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Insights into the molecular targets and emerging pharmacotherapeutic interventions for nonalcoholic fatty liver disease

Published:November 02, 2021DOI:https://doi.org/10.1016/j.metabol.2021.154925

      Abstract

      Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease worldwide. With no Food and Drug Administration approved drugs, current treatment options include dietary restrictions and lifestyle modification. NAFLD is closely associated with metabolic disorders such as obesity, type 2 diabetes, and dyslipidemia. Hence, clinically various pharmacological approaches using existing drugs such as antidiabetic, anti-obesity, antioxidants, and cytoprotective agents have been considered in the management of NAFLD and nonalcoholic steatohepatitis (NASH). However, several pharmacological therapies aiming to alleviate NAFLD-NASH are currently being examined at various phases of clinical trials. Emerging data from these studies with drugs targeting diverse molecular mechanisms show promising outcomes. This review summarizes the current understanding of the pathogenic mechanisms of NAFLD and provides an insight into the pharmacological targets and emerging therapeutics with specific interventional mechanisms. In addition, we also discuss the importance and utility of new approach methodologies and regulatory perspectives for NAFLD-NASH drug development.

      Abbreviations:

      AASLD (American Association for the Study of Liver Diseases), ACC (acetyl-CoA carboxylase), ALT (alanine aminotransferase), AOP (adverse outcome pathway), ASK1 (apoptosis signal-regulating kinase 1), AST (aspartate transaminase), BMI (body mass index), CCK (cholecystokinin), CCKR (CCK receptor), CCR2/CCR5 (C-C motif chemokine receptor type 2/5), COX-2 (cyclooxygenase-2), CYP7A1 (cholesterol 7 alpha-hydroxylase), DDP-4 (dipeptidyl peptidase-4), DGAT (diacylglycerol acyltransferase), DPP4 (dipeptidyl peptidase 4), EASD (European Association for the Study of Diabetes), EASL (European Association for the Study of the Liver), EASO (European Association for the Study of Obesity), EMA (European Medicines Agency), ER (endoplasmic reticulum), FAS (fatty acid synthase), FDA (Food and Drug Administration), FFA (free fatty acid), FGF (fibroblast growth factor), FXR (farnesoid X receptor), GC (glucocorticoid), GGT (gamma-glutamyl transferase), GHRH (growth hormone-releasing hormone), GIP (glucose-dependent insulinotropic polypeptide), GLP-1 (glucagon-like peptide-1), HbA1c (glycated hemoglobin), HCC (hepatocellular carcinoma), HDL-C (high-density lipoprotein cholesterol), HFD (high-fat diet), HSD17B13 (hydroxysteroid 17-beta dehydrogenase 13 protein), HVPG (hepatic venous pressure gradient), IBAT (ileal bile acid transporter), IFNs (interferons), IKK (inhibitor of IκB kinases), IL (interleukin), IND (investigational new drugs), iNOS (inducible nitric oxide synthase), JAK-STAT (Janus kinases-signal transducer and activator of transcription proteins), JNK (C-Jun N-terminal kinase), LDL-C (low-density lipoprotein cholesterol), LPS (lipopolysaccharide), LXR (liver X receptor), MAFLD (metabolic (dysfunction) associated fatty liver disease), MOA (mechanisms of action), MPC (mitochondrial pyruvate carrier), MRI-PDFF (magnetic resonance imaging-proton density fat fraction), NAFL/NAFLD (non-alcoholic fatty liver/disease), NASH (non-alcoholic steatohepatitis), NDA (New Drug Application), OECD (Organization for Economic Co-operation and Development), PNPLA3 (patatin-like phospholipase domain-containing 3 protein), PPAR (peroxisome proliferator-activated receptors), PRIME (Priority Medicine), SCD-1 (stearoyl-CoA desaturase), SGLT2 (sodium-glucose transport protein 2), siRNA (small interfering ribonucleic acid), T2DM (type 2 diabetes mellitus), TG (triglycerides), THR-β (thyroid hormone receptor), TIMP1 (tissue inhibitor of metalloprotease 1), TLR4 (toll-like receptor), TNFα (tumor necrosis factor α), VLDL (very-low-density lipoprotein)

