg, about 50 mg to about 600 mg, about 600 mg to about 750 mg, about 70 mg to about 600 mg, about 800 mg to about 800 mg, about 900 mg to about 1000 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, about 400 mg to about 500 mg, about 500 mg to about 600 mg, about The range may be from about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0149] In some embodiments, the dosage of the disclosed compound is about 1 mg to about 2,500 mg. In some embodiments, the dosage of the disclosed compound used in the compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0150] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the present disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a sigma receptor-associated disorder or disease in a patient.

[0151] The formulations may be used in admixture with conventional excipients, i.e., pharma- ceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration known in the art. The pharmaceutical preparations are sterilized and, if desired, may be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and / or aromatic substances, etc. These may, if desired, be combined with other active substances, such as other analgesics.

[0152] The route of administration of any of the compositions of the present disclosure includes oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.The compounds for use in the present disclosure may be formulated for administration by any suitable route, such as oral or parenteral, for example, transdermal, transmucosal (e.g. sublingual, lingual, (trans)oral, (trans)urethral, ​​vaginal (e.g. vaginal and perivaginal), (intravaginal) and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation and topical administration.

[0153] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelcaps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions useful in the present disclosure are not limited to the specific formulations and compositions described herein.

[0154] Oral route For oral application, tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps are particularly suitable. Compositions intended for oral use may be prepared according to any method known in the art, and such compositions may contain one or more substances selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulating and disintegrating agents such as corn starch; binding agents such as starch; and lubricants such as magnesium stearate. Tablets may be uncoated or may be coated by known techniques for elegance or to delay the release of the active ingredient. Preparations for oral use may be provided as hard gelatin capsules, in which the active ingredient is mixed with an inert diluent.

[0155] For oral administration, the compounds of the present disclosure may be in the form of tablets or capsules prepared by conventional means with pharma- ceutically acceptable excipients, such as binders (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., corn starch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, tablets may be coated using a suitable method and coating material, such as the OPADRY™ film coating system available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type, and OPADRY™ White, 32K18400). Liquid preparations for oral administration may be in the form of solutions, syrups, or suspensions. Liquid preparations may be prepared by conventional means with pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible oils); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

[0156] The present disclosure also includes multi-layer tablets, which include a layer that provides delayed release of one or more compounds of the present disclosure and an additional layer that provides immediate release of another medicament. A wax / pH-sensitive polymer mixture may be used to obtain a gastric insoluble composition in which the active ingredient is trapped and ensures its delayed release.

[0157] Parenteral Administration For parenteral administration, the compounds of the disclosure may be formulated for injection or infusion, e.g., intravenous, intramuscular or subcutaneous injection or infusion, or for bolus administration and / or continuous infusion. Suspensions, solutions or emulsions in oily or aqueous vehicles, optionally containing other formulatory substances such as suspending, stabilizing and / or dispersing agents, may be used.

[0158] Additional Dosage Forms Additional dosage forms of the present disclosure include those described in U.S. Patent Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of the present disclosure also include those described in U.S. Patent Application Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of the present disclosure include those described in PCT Application Publication Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0159] Controlled Release Formulations and Drug Delivery Systems In certain embodiments, the formulations of the present disclosure may be short-term, fast offset, and controlled, including, but not limited to, sustained release, delayed release, and pulsed release formulations.

[0160] The term sustained release is used in its ordinary sense to refer to a drug formulation that provides for gradual release of drug over an extended period of time and can, but does not necessarily, result in substantially constant blood levels of drug over an extended period of time, which may be as long as a month or more and should be a longer release than the same amount of drug administered in bolus form.

[0161] For sustained release, the compound may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compound.Thus, the compound for use in the method of the present disclosure may be administered, for example, in the form of microparticles by injection or in the form of wafers or disks by implantation.

[0162] In certain embodiments of the present disclosure, the compounds of the present disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0163] The term delayed release is used herein in its ordinary sense to refer to drug formulations that provide an initial release of drug after some delay following drug administration, including, but not necessarily, a delay of from about 10 minutes up to about 12 hours.

[0164] The term pulsatile release is used herein in its ordinary sense to refer to a drug formulation that provides release of drug in a manner that results in a pulsatile plasma profile of the drug following drug administration.

[0165] The term immediate release is used in its ordinary sense to refer to a drug formulation that provides release of the drug immediately following drug administration.