      Keywords

      1. Introduction

      Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide [
      • Godoy-Matos A.F.
      • Silva Júnior W.S.
      • Valerio C.M.
      NAFLD as a continuum: from obesity to metabolic syndrome and diabetes.
      ]. The two phenotypes of NAFLD are nonalcoholic fatty liver (NAFL), i.e., simple steatosis characterized by the presence of lipids in ≥5% of the hepatocytes, and nonalcoholic steatohepatitis (NASH) characterized by the presence of inflammation and hepatocellular damage [
      • Loomba R.
      • Sanyal A.J.
      The global NAFLD epidemic.
      ]. In certain cases, NAFL eventually progresses to NASH, which may subsequently advance to more severe liver disease such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) (Fig. 1). NAFLD has been regarded as the most common cause for HCC, which is the fifth most often diagnosed cancer worldwide [
      • Huang D.Q.
      • El-Serag H.B.
      • Loomba R.
      Global epidemiology of NAFLD-related HCC: trends, predictions, risk factors and prevention.
      ]. The global prevalence of NAFLD has increased drastically over the last few years. The estimated global prevalence is about 25% and keeps rising rapidly [
      • Golabi P.
      • Paik J.
      • Reddy R.
      • Bugianesi E.
      • Trimble G.
      • Younossi Z.M.
      Prevalence and long-term outcomes of non-alcoholic fatty liver disease among elderly individuals from the United States.
      ]. The highest prevalence was found in South America (30.45%) and Middle East (31.79%), whereas the lowest prevalence was reported in Africa (13.48%) [
      • Younossi Z.M.
      • Koenig A.B.
      • Abdelatif D.
      • Fazel Y.
      • Henry L.
      • Wymer M.
      Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes.
      ]. Although NAFLD may affect any age, sex, and ethnicity, a significant variation in NAFLD prevalence was reported amongst different gender, age, and ethnic groups [
      • Browning J.D.
      • Szczepaniak L.S.
      • Dobbins R.
      • Nuremberg P.
      • Horton J.D.
      • Cohen J.C.
      • et al.
      Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.
      ].
      Fig. 1
      Fig. 1Spectrum and risk factor for NAFLD-NASH.
      The spectrum of NAFLD ranges from simple steatosis to chronic steatohepatitis or NASH. Steatosis is usually benign; however, in some individuals, it may eventually progress into NASH, which can subsequently lead to more severe pathologies, including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Steatosis, NASH, and fibrosis can be reversed to a healthy state with timely and appropriate interventions, while later stages cannot be reversed. The established risk factor associated with NAFLD includes obesity, T2DM, dyslipidemia, metabolic syndrome. The other emerging conditions include obstructive sleep apnea, colorectal cancer, osteoporosis, psoriasis, endocrinopathies, hypothyroidism, hypopituitarism, hypogonadism, pancreatoduodenal resection, psoriasis, and polycystic ovary syndrome independent of obesity.
      Risk factors associated with the development and progression of NAFLD are multiple, including obesity, diabetes, insulin resistance, dyslipidemia, metabolic syndrome, and exposure to xenobiotics, environmental toxicants such as endocrine and/or metabolism disrupting chemicals [
      • Heindel J.J.
      • Blumberg B.
      • Cave M.
      • Machtinger R.
      • Mantovani A.
      • Mendez M.A.
      • et al.
      Metabolism disrupting chemicals and metabolic disorders.
      ]. Among the multifactorial etiology of NAFLD, aging has been suggested as one of the most common causes for its progression [
      • Bertolotti M.
      • Lonardo A.
      • Mussi C.
      • Baldelli E.
      • Pellegrini E.
      • Ballestri S.
      • et al.
      Nonalcoholic fatty liver disease and aging: epidemiology to management.
      ]. It has been suggested that NAFLD is a hepatic manifestation of metabolic diseases [
      • Marchesini G.
      • Brizi M.
      • Blanchi G.
      • Tomassetti S.
      • Bugianesi E.
      • Lenzi M.
      • et al.
      Nonalcoholic fatty liver disease: a feature of the metabolic syndrome.
      ], and several stressors, including environmental factors, genetics, epigenetics, and microbiota, can further increase the susceptibility [
      • Brunt E.M.
      • Wong V.W.S.
      • Nobili V.
      • Day C.P.
      • Sookoian S.
      • Maher J.J.
      • et al.
      Nonalcoholic fatty liver disease.
      ]. Hence, considering the complex heterogeneity of the disease, a consensus group of international experts has recently proposed to change the NAFLD acronym to MAFLD representing metabolic (dysfunction) associated fatty liver disease to accurately reflect the current knowledge of fatty liver diseases associated with metabolic dysfunction [
      • Eslam M.
      • Sanyal A.J.
      • George J.
      • Sanyal A.
      • Neuschwander-Tetri B.
      • Tiribelli C.
      • et al.
      MAFLD: a consensus-driven proposed nomenclature for metabolic associated fatty liver disease.
      ].
      NAFLD is a multisystem disease associated with extrahepatic comorbidities, including cardiovascular disease, diabetes, chronic kidney disease, polycystic ovary syndrome, osteoporosis, hypothyroidism, sleep apnea, colorectal cancer, endocrinopathies, and malignancies [
      • Byrne C.D.
      • Targher G.
      NAFLD: a multisystem disease.
      ,
      • Armstrong M.J.
      • Adams L.A.
      • Canbay A.
      • Syn W.K.
      Extrahepatic complications of nonalcoholic fatty liver disease.
      ]. With no Food and drug administration (FDA) approved drugs, current first-line treatments rely on dietary management, lifestyle modification, and physical exercise, whereas second-line treatment includes bariatric surgery [
      • Polyzos S.A.
      • Kang E.S.
      • Boutari C.
      • Rhee E.J.
      • Mantzoros C.S.
      Current and emerging pharmacological options for the treatment of nonalcoholic steatohepatitis.
      ]. Various pharmacological approaches using existing drugs have also been considered in the management of NAFLD and NASH. These attempts mainly focus on antidiabetics, anti-obesity drugs, antioxidants, and cytoprotective agents, including insulin sensitizers (e.g., metformin), thiazolidinediones (e.g., pioglitazone), glucagon-like peptide-1 (GLP-1) receptor agonists (e.g., liraglutide), a natural dihydroxy bile acid (e.g., ursodeoxycholic acid) or antioxidants (vitamin E) [
      • Polyzos S.A.
      • Kang E.S.
      • Boutari C.
      • Rhee E.J.
      • Mantzoros C.S.
      Current and emerging pharmacological options for the treatment of nonalcoholic steatohepatitis.
      ]. However, evidence-based practice guidelines and practice guidance issued by a committee of hepatology experts recommend only pioglitazone and vitamin E for clinical use in selected NASH patients (Table 1). The safety and efficacy of several monotherapies and drug combinations targeting various mechanisms of NAFLD pathogenesis are currently being evaluated in clinical trials. This review presents the current understanding of NAFLD pathogenesis, potential molecular targets, and emerging therapeutics with intended mechanisms of action (MOA). A highlight on recent regulatory perspectives for NAFLD-NASH drug development and approval process is also discussed.
      Table 1Summary of the current clinical practice guidelines for the management of NAFLD-NASH in the US, Europe, and the Asia Pacific regions.
      DrugsPrimary MOAGuidance statement
      AASLD [
      • Chalasani N.
      • Younossi Z.
      • Lavine J.E.
      • Charlton M.
      • Cusi K.
      • Rinella M.
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases.
      ]
      EASL, EASD, EASO [
      • Marchesini G.
      • Day C.P.
      • Dufour J.F.
      • Canbay A.
      • Nobili V.
      • Ratziu V.
      • et al.
      EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.
      ]
      Asia Pacific [
      • Chitturi S.
      • Wong V.W.-S.
      • Chan W.-K.
      • Wong G.L.-H.
      • Wong S.K.-H.
      • Sollano J.
      • et al.
      The Asia–Pacific working party on non-alcoholic fatty liver disease guidelines 2017—Part 2: management and special groups.
      ]
      PioglitazonePPARγ agonistPioglitazone can be considered in patients with and without T2DM with biopsy-proved NASHWhile no firm recommendations can be made, pioglitazone or vitamin E or their combination could be used for NASHNot recommended for general use in patients with NASH but could be considered for short-term use in patients with pre-diabetes or T2DM.

      Should be used with caution in cirrhotic patients.
      Vitamin EAntioxidantVitamin E 800 IU daily can be considered in nondiabetic patients with biopsy-proven NASH without cirrhosis.Further studies are needed before firm recommendations can be made.
      MetforminInsulin sensitizerNot recommended for the treatment of NAFLD or NASHInsufficient evidenceNot recommended as a treatment for NAFLD/NASH. Should remain the first-line anti-diabetic agent in patients with T2DM.
      Ursodeoxycholic acidHepatoprotective bile acidUrsodeoxycholic acid and Omega-3 fatty acids are not recommended for the treatment of NAFLD or NASH.Insufficient evidenceDo not appear to have beneficial effects on liver histology
      Omega-3 fatty acidsPolyunsaturated fatty acidOmega-3 fatty acids may be considered to treat hypertriglyceridemia in patients with NAFLD.Insufficient evidenceDo not appear to have beneficial effects on liver histology
      LiraglutideGLP-1 receptor agonistPremature to consider GLP-1 receptor agonistsInsufficient evidenceNot recommended as a treatment for NAFLD/NASH until more definitive data become available.
      PentoxifyllineNonspecific PDE inhibitorNot mentionedInsufficient evidenceNot recommended as a treatment for NAFLD/NASH.
      StatinsHMG-CoA reductase inhibitorsCan be used to treat dyslipidemia in patients with NAFLD and NASH.

      May be used in patients with NASH cirrhosis. Should be avoided in patients with decompensated cirrhosis.
      May be confidently used to reduce LDL cholesterol and prevent cardiovascular risk, with no benefits or harm on liver disease.May be administered to patients with NAFLD who have mild elevation of transaminases or compensated cirrhosis
      Obeticholic acidFXR agonistShould not be used off-label to treat NASHInsufficient evidenceNot recommended as a treatment for NAFLD/NASH
      Abbreviations: MOA, mechanisms of action; AASLD, American Association for the study of Liver Diseases; EASL, European Association for the Study of Liver; EASD, European Association for the study of Diabetes; EASO, European Association for the study of obesity; PPARγ, peroxisome proliferator-activated receptor γ, T2DM, type 2 diabetes mellitus, PDE, phosphodiesterase; HMG-CoA, β-Hydroxy β-methylglutaryl-CoA; FXR, farnesoid X receptor; LDL, low-density lipoprotein.