[0166] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any or all whole or partial increments thereof.

[0167] As used herein, rapid offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any and all whole or partial increments thereof.

[0168] Administration The therapeutically effective amount or dose of the compound of the present disclosure depends on the age, sex and weight of the patient, the patient's current medical condition and the progression of the sigma receptor-related disorder or disease of the patient under treatment. Those skilled in the art can determine the appropriate dose depending on these and other factors.

[0169] Suitable doses of the compounds of the present disclosure may range from about 0.01 mg to about 5,000 mg per day, such as about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as from about 5 mg to about 250 mg per day. The dose may be administered in a single dose, or in multiple doses, for example, 1 to 4 or more times per day. When multiple doses are used, the amount of each dose may be the same or different. For example, a 1 mg dose per day may be administered as two 0.5 mg doses with an interval of about 12 hours between doses.

[0170] It is understood that the amount of compound administered daily can be, in non-limiting examples, administered every day, every other day, every second day, every third day, every fourth day, or every fifth day. For example, every other day administration can begin with a 5 mg dose on one day on Monday, followed by a 5 mg dose on the first subsequent day on Wednesday, a 5 mg dose on the second subsequent day on Friday, etc.

[0171] If the patient's condition improves, at the physician's discretion, administration of the disclosed modulators may optionally be given continuously; alternatively, the dose of the administered drug may be temporarily reduced or temporarily discontinued for a specified period of time (i.e., a "drug holiday"). The length of the drug holiday may optionally vary between 2 days and 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays include, by way of example only, 10% to 100%, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0172] Once the improvement of the patient's condition occurs, a maintenance dose is administered as necessary.Then, the dose or frequency of administration, or both, is reduced according to the patient's condition until the improved disease is maintained at a level.In certain embodiments, the patient requires intermittent treatment on a long-term basis after any recurrence of symptoms and / or infection.

[0173] The compounds for use in the methods of the present disclosure may be formulated into unit dosage forms. The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dose for a patient undergoing treatment, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect, optionally together with a suitable pharmaceutical carrier. The unit dosage form may be a single daily dose, or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each administration.

[0174] The toxicity and therapeutic efficacy of such treatment regimens are 50 (a dose lethal to 50% of the population) and ED 50These may be measured in cell cultures or experimental animals, including, but not limited to, measuring the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 and ED 50 The therapeutic index is expressed as a ratio between ED and ED . Capsid assembly regulators that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies may optionally be used in formulating various dosages for use in humans. The dosage of such capsid assembly regulators is preferably within the range of ED with minimal toxicity. 50 The dosage may optionally vary within this range depending upon the dosage form employed and the route of administration utilized.

[0175] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this disclosure and are included in the claims appended hereto. For example, modifications of assay and / or reaction conditions using no more than routine experimentation with art-recognized substitutes should be understood to be within the scope of this application.

[0176] Whenever values ​​and ranges are provided herein, it should be understood that all values ​​and ranges that fall within these values ​​and ranges are to be included within the scope of the present disclosure. Moreover, all values ​​that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

[0177] The examples described herein further illustrate aspects of the present disclosure, but are in no way a limitation of the teachings or disclosure described herein.

[0178] The examples described herein are provided for illustrative purposes only and, unless specifically stated, are not intended to be limiting. Thus, the present disclosure should in no way be construed as limited to the examples described herein, but rather to encompass any and all variations that become evident as a result of the teachings provided herein.

[0179] Without further description, it is believed that one of ordinary skill in the art can, using the present description and illustrative examples, make and utilize the disclosed compounds and practice the claimed methods. As such, the working examples illustrate selected aspects of the disclosure, and are not to be construed as limiting in any way the remainder of the disclosure. EXAMPLES

[0180] Experimental Examples The present disclosure will now be described with reference to the following examples, which are provided for illustrative purposes only, and the present disclosure is not limited to these examples, but rather includes all variations that become apparent as a result of the teachings provided herein.

[0181] The materials and methods utilized in these experiments are now described.

[0182] Example 1: Increased SigmaR1 mRNA levels are associated with non-alcoholic fatty liver disease The role of sigmaR1 / sigma1 in liver physiology and pathophysiology was unclear. However, the data presented herein demonstrates that sigmaR1 / sigma1 plays a central role in liver metabolism and stress response. SigmaR1 transcript levels are notably 2-9 times higher in liver compared to other organs (Figure 1A). SigmaR1 transcript levels are more than 2-fold elevated in fibrotic NASH liver compared to normal healthy liver (Figure 1B). Interestingly, as fibrotic NASH liver progresses to cirrhosis and HCC, SigmaR1 transcript levels are rapidly decreased (Figure 1B). In certain embodiments, this indicates that the role of sigma1 changes as NAFLD progresses from NASH to HCC.

[0183] The on-target action of sigma 1 regulators does not induce side effects (including weight loss and behavioral abnormalities), and selective sigma 1 antagonists / inhibitors do not show cytotoxicity in animal studies. Indeed, sigma R1 knockout (KO) mice are viable, fertile, and do not show any obviously different phenotype from wild-type mice (Langa, et al., 2003, Eur J Neurosci 18:2188-2196), suggesting that inhibition of sigma 1 has only minor effects on normal tissues. Thus, sigma 1 targeting drugs can be used safely and effectively as single agents, and can also be used to supplement or enhance therapeutic effects.

[0184] Example 2: SigmaR1 knockout prevents high-fat diet-induced fatty liver In certain embodiments, the progression of NAFL to NASH and HCC in sigmaR1 knockout (KO) mice fed a high-fat Western diet (HFWD) was characterized compared to wild-type (WT) mice. In this study, a full sigmaR1 knockout (KO) C57BL / 6 mouse model fed HFWD+CCl4 (Tsuchida, et al., 2018, J Hepatol 69:385-395) is used to evaluate key biomarkers of disease progression: steatosis, inflammation, hepatocyte ballooning, fibrosis, hepatic stellate cell (HSC) activation, serum liver enzymes, insulin resistance / glucose tolerance, and HCC lesion formation. In WT C57BL / 6 mice, the status of sigmaR1 transcript and sigma1 protein levels, as well as liver immunostaining patterns at all stages of disease progression are monitored.

[0185] Complete SigmaR1 knockout (KO) C57BL / 6 mice were generated. C57BL / 6 mice are a mouse strain for diet-induced obesity and NAFLD studies. Two-year-old male wild-type SigmaR1 and SigmaR1 KO C57BL / 6 mice were fed either a normal diet (ND) or a high-fat diet (HFD) for 8 weeks (Figure 2A-2E), after which livers were harvested and assessed for body weight, histology, and steatosis. By 8 weeks on HFD, WT mice had a median liver weight of 3.9 grams compared to 2.2 grams for SigmaR1 KO mouse livers (Figure 2A). In contrast, liver weights of WT and SigmaR1 KO mice fed a normal diet (ND) were comparable (Figure 2A). Body weights were similar at approximately 56 grams for both WT and SigmaR1 KO mice. Livers from HFD-fed WT and Sigma R1 KO mice were visibly different in size, color, and texture (Figure 2D). Hematoxylin and eosin (H&E) staining showed a markedly different histology, with large voids representing lipid droplets, as well as evidence of increased immune cell infiltration in the livers of WT mice (Figure 2B-2C). Accumulation of neutral lipids / lipid droplets was confirmed by Oil-Red-O staining, showing markedly elevated neutral lipid levels in the livers of HFD-fed WT mice (Figure 2E).

[0186] In mammals, white adipose tissue (WAT) stores and releases lipids, whereas brown adipose tissue (BAT) oxidizes lipids to fuel thermogenesis. In obese individuals, BAT content and activity are reduced as a result of the conversion of brown adipocytes into white-like unilocular cells. As shown in Figure 3A-3C, in wild-type mice, a high-fat diet (HFD) induced whitening of brown adipose tissue (BAT) (Figure 3A), which was accompanied by both an increase in total lipid area (Figure 3B) and an increase in average lipid droplet size (Figure 3C). SigmaR1 KO significantly rescued the whitening (Figure 3A), increase in lipid area (Figure 3B) and increase in lipid droplet size (Figure 3C) induced by HFD. TIFF2024504632000016.tif36128

[0187] Example 3: Downregulation of Sigma-1 expression reduced lipid droplet accumulation in cells With reference to Figures 4A-4B, in this study, we found that downregulation of sigma 1 (σ1) expression using sigma R1-targeting shRNA significantly reduced lipid droplet accumulation in cells. The shRNA was delivered using a lentiviral vector. As shown in Figure 4B, it was confirmed that two shRNAs, namely, shRNA #4 (σ1 shR #4 = MISSION / SigmaAldrich clone TRCN0000061010;) and shRNA #5 (σ1 shR #5 = MISSION / SigmaAldrich clone TRCN0000061008), could downregulate the expression of sigma 1. As shown in Figure 4A, treatment of LNCaP cells with dihydrotestosterone (DHT) (and non-specific control shRNA) for 72 hours (72h) resulted in the prominent formation and accumulation of lipid droplets. However, both shRNA #4 and shRNA #5, which specifically reduce sigma-1 expression, almost completely abolished dihydrotestosterone-induced LD formation and accumulation.