      2. Pathogenesis of NAFLD

      The pathophysiology of NAFLD is multifactorial, and several mechanisms have been proposed. The widely known two-hit hypothesis considers the accumulation of lipids (steatosis) as the first hit, leading to metabolic and biological alterations that can easily be influenced by other factors, such as inflammatory cytokines or oxidative stress, as the second hit required for the development of NASH and fibrosis [
      • Day C.P.
      • James O.F.W.
      Steatohepatitis: a tale of two “Hits”?.
      ]. In contrast, the multiple parallel hit hypothesis emphasizes several hits (factors) contributing simultaneously to the development and progression of NAFLD [
      • Tilg H.
      • Moschen A.R.
      Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis.
      ].
      The accumulation of lipids in the hepatocytes or hepatic steatosis is a characteristic histological feature of NAFLD, which results from the disbalance between fatty acid influx to the liver and subsequent utilization [
      • Neuschwander-Tetri B.A.
      Non-alcoholic fatty liver disease.
      ]. The major sources of free fatty acids (FFA) in the liver are i) increased lipolysis (60%), ii) de novo lipogenesis (26%), and iii) excessive dietary intake (14%) [
      • Donnelly K.L.
      • Boldt M.D.
      • Parks E.J.
      • Smith C.I.
      • Schwarzenberg S.J.
      • Jessurun J.
      Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.
      ]. This complex disease involves an interplay between several factors; however, systemic insulin resistance is a major driver of hepatic steatosis in NAFLD [
      • Parthasarathy G.
      • Revelo X.
      • Malhi H.
      Pathogenesis of nonalcoholic steatohepatitis: an overview.
      ].
      Hepatocyte lipotoxicity and immune-mediated inflammation play a key role in the development and progression of NAFLD. The lipotoxicity of accumulated lipids and FFAs results in hepatocellular injury characterized by oxidative stress, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, apoptosis, and subsequent expression of proinflammatory cytokines and inflammatory factors [
      • Reccia I.
      • Kumar J.
      • Akladios C.
      • Virdis F.
      • Pai M.
      • Habib N.
      • et al.
      Non-alcoholic Fatty Liver Disease: a Sign of Systemic Disease.
      ] (Fig 2). The cellular injury further leads to the activation of immune and apoptotic cell death pathways, which represent distinctive characteristics in the pathophysiology of NASH. The lipotoxic lipids can activate both the intrinsic- and extrinsic-mediated (death receptor) apoptotic pathway in hepatocytes through the transcriptional up-regulation of proapoptotic and downregulation of antiapoptotic proteins [
      • Malhi H.
      • Gores G.J.
      Molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease.
      ]. Hepatocyte apoptosis or other forms of hepatocyte cell death play an essential role in the activation of immune responses associated with the progression of NAFLD to a more severe state, e.g., fibrosis and cirrhosis development [
      • Parthasarathy G.
      • Revelo X.
      • Malhi H.
      Pathogenesis of nonalcoholic steatohepatitis: an overview.
      ].
      Fig. 2
      Fig. 2Pathophysiology of NAFLD.
      The interplay between the diet, microbiota, and host genetics plays a crucial role in the complex pathogenesis of NAFLD through a variety of mechanisms. Fatty acids derived from dietary sources, de novo lipogenesis, and lipolysis in the peripheral tissues are delivered to the liver through blood circulations. The FFAs in the hepatocytes either undergo mitochondrial β-oxidation or esterification to form TGs. TGs can be exported into the blood as VLDL or accumulate in the hepatocytes as lipid droplets. Lipid droplets further undergo regulated lipolysis to release fatty acids back into the hepatocyte FFA pool. Increased synthesis and accumulation of TGs and lipotoxic lipids in the liver hampers mitochondrial β-oxidation, leading to mitochondrial dysfunction with oxidative stress and endoplasmic stress, further promoting hepatic injury and inflammation. The cellular injury further leads to the activation of immune and apoptotic cell death pathways leading to the progression of NAFLD to a more severe state.
      Moreover, mitochondrial dysfunction and associated oxidative stress are the hallmark feature of NAFLD and NASH development [
      • Longo M.
      • Meroni M.
      • Paolini E.
      • Macchi C.
      • Dongiovanni P.
      Mitochondrial dynamics and nonalcoholic fatty liver disease (NAFLD): new perspectives for a fairy-tale ending?.
      ]. Mitochondrial stress promotes cell death, liver fibrogenesis, inflammation, and innate immune responses, such as the accumulation of mitochondrial DNA (mtDNA) leading to enhanced production of inflammatory cytokines, e.g., interferons (IFNs) that are responsible for the development and progression of NAFLD as reviewed elsewhere [
      • Mansouri A.
      • Gattolliat C.H.
      • Asselah T.
      Mitochondrial dysfunction and signaling in chronic liver diseases.
      ,
      • Negi C.K.
      • Khan S.
      • Dirven H.
      • Bajard L.
      • Bláha L.
      Flame retardants-mediated interferon signaling in the pathogenesis of nonalcoholic fatty liver disease.
      ]
      Accumulating evidence also suggests the circadian clock (mammalian ≈24-h endogenous timing mechanism) as a major regulator of critical physiological functions, including hepatic metabolic processes, such as glucose, lipid, or cholesterol/bile acids metabolism [
      • Mukherji A.
      • Dachraoui M.
      • Baumert T.F.
      Perturbation of the circadian clock and pathogenesis of NAFLD.
      ]. Disruption of the circadian clock has been shown to play an important role in increasing the incidence of metabolic dysregulation, significantly contributing to the development of metabolic syndrome and NAFLD [
      • Mukherji A.
      • Bailey S.M.
      • Staels B.
      • Baumert T.F.
      The circadian clock and liver function in health and disease.
      ].

      3. Potential targets and emerging pharmacotherapeutics for NAFLD

      A better understanding of the NAFLD pathogenesis has led to the clinical translation of various potential therapeutics which are currently under clinical evaluation (Table 2 and Fig. 3). These investigational drugs target several aspects of metabolic disruption, oxidative stress, inflammation, and/or inflammatory signaling. According to their intended mechanisms, these therapies can be broadly classified into five categories: i) metabolic targeted therapies, ii) oxidative stress targeted therapies, iii) inflammation targeted therapies, iv) apoptosis targeted therapies, and v) fibrosis targeted therapies. Drugs targeting each of these pharmacological targets can provide multiple benefits and act through several mechanisms (Fig. 4).
      Table 2Interventional clinical trials registered on the clinical trial database (https://www.clinicaltrials.gov) for the treatment of NAFLD-NASH.
      DrugMechanismConditionPhaseSponsorCT Identifier
      Monotherapy
      EDP-297FXR agonistNASH With Liver FibrosisI (Recruiting)Enanta PharmaceuticalsNCT04559126
      EDP-305FXR agonistNASHII (Recruiting)Enanta PharmaceuticalsNCT04378010
      MET642FXR agonistNASHII (Not yet recruiting)Metacrine, Inc.NCT04773964
      Obeticholic AcidFXR agonistNASH, Compensated CirrhosisIII (Active, Not recruiting)Intercept PharmaceuticalsNCT02548351

      NCT03439254
      MGL-3196 (Resmetirom)THR-β agonistHepatic impairment, NASH, NAFLD CirrhosisI (Recruiting)

      III (Recruiting)

      III (Recruiting)
      Madrigal Pharmaceuticals, Inc.NCT04643795

      NCT03900429

      NCT04197479

      NCT04951219
      VK2809THR-β agonistNASHII (Recruiting)Viking Therapeutics, Inc.NCT04173065
      LanifibranorPan-PPAR agonistNAFLD, T2DM, FibrosisII (Recruiting)

      III (Recruiting)
      University of Florida, Inventiva PharmaNCT03459079

      NCT04849728
      PioglitazonePPARα agonistT2DM, NASHII (Recruiting)University of Florida, NIDDKNCT04501406
      Saroglitazar MagnesiumPPARα, γ agonistsNASH, FibrosisII (Recruiting)Zydus Discovery DMCCNCT05011305
      MSDC-0602KMPC inhibitorT2DM, NASH, NAFLDIII (Not yet recruiting)Cirius Therapeutics, Inc.NCT03970031
      PXL065MPC inhibitorNASHII (Recruiting)Poxel SANCT04321343
      OltiprazLXR-α inhibitorNAFLDIII (Recruiting)PharmaKingNCT04142749
      EfruxiferminFGF21 analogNASHII (Active, Not recruiting)

      II (Recruiting)
      Akero Therapeutics Inc.NCT03976401

      NCT05039450

      NCT04767529
      BIO89-100FGF21 analogNASHI, II (Active, Not recruiting)

      I, II (Recruiting)
      89bio, Inc.NCT04048135

      NCT04929483

      NCT05022693
      BOS-580FGF21 analogNASHII (Not yet recruiting)Boston PharmaceuticalsNCT04880031
      BMS-986036Pegylated FGF21 analogLiver Fibrosis, NAFLD, NASH, Hepatic CirrhosisII (Active, Not recruiting)Bristol-Myers SquibbNCT03486899

      NCT03486912

      NCT04267393
      NGM282FGF19 analogNASHII (Active, Not recruiting)NGM Biopharmaceuticals, IncNCT03912532
      AldaferminFGF19 analogCompensated Cirrhosis, NASHII (Recruiting)NGM Biopharmaceuticals, IncNCT04210245
      BFKB8488ABispecific anti-FGFR1/KLB agonist antibodyNASHII (Recruiting)Genentech, Inc.NCT04171765
      MK-3655ΒKlotho/FGFR1c receptor-selective agonist antibodyNASHII (Recruiting)Merck Sharp & Dohme Corp.NCT04583423
      TirzepatideGIP/GLP-1 receptor agonistNASHII (Recruiting)Eli Lilly and CompanyNCT04166773
      XW003GLP-1 analogueT2DM, NASHI (Recruiting)Sciwind Biosciences, NovotechNCT04389775
      HM15211GLP-1/GIP/glucagon agonistNASHII (Recruiting)Hanmi Pharmaceutical Company Ltd.NCT04505436
      ALT-801GLP-1/glucagon receptor AgonistNASHI (Recruiting)

      I (Not yet recruiting)
      Altimmune, Inc.NCT04561245

      NCT04972396

      NCT05006885
      SemaglutideGLP-1 receptor agonistNAFLD, HIV InfectionsII (Recruiting)NIAID, The University of Texas Health Science CenterNCT04216589

      NCT03884075
      SemaglutideGLP-1 receptor agonistNASHIII (Recruiting)Novo Nordisk A/SNCT04822181
      EfinopegdutideGLP-1/glucagon receptor agonistNAFLD, NASHII (Recruiting)Merck Sharp & Dohme Corp.NCT04944992
      BI 456906GLP-1 receptor agonistNASHII (Recruiting)Boehringer IngelheimNCT04771273
      DD01GLP-1/glucagon agonistT2DM, NAFLDI (Recruiting)Neuraly, Inc.NCT04812262
      HEC88473GLP-1/FGF21 dual agonistNASHI (Recruiting)Dongguan HEC Biopharmaceutical R&D Co. Ltd.NCT04829123
      TVB-2640FAS inhibitorNAFLDII (Recruiting)Sagimet Biosciences Inc.NCT03938246

      NCT04906421
      AramcholSCD-1 inhibitorNASHIII (Recruiting)Galmed PharmaceuticalsNCT04104321
      PF-07202954DGAT2 inhibitorNAFLD, Liver FibrosisI (Recruiting)PfizerNCT04857437
      ION224DGAT2 inhibitorNASHII (Recruiting)Ionis Pharmaceuticals, Inc.NCT04932512
      SNP-610DGAT2 inhibitorNASHII (Not yet recruiting)Sinew Pharma Inc.NCT03468556
      SNP-612Enzyme modulatorsNASHNA (Recruiting)Sinew Pharma Inc.NCT03868566
      SNP-630Enzyme modulatorsNASHI (Not yet recruiting)Sinew Pharma Inc.NCT04808154
      ORMD-0801 (Insulin)Oral insulinT2DM, NASHII (Recruiting)

      II (Not yet recruiting)
      Oramed, Ltd., IntegriumNCT04618744

      NCT04616014
      TesamorelinGHRH analogNAFLD, obesityII (Recruiting)Massachusetts General HospitalNCT03375788
      DapagliflozinSGLT2 inhibitorNASHIII (Recruiting)Nanfang Hospital of Southern Medical UniversityNCT03723252
      ProglumideCCK receptor antagonistNASHI (Recruiting)Georgetown UniversityNCT04152473
      Vitamin EAntioxidantNAFLD, NASHNA (Active, Not recruiting)Zhejiang MedicineNCT02962297
      Vitamin EAntioxidantNAFLD, NASH

      HIV Infection
      II (Recruiting)Indiana UniversityNCT03669133
      Alpha-tocopherolAntioxidantDiabetes, Fatty Liver, ObesityI (Recruiting)NIDDKNCT00862433
      TocotrienolAntioxidantNASH, NAFLDII (Recruiting)Indiana UniversityNCT02581085
      D-alpha-tocopherolAntioxidantNASH, NAFLDII (Not yet recruiting)NIDDKNCT04801849
      IdebenoneAntioxidantNASHI, II (Recruiting)Stanford University, NIDDKNCT04669158
      LeronlimabCCR5 receptor antagonistNASHII (Recruiting)CytoDyn, Inc., Amarex Clinical ResearchNCT04521114
      CC-90001JNK inhibitorNAFLD, Liver CirrhosisII (Not yet recruiting)CelgeneNCT04048876
      HPN-01IKK inhibitorNASHI (Recruiting)Hepanova Inc.NCT04481594
      JKB-122TLR4 antagonistNASH With FibrosisII (Not yet recruiting)TaiwanJ Pharmaceuticals Co., LtdNCT04255069
      BelapectinGalectin-3 inhibitorNASH, CirrhosisII, III (Recruiting)Galectin Therapeutics Inc.NCT04365868
      SRT-015ASK1 inhibitorNASHI (Recruiting)Syneos HealthNCT04887038
      IcosabutateFatty acidNASHII (Recruiting)NorthSea Therapeutics BV.NCT04052516
      ALS-L1023Angiogenesis inhibitorNASHII (Recruiting)AngioLab, Inc.NCT04342793
      ALN-HSDHSD17B13 inhibitorNASHI (Recruiting)Alnylam PharmaceuticalsNCT04565717
      ARO-HSDHSD17B13 inhibitorNASHI (Recruiting)Arrowhead PharmaceuticalsNCT04202354
      ION839PNPLA3 inhibitorNASHI (Recruiting)AstraZeneca, ParexelNCT04483947
      AMG 60PNPLA3 inhibitorNAFLDI (Recruiting)AmgenNCT04857606
      CRV431Cyclophilin inhibitorNASH, NAFLD, Fibrosis,II (Recruiting)Hepion Pharmaceuticals, Inc.NCT04480710
      AspirinNSAIDsNAFLD, NASHI, II (Recruiting)Massachusetts General HospitalNCT04031729
      AtorvastatinHMG-CoA reductase inhibitorNASHII (Not yet recruiting)Duke UniversityNCT04679376
      SpironolactoneMR antagonistNASHI, II (Recruiting)University of California, San FranciscoNCT03576755
      VigabatrinGABA-T inhibitorNAFLD, obesityII (Not yet recruiting)Washington University School of MedicineNCT04321395
      LisinoprilACE inhibitorNASHII (Recruiting)National Cancer InstituteNCT04550481
      Digoxin tabletNa+/K+ ATPase inhibitorNASHII (Not yet recruiting)The Affiliated Nanjing Drum Tower Hospital of NanjingNCT04216693
      LPCN 1144Bioidentical testosteroneNASHII (Active, Not recruiting)Lipocine Inc.NCT04685993 NCT04134091
      ZSP1601NANASHI, II (Recruiting)Guangdong Raynovent Biotech Co., LtdNCT04140123
      HU6Mitochondrial uncouplerNASHII (Recruiting)Rivus Pharmaceuticals, Inc.NCT04874233
      NamodenosonA3 adenosine receptor agonistNASHII (Not yet recruiting)Can-Fite BioPharmaNCT04697810
      FM101A3 adenosine receptor agonistNAFLD, NASHII (Recruiting)Future MedicineNCT04710524
      CilostazolPDE3 inhibitorNAFLDI, II (Recruiting)Sadat City UniversityNCT04761848
      TERN-201VAP1 inhibitorNASHI (Active, Not recruiting)Terns, Inc.NCT04897594
      Combination therapy
      Elobixibat Cholestyramine powderElobixibat: BAT inhibitor

      Cholestyramine: Decrease bile acid recirculation
      NAFLD, NASHII (Active, Not recruiting)Yokohama City University, EA Pharma Co., Ltd.NCT04235205
      Saroglitazar

      Vitamin E
      Saroglitazar: PPARα, γ agonist

      Vitamin E: Antioxidant
      NAFLDIII (Recruiting)Asian Institute of Gastroenterology, IndiaNCT04193982
      PF-06865571

      PF-05221304
      PF-06865571: DGAT2 inhibitor

      PF-05221304: ACC inhibitor
      NASH, NAFLD, FibrosisII (Recruiting)PfizerNCT04399538 NCT04321031
      LYS006

      Tropifexor
      LYS006: leukotriene A4 hydrolase inhibitor, Tropifexor: FXR agonistNAFLD, NASHII (Recruiting)Novartis PharmaceuticalsNCT04147195
      MET409