[0188] Example 4: Pharmaceutical inhibition of sigma1 reduced the levels of high-fat diet-induced hepatic steatosis In this study, we evaluated the impact of sigma 1 targeted drug therapy in preventing and reversing steatosis. In certain embodiments, small molecule sigma 1 modulators phenocopy key aspects of the hepatoprotective effects of sigmaR1 KO mice.

[0189] With reference to Figures 5 and 6A-6C, pharmacological inhibition of sigma1 phenocopies certain aspects of sigma1 knockdown. Oleate- and glucose-induced lipid droplets fail to form in sigmaR1 KO MEFs. Also, the small molecule inhibitor IPAG eliminates oleate-, cholesterol-, and glucose-induced lipid droplets by lipophagocytosis (Figure 5). With reference to Figures 6A-6C, HFD-fed mice orally administered the small molecule inhibitor CT-189 (also referred to herein as "compound G" or "CT-110") showed significantly reduced liver fat levels (Figure 6B), and significantly reduced brown adipose tissue whitening levels (Figure 6C), compared to vehicle-administered mice.

[0190] The sigma 1i lead compound CT189 [1-(4-chlorophenyl)-3-(3(4-fluorophenoxy)propyl)guanidine, also referred to herein as compound G or CT-110], is orally bioavailable, binds to sigma 1 with high affinity (Ki = 38 nM), and has limited off-target propensity based on the CEREP Safety 44 panel screen. CT189 is safe and does not induce liver toxicity, even at doses exceeding those proposed herein (Salvino et al. 2017 (PMID: 28385503); Thomas et al. 2017 (PMID: 28235766); Kim and Maher 2017 (PMID: 28744586)). CT189 has been used in tumor xenograft experiments, where daily oral dosing for >4 weeks did not produce detectable changes in mouse weight or behavior. Therefore, this compound, through its inhibition of sigma-1, may elicit dual antineoplastic and adiposity-reducing properties.

[0191] Example 5: Pharmaceutical inhibition of sigma-1 reduces expression of fibrosis-associated pro-inflammatory cytokines Often, NASH patients are identified / diagnosed after they already have fibrotic disease, and this study will help determine whether sigma-1 medications can block the progression of liver fibrosis.

[0192] Referring to Figure 7, inhibition of sigma 1 suppresses the levels of major fibrosis-related pro-inflammatory cytokines, including soluble IL-6, TNFα and IL-17 (Figure 7). Sigma 1 regulators have immunomodulatory and anti-inflammatory properties, as well as anti-fibrotic properties. Furthermore, some sigma 1 inhibitors suppress HCC cell proliferation and induce apoptosis in vitro.

[0193] This study will show whether sigma 1 targeted drug therapy can halt the progression of NAFLD and promote regression and recovery after cessation of HFWD following the development of NASH with steatosis and / or fibrosis. It can be determined whether sigma 1i drugs can be used to enhance / promote recovery from steatosis and / or fibrosis following dietary modification.

[0194] Example 6: Steatosis This study shows whether Sigma 1-targeted drug therapy attenuates / mitigates steatosis while mice are still receiving HFWD. After mice are returned to a normal diet (ND), it can be determined whether Sigma 1-targeted drug therapy promotes and enhances the degree of steatosis regression. Marked steatosis develops at 4-8 weeks on HFD. Data obtained herein show high-fat diet-induced elimination of lipid droplets (Figures 2A-2E). Changes in steatosis are measured using Oil Red-O and H&E staining as described elsewhere herein (Figures 2A-2E).