      Empagliflozin
      MET409: FXR agonist

      Empagliflozin: SGLT2 inhibitor
      T2DM, NASHII (Not yet recruiting)Metacrine, Inc.NCT04702490
      NNC0194 0499 SemaglutideNNC0194 0499: FGF receptor agonist

      Semaglutide: GLP-1 receptor agonist
      NASHII (Recruiting)Novo Nordisk A/SNCT05016882
      Tropifexor

      Licogliflozin
      Tropifexor: FXR agonist

      Licogliflozin: SGLT1/2 inhibitor
      NASHII (Recruiting)Novartis PharmaceuticalsNCT04065841
      Semaglutide

      Cilofexor Firsocostat
      Semaglutide GLP-1 receptor agonists

      Cilofexor: FXR agonist

      Firsocostat: ACC inhibitor
      NASHII (Recruiting)Gilead Sciences| Novo Nordisk A/SNCT04971785
      Abbreviations: NIDDK, National Institute of Diabetes and Digestive and Kidney Diseases; NIAID, National Institute of Allergy and Infectious Diseases; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; Na+/K+ ATPase, sodium-potassium adenosine triphosphatase; PDE3, phosphodiesterase type 3; AMPK, adenosine monophosphate-activated protein kinase; FXR, farnesoid X receptor; SGLT1/2, sodium-glucose cotransporter type 1/2; THR-β, thyroid receptor β; PPAR, peroxisome proliferator-activated receptor alpha; MPC, mitochondrial pyruvate carrier; KHK, ketohexokinase; FGF21, fibroblast growth factor 21; GIP/GLP-1, glucose-dependent insulinotropic peptide/glucagon like peptide-1; ACC, acetyl-coenzyme A carboxylase; SCD1, stearoyl-CoA; DGAT2, diacylglycerol O-acyltransferase desaturase-2; CCR5, CC chemokine receptor 5; JNK, C-Jun N-terminal kinase; BATi, bile acid transporter inhibitor; PNPLA3, patatin-like phospholipase domain-containing protein 3; GR, glucocorticoid receptor; HSD17B13, 17β-hydroxysteroid dehydrogenase type 13; NSAIDs, non-steroidal anti-inflammatory drugs, TLR4, toll like receptor 4; FASN, fatty acid synthase; GHRH, growth hormone-releasing hormone; MR, mineralocorticoid receptor; GABA-T, gamma-aminobutyric acid transaminase; ACE, angiotensin-converting enzyme; VAP-1, vascular adhesion protein-1.
      Fig. 3
      Fig. 3Emerging pharmacotherapeutics for NAFLD/NASH at various phases of the clinical trial.
      The outer circle (orange) represents drugs at a phase I trial, the circle in the middle (blue) represents drugs at a phase II trial, and the innermost circle (pink) represents drugs at a phase III trial, as per the clinical trial database- https://www.clinicaltrials.gov. Terminated trials that failed due to the lack of efficacy endpoints are highlighted in red.
      Fig. 4
      Fig. 4Overview of mechanisms of action of emerging pharmacotherapeutics. Major targets of pharmacological interventions for NAFLD/NASH in clinical trials.

      3.1 Metabolic targeted therapies

      3.1.1 Peroxisome proliferator-activated receptor (PPAR) agonists

      PPARs are ligand-activated transcription factor nuclear receptors. The isotypes of PPAR- PPARα, PPARβ/δ, and PPARγ are widely expressed in various tissues and regulate several functions such as lipid metabolism, glucose metabolism, and inflammation via modulation of distinct target gene expression [
      • Fougerat A.
      • Montagner A.
      • Loiseau N.
      • Guillou H.
      • Wahli W.
      Peroxisome proliferator-activated receptors and their novel ligands as candidates for the treatment of non-alcoholic fatty liver disease.
      ]. Increasing evidence suggests the involvement of PPARs in NAFLD, and multiple observations support PPARs modulation as a potential therapeutic target for NAFLD or NASH [
      • Francque S.
      • Verrijken A.
      • Caron S.
      • Prawitt J.
      • Paumelle R.
      • Derudas B.
      • et al.
      PPARα gene expression correlates with severity and histological treatment response in patients with non-alcoholic steatohepatitis.
      ,
      • Zarei M.
      • Barroso E.
      • Palomer X.
      • Dai J.
      • Rada P.
      • Quesada-López T.
      • et al.
      Hepatic regulation of VLDL receptor by PPARβ/δ and FGF21 modulates non-alcoholic fatty liver disease.
      ]. PPAR targeting drugs, including thiazolidinediones, such as pioglitazone (a synthetic ligand of PPARγ), are clinically used to treat type 2 diabetes mellitus (T2DM). As per the proof of concept study, administration of pioglitazone led to metabolic and histologic improvement (reductions in steatosis, inflammation, and ballooning necrosis) in subjects with NASH [
      • Belfort R.
      • Harrison S.A.
      • Brown K.
      • Darland C.
      • Finch J.
      • Hardies J.
      • et al.
      A Placebo-controlled Trial of Pioglitazone in Subjects with Nonalcoholic Steatohepatitis.
      ]. In nondiabetic subjects with NASH, pioglitazone therapy lasting over 12 months improved metabolic and histologic parameters and remarkably reduced liver injury and fibrosis [
      • Aithal G.P.
      • Thomas J.A.
      • Kaye P.V.
      • Lawson A.
      • Ryder S.D.
      • Spendlove I.
      • et al.
      Randomized, placebo-controlled trial of pioglitazone in nondiabetic subjects with nonalcoholic steatohepatitis.
      ]. In the PIVENS trial, pioglitazone was associated with highly significant reductions in hepatic steatosis, lobular inflammation, and hepatocellular ballooning, also improved insulin resistance and liver-enzyme levels. However, it did not meet the prespecified significance level for the primary outcome (improvement in the histologic features of NASH) and increased the weight gain in NASH patients [
      • Sanyal A.J.
      • Chalasani N.
      • Kowdley K.V.
      • McCullough A.
      • Diehl A.M.
      • Bass N.M.
      • et al.
      Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis.
      ]. A long-term (3 years) pioglitazone treatment in patients with NASH and prediabetic or T2DM patients was safe, well-tolerated, and effective in improving the metabolic and histological parameters of NASH without any major drug-related adverse events, except weight gain [
      • Cusi K.
      • Orsak B.
      • Bril F.
      • Lomonaco R.
      • Hecht J.
      • Ortiz-Lopez C.
      • et al.
      Long-term pioglitazone treatment for patients with nonalcoholic steatohepatitis and prediabetes or type 2 diabetes mellitus a randomized trial.
      ]. It must be mentioned that pioglitazone is contraindicated in patients with established heart failure or with an increased risk of heart failure because it can lead to the progression of heart failure [
      • Gastaldelli A.
      • Cusi K.
      From NASH to diabetes and from diabetes to NASH: mechanisms and treatment options.
      ,
      • Francque S.
      • Szabo G.
      • Abdelmalek M.F.
      • Byrne C.D.
      • Cusi K.
      • Dufour J.-F.
      • et al.
      Nonalcoholic steatohepatitis: the role of peroxisome proliferator-activated receptors.
      ]. Presently, pioglitazone is undergoing a phase II trial (NCT04501406).
      Saroglitazar is also a dual (PPARα/γ) agonist which significantly reduced serum ALT levels, insulin resistance, dyslipidemia, liver fat content, markers of hepatocellular injury, and fibrosis in patients with NAFLD/NASH in a recent phase II trial (EVIDENCES IV) [
      • S. G.
      • M. N.
      • N. L.
      • R. M.
      • V. A.
      • K. C.
      • et al.
      Saroglitazar, a PPAR-α/γ agonist, for treatment of NAFLD: a randomized controlled double-blind phase 2 Trial.
      ]. Saroglitazar is now being evaluated in NASH and fibrosis patients, with primary histological endpoints- resolution of NASH with no worsening of fibrosis (NCT05011305).
      Several other PPAR agonists have not shown any significant improvements in NAFLD- NASH resolution. For instance, pemafibrate (K-877), a selective agonist of PPARα, did not reduce the liver fat content; however, liver stiffness and ALT levels were significantly reduced in the recent phase II clinical trial in NAFLD patients [
      • Nakajima Atsushi
      • Eguchi Yuichiro
      • Imajo Kento
      • Tamaki Nobuharu
      • Suganami Hideki
      • Nojima Toshiaki
      • et al.
      Parallel sessions.
      ]. Pemafibrate-mediated decrease in serum transaminase activities and lipid profiles (without any major adverse effects) was also reported in a recent clinical trial in patients with dyslipidemia [
      • Ishibashi S.
      • Yamashita S.
      • Arai H.
      • Araki E.
      • Yokote K.
      • Suganami H.
      • et al.
      Effects of K-877, a novel selective PPARα modulator (SPPARMα), in dyslipidaemic patients: a randomized, double blind, active- and placebo-controlled, phase 2 trial.
      ].
      Another selective activator of PPARγ rosiglitazone did not improve NASH but reduced hepatic steatosis and transaminase levels as reported in the short and long-term phase II (FLIRT) trial [
      • Ratziu V.
      • Charlotte F.
      • Bernhardt C.
      • Giral P.
      • Halbron M.
      • Lenaour G.
      • et al.
      Long-term efficacy of rosiglitazone in nonalcoholic steatohepatitis: results of the fatty liver improvement by rosiglitazone therapy (FLIRT 2) extension trial.
      ,
      • Roden M.
      Mechanisms of disease: hepatic steatosis in type 2 diabetes - pathogenesis and clinical relevance.
      ]. In a 52-week phase II trial in NASH patients, a selective PPARβ/δ agonist seladelpar (MBX-8025) reduced the liver fat content, improved liver biochemistry, and increased fatty acid oxidation. However, it failed to meet the primary endpoint, i.e., a change in liver fat content confirmed by magnetic resonance imaging- proton density fat fraction (MRI-PDFF) at week 12 [
      • Harrison S.A.
      • Gunn N.T.
      • Khazanchi A.
      • Guy C.D.
      • Brunt E.M.
      • Moussa S.
      • et al.
      A 52-week multi-center double-blind randomized phase 2 study of seladelpar, a potent and selective peroxisome proliferator-activated receptor delta (PPAR-delta) agonist, in patients with nonalcoholic steatohepatitis (NASH).
      ].
      The dual PPARα/δ agonist elafibranor also failed to reach the primary histological endpoint of NASH resolution without worsening of fibrosis, according to the interim report on the RESOLVE-IT phase III trial [
      • GENFIT
      Announces Results from Interim Analysis of RESOLVE-IT Phase 3 Trial of Elafibranor in Adults with NASH and Fibrosis | GENFIT.
      ]. In a previous study, elafibranor did not meet the predefined end point in the intention to treat the population. However, it resolved NASH without worsening of fibrosis in the intention-to-treat analysis. Moreover, elafibranor was well tolerated and improved the cardiometabolic risk profile [
      • Ratziu V.
      • Harrison S.A.
      • Francque S.
      • Bedossa P.
      • Lehert P.
      • Serfaty L.
      • et al.
      Elafibranor, an agonist of the peroxisome proliferator−activated receptor−α and −δ, induces resolution of nonalcoholic steatohepatitis without fibrosis worsening.
      ].
      Although some studies have shown a limited efficacy of activation of individual PPARs (PPARα and PPARγ), ongoing clinical trials suggest that dual and/or pan-PPAR agonists might have a broader and more efficacious therapeutic potential against NASH by targeting multiple interrelated pathological mechanisms. Lanifibranor (IVA337) is a pan-PPAR agonist with potent but moderate and well-balanced effects on the three isotypes of PPAR. In the preclinical animal models of NASH, lanifibranor improved hepatic steatosis, inflammation, and all histological features of NASH, including liver fibrosis [
      • Wettstein G.
      • Luccarini J.-M.
      • Poekes L.
      • Faye P.
      • Kupkowski F.
      • Adarbes V.
      • et al.
      The new-generation pan-peroxisome proliferator-activated receptor agonist IVA337 protects the liver from metabolic disorders and fibrosis.
      ]. Moreover, in phase II NATIVE trial, lanifibranor met both the primary and secondary efficacy endpoint to reduce NASH and fibrosis in patients with non-cirrhotic NASH. Lanifibranor is the first drug candidate to achieve statistically significant effects on NASH resolution with no worsening of fibrosis and improvement of fibrosis with no worsening of NASH [
      • InventivaPharma
      PRESS RELEASEInventiva’s lanifibranor meets the primary and key secondary endpoints in the Phase IIb NATIVE clinical trial in non-alcoholic steatohepatitis (NASH).
      ]. At present, lanifibranor is undergoing a phase III clinical trial (NCT04849728).