[0195] inflammation Several sigma 1 ligands have anti-inflammatory properties, and in vitro data with CT189 demonstrate that this compound can suppress IL-17, IL-6, and TNFα, thus indicating that this compound series can effectively suppress inflammation in the liver (Figure 7). To test whether sigma 1i can prevent inflammation in fatty liver, treatment is initiated at weeks 4-8 and continued until weeks 8-12. To test whether sigma 1i enhances and promotes regression of inflammation, treatment is initiated immediately after mice are returned to a normal diet (ND) and continued for 4 weeks during the recovery period, and mouse livers are harvested weekly during this period, i.e., at four data points, to measure the rate of recovery. The extent of recovery is quantified by NAFLD score, and changes in immune cell infiltration are quantified by H&E, CD45+, CD68+ cells, and specifically stained for CD3+, CD4+, and CD8+ cells in selected sample sets, as described elsewhere herein.

[0196] Fibrosis The factors involved in the natural history of NAFLD are not well defined, so it remains unclear what the relative contributions of steatosis, inflammation, associated oxidative stress, and other metabolic changes are to the development and progression of NAFLD. However, it appears that the convergence of most or all of these is necessary. The present data demonstrate that sigma1i can prevent steatosis and can also attenuate the progression of steatosis. Therefore, we test whether sigma1i with CT189 can prevent fibrosis when mice are treated after the onset of steatosis but before the onset of fibrosis; in this case, treatment is started at week 8 and daily dosing is continued until week 12 or until vehicle-treated mice develop fibrosis. We also test whether CT189 eliminates or prevents the progression of fibrotic lesions in mice that have already developed fibrosis; in this case, treatment is started approximately between week 12 and week 16 or until vehicle-treated mice develop the equivalent of stage 4 fibrosis. Finally, we evaluate drug-assisted recovery when HFD is discontinued and mice are returned to ND at time point 12 (NASH with fibrosis). Fibrotic lesions are detected and measured with pico-Sirius Red, a molecular analysis of fibrogenic gene expression.

[0197] Blood-Based Biomarkers of Hepatic Response to Sigma-1i Targeted Drug Therapy: Serum Levels of ALT and AST Serum levels of ALT and AST will be monitored in response to Sigma 1i treatment to determine whether changes in serum levels of ALT and AST track / correspond to histological improvement. Serum ALT and AST levels will be measured from tail vein bleeds using materials and methods described elsewhere herein.

[0198] Changes in biomarkers of metabolic comorbidities in response to sigma-1i targeted drug therapy: serum levels of insulin, glucose, triglycerides (TG), and cholesterol During each treatment time frame described elsewhere herein, serum levels of glucose, insulin, triglycerides and cholesterol in response to Sigma1i treatment are monitored using insulin kits, glucose detectors, total TG and cholesterol kits or by LC-MS / MS.

[0199] Hepatocellular carcinoma The present data indicate that sigma1i has antitumor properties and that HCC cells are responsive / sensitive to sigma1i (Kim et al., 2012 (PMID: 22925888); Schrock et al., 2013 (PMID: 24006496); Kim and Maher, 2017 (PMID: 28744586); Oyer et al., 2019 (PMID: 31695608)). Therefore, we test whether sigma1i with CT189 can prevent HCC when mice are treated after the development of steatosis but before the appearance of HCC lesions. In this case, treatment will begin at week 20 and daily dosing will continue until week 24 or until vehicle-treated mice develop HCC lesions. We also test whether CT189 eliminates or abrogates the progression of these neoplastic lesions using mice that have already developed HCC lesions. In this case, treatment is initiated approximately between weeks 24 and 28, or until vehicle-treated mice develop the equivalent of end-stage tumor burden. The effect of HFD continuation is assessed by returning mice to ND at weeks 20 (preventative protocols) and 24 (curative protocols) prior to initiation of treatment.

[0200] Example 7: Materials and Methods Full sigmaR1 knockout (KO) C57BL / 6 mice can be used in comparison to WT sigmaR1 C57BL / 6 mice to assess the requirement of sigmaR1 / sigma1 in the progression from healthy liver through the following NAFLD stages: Hepatic steatosis (8 weeks), accelerating and amplifying HFWD-induced NASH with associated fibrosis. NASH with inflammatory fibrosis development due to high-fat Western diet (HFWD, WD containing 21.1% fat, 41% sucrose and 1.25% cholesterol by weight (Teklad diets, TD.120528) and high sugar diet (23.1 g / L D-fructose (Sigma-Aldrich, F0127) and 18.9 g / L D-glucose (Sigma-Aldrich, G8270)) alone compared to HFWD+CCl4 diet (administered weekly). (12 weeks), progression to cirrhosis (24 weeks) and increase in frequency, number and size of HCC lesions (24 weeks). All groups are compared to a normal chow diet (ND, lab diet, Rodent diet 20, #5053).

[0201] Histological markers of NAFL disease progression in the absence of sigma-1 Formalin-fixed, paraffin-embedded liver sections were stained with hematoxylin and eosin (H&E) for evaluation of tissue architecture and liver histology, Sirius Red (Sigma, 365548-5G) / Fast Green (Sigma, F258) for collagen to evaluate fibrosis, Oil Red-O for neutral lipid accumulation in the liver, and Periodic Acid Schiff (PAS) for glycogen accumulation. NAFLD activity scores and fibrosis stages were measured by an experienced pathologist blinded to treatment conditions using the NASH CRN scoring system.

[0202] Molecular markers of fibrosis Changes in expression of fibrogenic genes collagen-α1, β-PDGFR, TGF-β, and Timp-1 will be assessed by qRT-PCR. Additionally, hepatic stellate cell (HSC) activation will be assessed by immunostaining for desmin (to identify HSC) and α-SMA (to detect activated HSC). Hepatocyte apoptosis will be monitored by cleaved caspase and / or TUNEL staining.

[0203] Inflammatory markers Inflammation is detected by immunostaining for CD45 (pan-leukocyte antigen) and CD68 (monocyte / macrophage infiltration). CD45-positive sections show CD3 + , CD4 + and CD8 + Increased infiltration of positive immune cells is monitored.

[0204] Our data show that sigma-1 modulation can suppress pro-inflammatory IL-17, IL-6, and TNFα (Figure 7). Monitor soluble IL-17 (sIL-17) because CD4+ T lymphocytes in the liver produce high levels of IL-17 in NAFLD. Also monitor IL-6 and TNFα because dietary and genetic obesity promotes hepatic inflammation and tumorigenesis by enhancing IL-6 and TNFα expression.

[0205] Liver enzymes Elevated serum ALT (alanine transaminase) and AST (aspartate transaminase) are biomarkers of NAFLD progression / liver dysfunction. Serum ALT and AST activities are monitored with a fluorometric kit (Biovision) and / or measured using VITROS 5,1 FS (Ortho Clinical Diagnostics). Total serum triglyceride (TG) and cholesterol levels are measured using commercial test kits and / or by LC-mass spectrometry and / or using VITROS 5,1 FS (Ortho Clinical Diagnostics).

[0206] Insulin Resistance and Glucose Tolerance Insulin resistance and glucose tolerance are symptoms / indicators of metabolic syndrome and NAFLD. Biomarkers related to metabolic syndrome are monitored. Fasting and non-fasting plasma insulin levels are tested with Ultrasensitive Mouse Insulin ELISA kit (Crystal Chem, 90080). Fasting and non-fasting glucose levels are monitored with One Touch Ultra (Life Scan). In animals treated with CCl4, blood is analyzed 7 days after the most recent treatment. For evaluation of insulin resistance in non-fasting homeostatic models, QUICKI is calculated as described elsewhere (Lee, et al., 2008, Amer. J. Physiol Endocrinol. Metab. 294, E261-270; Bowe, et al., 2014, J. Endocrinol. 222:G13-25).

[0207] Sigma R1 / Sigma 1 state In WT C57BL / 6 mice, sigmaR1 mRNA transcript status will be monitored and assessed by qRT-PCR, and sigma1 protein levels and distribution patterns will be monitored and assessed by immunohistochemistry (IHC) staining in liver tissue at the key stages of disease progression: steatosis (8 weeks), steatohepatitis with inflammation and fibrosis (12 weeks), and HCC lesions (24 weeks).

[0208] The disclosures of any and all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties.

[0209] While the present disclosure has been disclosed with reference to particular embodiments, it will be apparent that other embodiments and variations of the present disclosure may be devised by those skilled in the art without departing from the true spirit and scope of the present disclosure, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A pharmaceutical composition for treating, ameliorating and / or preventing lipid droplet (LD) accumulation in cells, comprising an agent that reduces and / or suppresses sigma R1 transcription in cells and / or reduces and / or inhibits sigma 1 activity and / or expression.