      3.1.2 Mitochondrial pyruvate carrier (MPC) inhibitor

      MPC transports the pyruvate generated during glycolysis or from lactate into the inner mitochondrial membrane matrix for metabolism and promotes gluconeogenesis and de novo lipogenesis [
      • McCommis K.S.
      • Finck B.N.
      Treating hepatic steatosis and fibrosis by modulating mitochondrial pyruvate metabolism.
      ]. Targeted MPC inhibition has been reported to restrict pyruvate transport and metabolism in the hepatocytes, resulting in the disruption of gluconeogenesis, de novo lipogenesis, and enhanced fatty acid oxidation [
      • McCommis K.S.
      • Finck B.N.
      Treating hepatic steatosis and fibrosis by modulating mitochondrial pyruvate metabolism.
      ]. In a recent phase IIb study (EMMINENCE) conducted on patients with biopsy-confirmed NASH and fibrosis (F1-F3), MSDC-0602K did not demonstrate statistically significant effects on primary and secondary liver histology endpoints (i.e., reversal of NASH or fibrosis) but reduced serum levels of fasting glucose, fasting insulin, and glycated hemoglobin (HbA1c). It was, however, also associated with an increased incidence of weight gain [
      • Harrison S.A.
      • Alkhouri N.
      • Davison B.A.
      • Sanyal A.
      • Edwards C.
      • Colca J.R.
      • et al.
      Insulin sensitizer MSDC-0602K in non-alcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase IIb study.
      ]. MSDC-0602K is a second-generation thiazolidinedione designed to minimize direct binding to PPAR-γ while still producing insulin-sensitizing effects. However, the increase in weight gain indicates a significant PPAR-γ effect on adipose tissue that led to metabolic improvement (lower glucose levels), but it showed no advantage over other PPAR-γ agonists, e.g., pioglitazone.

      3.1.3 Ketohexokinase inhibitor

      Ketohexokinase enzyme phosphorylates fructose into fructose-1-phosphate, promoting gluconeogenesis and de novo lipogenesis. Fructose may also directly or indirectly induce hepatic insulin resistance leading to hyperinsulinemia, which can further stimulate hepatic lipogenesis [
      • Softic S.
      • Stanhope K.L.
      • Boucher J.
      • Divanovic S.
      • Lanaspa M.A.
      • Johnson R.J.
      • et al.
      Fructose and hepatic insulin resistance.
      ]. Ketohexokinase inhibition by PF-06835919 improved NASH by reducing fructose-induced steatosis, fibrogenesis, and liver injury in mouse and human liver cell cultures [
      • Shepherd E.L.
      • Saborano R.
      • Northall E.
      • Matsuda K.
      • Ogino H.
      • Yashiro H.
      • et al.
      Ketohexokinase inhibition improves NASH by reducing fructose-induced steatosis and fibrogenesis.
      ,
      • Futatsugi K.
      • Smith A.C.
      • Tu M.
      • Raymer B.
      • Ahn K.
      • Coffey S.B.
      • et al.
      Discovery of PF-06835919: a potent inhibitor of ketohexokinase (khk) for the treatment of metabolic disorders driven by the overconsumption of fructose.
      ]. PF-06835919 administration to NAFLD subjects showed a reduction in hepatic fat content and insulin resistance, ALT, AST, and gamma-glutamyl transferase (GGT) [
      • Calle R.
      • Bergman A.
      • Somayaji V.
      • Chidsey K.
      • Kazierad D.
      PS-110-Ketohexokinase inhibitor PF-06835919 administered for 6 weeks reduces whole liver fat as measured by magnetic resonance imaging-proton density fat fraction in subjects with non-alcoholic fatty liver disease.
      ]. In a recent phase II study in participants with NAFLD, a significant reduction in the whole liver fat and improvement in inflammatory markers were observed in participants receiving PF-06835919300, while no serious adverse effects were reported [
      • Kazierad D.J.
      • Chidsey K.
      • Somayaji V.R.
      • Bergman A.J.
      • Birnbaum M.J.
      • Calle R.A.
      Inhibition of ketohexokinase in adults with NAFLD reduces liver fat and inflammatory markers: a randomized phase 2 trial.
      ].