2. A pharmaceutical composition for treating, ameliorating and / or preventing inflammation associated with hepatic steatosis (fatty liver) and / or steatohepatitis in a subject, comprising an effective amount of an agent that reduces and / or suppresses sigma R1 transcription in the subject and / or reduces and / or inhibits sigma 1 activity and / or expression.

3. A pharmaceutical composition for treating, ameliorating and / or preventing non-alcoholic fatty liver disease (NAFLD) in a subject, comprising an effective amount of an agent that reduces and / or suppresses sigma R1 transcription in the subject and / or reduces and / or inhibits sigma 1 activity and / or expression.

4. A pharmaceutical composition for treating, ameliorating and / or preventing non-alcoholic steatohepatitis (NASH) in a subject, comprising an effective amount of an agent that reduces and / or suppresses sigma R1 transcription in the subject and / or reduces and / or inhibits sigma 1 activity and / or expression.

5. A pharmaceutical composition for reversing, slowing or preventing the progression of NAFLD to NASH in a subject suffering from NAFLD, comprising an effective amount of an agent that reduces and / or suppresses sigma R1 transcription in the subject and / or reduces and / or inhibits sigma 1 activity and / or expression.

6. A pharmaceutical composition for reversing, slowing or preventing the progression of NASH to hepatocellular carcinoma (HCC) in a subject suffering from NASH, comprising an effective amount of an agent that regulates sigma R1 transcription and / or regulates sigma 1 activity and / or expression in a subject.

7. 2. A method for assisting in determining and / or assessing the risk of progressing to NASH in a subject suffering from NAFLD, comprising: Measuring the level of sigmaR1 transcription and / or sigma1 activity / expression in the subject; and Comparing the measured levels with the levels of sigmaR1 transcription and / or sigma1 activity / expression in a control sample.

8. 1. A method for assisting in determining and / or assessing the risk of progression to HCC in a subject suffering from NASH, comprising: Measuring the level of sigmaR1 transcription and / or sigma1 activity / expression in the subject; and Comparing the measured levels with the levels of sigmaR1 transcription and / or sigma1 activity / expression in a control sample.

9. 9. The method of claim 7 or 8, wherein the control sample is derived from at least one of the following: subjects with NAFLD; subjects without NAFLD; Subjects with NASH; subjects without NASH; Subjects with HCC; subjects without HCC; Subjects with NAFLD who are not known to have progressed to NASH and / or are not at risk of progressing to NASH; Subjects with NAFLD who are known to have progressed to and / or are at risk of progressing to NASH; Subjects with NASH who have not progressed to HCC and / or are known to be at no risk of progressing to HCC; Subjects with NASH known to have progressed to and / or be at risk for progressing to HCC.

10. The pharmaceutical composition of any one of claims 2 to 6, which reverses, prevents and / or ameliorates at least one of the following: steatosis, inflammation, hepatocyte ballooning, fibrosis, hepatic stellate cell (HSC) activation, increased serum liver enzymes, insulin resistance / glucose tolerance, and HCC lesion formation.

11. The pharmaceutical composition of any one of claims 1 to 6 and 10, wherein the agent comprises a CRISPR system against sigma R1 and / or a nucleic acid against sigma R1 selected from siRNA, shRNA, microRNA or antisense polynucleotide.

12. The pharmaceutical composition of claim 11, wherein a viral vector expressing the nucleic acid is administered to the subject.

13. The pharmaceutical composition of claim 12, wherein the nucleic acid is expressed in the liver of the subject.

14. The pharmaceutical composition of any one of claims 1 to 6 and 10, wherein the agent is selected from the group consisting of: (i) Formula (I): A compound of the formula: Ring A is a monocyclic or bicyclic aryl ring or a monocyclic or bicyclic heteroaryl ring, where the aryl or heteroaryl ring is selected from 0 to 4 R 1 may be substituted with a group; R 1 Each of the -C 1 ~C 6 Alkyl, -C 1 ~C 6 Fluoroalkyl, -C 1 ~C 6 Heteroalkyl, F, Cl, Br, I, -CN, -NO 2 , -OR 3 , -SR 3 , -S(=O)R 3 , -S(=O) 2 R 3 , -NHS(=O) 2 R 3 , -C(=O)R 3 , -OC(=O)R 3 , -CO 2 R 3 , -OCO 2 R 3 , -CH(R 3 ) 2 , -N(R 3 ) 2 , -C(=O)N(R 3 ) 2 , -OC(=O)N(R 3 ) 2 , -NHC(=O)NH(R 3 ), -NHC(=O)R 3 , -NHC(=O)OR 3 , -C(OH)(R 3 ) 2 , and -C(NH 2 )(R 3 ) 2 are independently selected from the group consisting of: R 2 Each of these is H, C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, and -C 1 ~C 3 Alkyl-(C 3 ~C 6 cycloalkyl), where the alkyl, heteroalkyl or cycloalkyl group is selected from the group consisting of 0 to 5 R 1 or may be substituted with X 3 and R 2 together, 0 to 2 R 1 may be substituted with a group (C 3 ~C 7 ) forming a heterocycloalkyl group; R 3 Each of these is H, C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, Aryl, and -C 1 ~C 3 Alkyl-(C 3 ~C 6 cycloalkyl), where the alkyl, heteroalkyl, aryl, or cycloalkyl group is selected from 0 to 5 R 1 may be substituted with a group; X 1 Ha-CH 2 -, -S-, -O- or -(NR 2 )- and; X 2 HA=CH 2 , =S, =O or =NR 2 and X 3 is -S-, -O-, or -NR 2 - a compound; (ii) Formula (II): R A -R B (II) A compound of the formula: R A teeth, selected from the group consisting of; X 4 is selected from the group consisting of methoxy, F, Cl, Br, and I; and R B teeth, A compound selected from the group consisting of: (iii) Formula (III): A compound of the formula: R 1 and R 2 Each of the -C 1 ~C 6 Alkyl, -C 1 ~C 6 Fluoroalkyl, -C 1 ~C 6 Heteroalkyl, F, Cl, Br, I, -CN, -NO 2 , -OR 5 , -SR 5 , -S(=O)R 5 , -S(=O) 2 R 5 , -NHS(=O) 2 R 5 , -C(=O)R 5 , -OC(=O)R 5 , -CO 2 R 5 , -OCO 2 R 5 , -CH(R 5 ) 2 , -N(R 5 ) 2 , -C(=O)N(R 5 ) 2 , -OC(=O)N(R 5 ) 2 , -NHC(=O)NH(R 5 ), -NHC(=O)R 5 , -NHC(=O)OR 5 , -C(OH)(R 5 ) 2 , and -C(NH 2 )(R 5 ) 2 are independently selected from the group consisting of: R 3 -C 1 ~C 6 Alkyl, -C 1 ~C 6 Fluoroalkyl, -C 1 ~C 6 selected from the group consisting of alkoxy, F, Cl, Br, and I; R 4 -C 1 ~C 6 Alkyl, -C 1 ~C 6 selected from the group consisting of alkoxy, F, Cl, Br, and I; R 5 Each of these is H, C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, Aryl, and -C 1 ~C 3 Alkyl-(C 3 ~C 6 cycloalkyl), where the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted; X is CH 2 , C═O, or O; n is an integer from 1 to 3; x is an integer from 0 to 4; and wherein y is an integer from 0 to 4; a salt, solvate, or N-oxide thereof; and any combination thereof.

15. 15. The pharmaceutical composition of claim 14, wherein the compound is selected from the group consisting of: 1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A); 1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (compound B); 1-(n-propyl)-3-(4-iodophenyl)guanidine (compound C); 1-(n-propyl)-3-(4-methoxyphenyl)guanidine (compound D); 1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (compound F); 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (compound G); Salts, solvates or N-oxides thereof, and any combination thereof.

16. 15. The pharmaceutical composition of claim 14, wherein the compound is selected from the group consisting of: 1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (compound E); 1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine (compound H); Salts, solvates or N-oxides thereof, and any combination thereof.

17. A pharmaceutical composition according to any one of claims 14 to 16, further comprising a pharma- ceutically acceptable carrier.

18. A pharmaceutical composition described in any one of claims 14 to 17, used in combination with at least one additional agent that treats, prevents and / or ameliorates one of NAFDL, NASH and / or HCC.

19. The pharmaceutical composition of any one of claims 14 to 18, wherein the composition is administered by a route including oral, nasal, rectal, vaginal, parenteral, buccal, sublingual or topical.

20. The pharmaceutical composition of any one of claims 14 to 19, wherein the subject is a mammal.

21. 21. The pharmaceutical composition of claim 20, wherein the mammal is a human.

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