      3.1.4 Farnesoid X receptor (FXR) agonist

      FXR is a ligand-activated nuclear receptor transcription factor, abundantly expressed in the liver, intestine, and kidney. It regulates several metabolic functions, including bile acid synthesis, glucose homeostasis, and lipid metabolism [
      • Kim S.G.
      • Kim B.K.
      • Kim K.
      • Fang S.
      Bile acid nuclear receptor farnesoid X receptor: therapeutic target for nonalcoholic fatty liver disease.
      ]. Activation of FXR by ligands induces a small heterodimer partner, which suppresses CYP7A1 gene expression. CYP7A1 is a rate-limiting enzyme that converts cholesterol to bile acids, inhibition of which results in the reduced rate of bile synthesis in the liver. Activation of FXR in both hepatocytes and enterocytes negatively regulates the bile acid synthesis and improves hepatic steatosis and inflammation [
      • Hoofnagle J.H.
      FXR Agonists as therapy for liver disease.
      ].
      Obeticholic acid, a synthetic derivative of chenodeoxycholic acid, is a potent and specific FXR agonist, already approved for primary biliary cholangitis by the US FDA. In a recent FLINT trial in NASH patients without cirrhosis, obeticholic acid improved the histological features of NASH and fibrosis. However, treatment was associated with pruritus in 23% of patients, along with increased total cholesterol and low-density lipoprotein cholesterol (LDL-C) levels and decreased high-density lipoprotein cholesterol (HDL-C) [
      • Neuschwander-Tetri B.A.
      • Loomba R.
      • Sanyal A.J.
      • Lavine J.E.
      • Van Natta M.L.
      • Abdelmalek M.F.
      • et al.
      Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial.
      ]. The subsequent phase III clinical trial (REGENERATE) treatment with obeticholic acid 25 mg showed improvement in fibrosis with no worsening of NASH in patients with stage F2/F3 fibrosis at the month-18 interim analysis. In addition, a posthoc analysis showed that obeticholic acid treatment resulted in NASH resolution with no worsening of fibrosis. However, pruritis was the most common adverse effect, and the treatment was also associated with a 20% increase in LDL-C [
      • Younossi Z.M.
      • Ratziu V.
      • Loomba R.
      • Rinella M.
      • Anstee Q.M.
      • Goodman Z.
      • et al.
      Articles obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial.
      ]. Based on the surrogate endpoints, the New Drug Application (NDA) for marketing authorization of obeticholic acid did not receive approval by US FDA because the predicted benefits of obeticholic acid remained uncertain and did not sufficiently outweigh the potential risks []. However, the agency recommended additional post interim analysis on the efficacy and safety of obeticholic acid from the REGENERATE trial. The safety and efficacy of obeticholic acid for the treatment of compensated cirrhosis due to NASH in phase III REVERSE (NCT03439254) is currently being evaluated.
      Tropifexor (LJN452) is a highly potent non-bile acid FXR agonist which demonstrated a robust reduction in the hepatic fat fraction, ALT, and body weight with good safety and tolerability after 12 weeks of treatment. No significant effects on NASH resolution and fibrosis improvement were observed. Like other FXR agonists, tropifexor was also associated with mild pruritis and a minor dose-related increase in LDL-C as presented in the interim results of phase IIb FLIGHT-FXR trial [
      • Lucas K.J.
      • Lopez P.
      • Lawitz E.
      • Sheikh A.
      • Aizenberg D.
      • Hsia S.
      • et al.
      Tropifexor, a highly potent FXR agonist, produces robust and dose-dependent reductions in hepatic fat and serum alanine aminotransferase in patients with fibrotic NASH after 12 weeks of therapy: FLIGHT-FXR Part C interim results.
      ], which is now terminated (NCT02855164). However, a phase II trial of tropifexor as a monotherapy and combination with leukotriene A4 hydrolase inhibitor- LYS006 (NCT04147195) and with licogliflozin (a novel SGLT1/2 inhibitor) is ongoing for NAFLD-NASH or NASH/fibrosis patients to evaluate the safety and efficacy profile (NCT04065841).
      Cilofexor, another FXR agonist, was reported to reduce hepatic steatosis, serum bile acids level and improve liver biochemistry without any significant improvements in fibrosis in a phase II trial in noncirrhotic NASH patients. Treatment was associated with moderate to severe pruritis [
      • Patel K.
      • Harrison S.A.
      • Elkhashab M.
      • Trotter J.F.
      • Herring R.
      • Rojter S.E.
      • et al.
      Cilofexor, a nonsteroidal FXR agonist, in patients with noncirrhotic NASH: a phase 2 randomized controlled trial.
      ].
      Nidufexor (LMB763), an FXR agonist, also decreased ALT and hepatic fat fraction in patients with NASH after 12 weeks of treatment. Although HDL-C levels were decreased, no meaningful changes in total cholesterol, LDL-C, or TG were observed, and pruritis was the most frequent adverse effect with a 100 mg dose [
      • Aspinall R.
      • Shennak M.
      • Stocia G.
      • Quinn D.
      • Javashvili L.
      • Molteni V.
      • et al.
      Nidufexor, a non-bile acid FXR agonist, decreases ALT and hepatic fat fraction in patients with NASH after 12 weeks dosing.
      ]. A phase II clinical trial of Nidufexor in patients with NASH has recently been terminated as per the clinical trial database (NCT02913105).
      EDP-297 and EDP-305 are potent non-bile acid FXR agonists, the potential of which against NASH is being evaluated in phase I and phase II clinical trials, respectively. EDP-297 has shown potent anti-fibrotic effects and significantly reduced liver fat contents in a diet-induced rodent model of NASH, while EDP-305 reduced fibrosis liver injury in murine biliary and metabolic models of liver disease [
      • An P.
      • Wei G.
      • Huang P.
      • Li W.
      • Qi X.
      • Lin Y.
      • et al.
      A novel non-bile acid FXR agonist EDP-305 potently suppresses liver injury and fibrosis without worsening of ductular reaction.
      ,
      • Erstad D.J.
      • Farrar C.T.
      • Ghoshal S.
      • Masia R.
      • Ferreira D.S.
      • Chen Y.I.
      • et al.
      Molecular magnetic resonance imaging accurately measures the antifibrotic effect of EDP-305, a novel farnesoid X receptor agonist.
      ].
      Another novel non-bile acid FXR agonist, MET409, has recently been shown to significantly reduce the liver fat content after 12 weeks of treatment in patients with NASH. It also improved the biochemical parameters of liver function. However, treatment was associated with pruritus, decreased HDL-C, and increased LDL-C levels [
      • Harrison S.A.
      • Bashir M.R.
      • Lee K.-J.
      • Shim-Lopez J.
      • Lee J.
      • Wagner B.
      • et al.
      A structurally optimized FXR agonist, MET409, reduced liver fat content over 12 weeks in patients with non-alcoholic steatohepatitis.
      ].
      EYP001a is an orally bioavailable synthetic non-steroidal, non-bile acid FXR agonist currently being evaluated in a phase II trial (NCT03812029). MET642 is also a potent non-bile acid FXR agonist and is undergoing a phase II trial in NASH patients (NCT04773964).

      3.1.5 Liver X receptor (LXR) alpha-inhibitor

      LXR is a ligand-activated transcriptional nuclear receptor that has been studied for several decades as a possible therapeutic target for combating metabolic diseases, including NAFLD [
      • Ni M.
      • Zhang B.
      • Zhao J.
      • Feng Q.
      • Peng J.
      • Hu Y.
      • et al.
      Biological mechanisms and related natural modulators of liver X receptor in nonalcoholic fatty liver disease.
      ]. Among two subtypes of LXR (LXRα and LXRβ), LXRα was reported to be overexpressed in the liver of patients with NAFLD [
      • Lima-Cabello E.
      • García-Mediavilla M.V.
      • Miquilena-Colina M.E.
      • Vargas-Castrillón J.
      • Lozano-Rodríguez T.
      • Fernández-Bermejo M.
      • et al.
      Enhanced expression of pro-inflammatory mediators and liver X-receptor-regulated lipogenic genes in non-alcoholic fatty liver disease and hepatitis C.
      ], and its expression was positively correlated with the hepatic fat content, inflammation, and fibrosis in NAFLD [
      • Ahn S.B.
      • Jang K.
      • Jun D.W.
      • Lee B.H.
      • Shin K.J.
      Expression of liver X receptor correlates with intrahepatic inflammation and fibrosis in patients with nonalcoholic fatty liver disease.
      ]. LXR activation can promote de novo lipogenesis by increasing the expression of several genes responsible for glycolysis, lipogenesis, inflammation, and innate immunity responses leading to steatosis, inflammation, and fibrosis [
      • Ni M.
      • Zhang B.
      • Zhao J.
      • Feng Q.
      • Peng J.
      • Hu Y.
      • et al.
      Biological mechanisms and related natural modulators of liver X receptor in nonalcoholic fatty liver disease.
      ,
      • Liu Y.
      • de K. Qiu
      • Ma X.
      Liver X receptors bridge hepatic lipid metabolism and inflammation.
      ]. Inhibition of LXRα by antagonists such as oltipraz (a synthetic dithiolethione) reduced the liver fat content in a 24-week phase II study with NAFLD patients. However, it significantly increased total cholesterol and HDL-C levels. No significant change was observed in insulin sensitivity, adipokine, ALT, AST, GGT, insulin, glucose, TG, LDL-C, TNFα, and IL-6 [
      • Kim W.
      • Kim B.G.
      • Lee J.S.
      • Lee C.K.
      • Yeon J.E.
      • Chang M.S.
      • et al.
      Randomised clinical trial: the efficacy and safety of oltipraz, a liver X receptor alpha-inhibitory dithiolethione in patients with non-alcoholic fatty liver disease.
      ].

      3.1.6 Fibroblast growth factors (FGF) analogs/activators

      FGF19, FGF21, and FGF23 are metabolic hormones with either autocrine or paracrine actions [
      • Maratos-Flier E.
      Fatty liver and FGF21 physiology.
      ]. FGF21 synthesized predominantly in the liver has been identified as a critical regulator of metabolic functions, including glucose metabolism, lipid metabolism, energy homeostasis, and insulin sensitivity [
      • Li H.
      • Zhang J.
      • Jia W.
      Fibroblast growth factor 21: a novel metabolic regulator from pharmacology to physiology.
      ,
      • Cuevas-Ramos D.
      • Almeda-Valdes P.
      • Aguilar-Salinas C.
      • Cuevas-Ramos G.
      • Cuevas-Sosa A.
      • Gomez-Perez F.
      The role of Fibroblast Growth Factor 21 (FGF21) on energy balance, glucose and lipid metabolism.
      ]. In addition, FGF21 inhibits the lipolysis of white adipose tissue, reduces the delivery of FFAs to the liver [
      • Liu J.
      • Xu Y.
      • Hu Y.
      • Wang G.
      The role of fibroblast growth factor 21 in the pathogenesis of non-alcoholic fatty liver disease and implications for therapy.
      ], promotes PPARα mediated hepatic fatty acid β-oxidation, and mitigates lipotoxicity [
      • Badman M.K.
      • Pissios P.
      • Kennedy A.R.
      • Koukos G.
      • Flier J.S.
      • Maratos-Flier E.
      Hepatic fibroblast growth factor 21 Is regulated by PPARα and is a key mediator of hepatic lipid metabolism in ketotic states.
      ], attenuates hepatic ER stress, inflammation, and apoptosis [
      • Zarei M.
      • Pizarro-Delgado J.
      • Barroso E.
      • Palomer X.
      • Vázquez-Carrera M.
      Targeting FGF21 for the treatment of nonalcoholic steatohepatitis.
      ]. FGF21 acts by binding to its receptor complex consisting of FGF receptor FGFR1c and a coreceptor called β-Klotho, which is restrictedly expressed in hepatocytes and adipose tissue [
      • Kobayashi K.
      • Tanaka T.
      • Okada S.
      • Morimoto Y.
      • Matsumura S.
      • Manio M.C.C.
      • et al.
      Hepatocyte β-Klotho regulates lipid homeostasis but not body weight in mice.
      ]. FGF21 analogs have demonstrated efficacy in animal models and humans with NASH, and several clinical trials with FGF21 analogs are currently underway (Table 2).
      BMS-986036 (pegbelfermin) is a pegylated FGF21 analog. The safety of pegbelfermin was demonstrated in a phase IIa study with NASH patients where subcutaneous treatment of pegbelfermin for 16 weeks was well tolerated and significantly reduced hepatic fat, improved biomarkers of metabolic function (adiponectin and lipid concentrations), markers of hepatic injury (ALT, AST), and biomarkers of fibrosis in patients with NASH [
      • Sanyal A.
      • Charles E.D.
      • Neuschwander-Tetri B.A.
      • Loomba R.
      • Harrison S.A.
      • Abdelmalek M.F.
      • et al.
      Pegbelfermin (BMS-986036), a PEGylated fibroblast growth factor 21 analogue, in patients with non-alcoholic steatohepatitis: a randomised, double-blind, placebo-controlled, phase 2a trial.
      ]. The efficacy of pegbelfermin is now being evaluated in phase II clinical trials in patients with NASH and stage 3 liver fibrosis (FALCON 1; NCT03486899) or compensated cirrhosis (FALCON 2; NCT03486912). Another pegylated novel FGF21 analog molecule, BIO89-100, is being evaluated in phase I/II trial in subjects with NASH or NAFLD (NCT04048135).
      MK-3655 (NGM313) is a monoclonal agonistic antibody that selectively activates the FGF receptor 1/Klothoβ (FGFR1/KLB). A single dose of NGM313 has improved insulin resistance and reduced hepatic fat content in obese subjects with a significant increase in glucose disposal rate, HbA1c, TG, LDL-C, and HDL-C [
      • Depaoli Alex
      • Phung Van
      • Bashir Mustafa R.
      • Morrow Linda
      • Beysen Carine
      • Yan Andrew
      • et al.
      140-LB: NGM313, a novel activator of b-Klotho/FGFR1c, improves insulin resistance and reduces hepatic fat in obese, nondiabetic subjects.
      ]. MK-3655 is currently processed for a phase II trial (NCT04583423).
      BFKB8488A is a humanized bispecific FGFR1/KLB agonistic antibody that was active and well-tolerated in humans. A single administration of BFKB8488A resulted in metabolically beneficial effects in humans by improving cardiometabolic functions such as increased total serum adiponectin HDL-C and decreased serum TG, LDL-C, fasting insulin, and transient bodyweight. However, nausea was the most common adverse effect [
      • Baruch A.
      • Wong C.
      • Chinn L.W.
      • Vaze A.
      • Sonoda J.
      • Gelzleichter T.
      • et al.
      Antibody-mediated activation of the FGFR1/Klothoβ complex corrects metabolic dysfunction and alters food preference in obese humans.
      ]. In a recent phase II study, multiple doses of BFKB8488A have been reported to reduce hepatic fat fraction and improve liver health in NAFLD patients [
      • Kunder R.
      • Yeh F.
      • Chinn L.W.
      • et al.
      Multiple doses of an anti-FGFR1/KLB bispecific antibody (BFKB8488A) are associated with a decrease in hepatic fat in patients with NAFLD.
      ]. Further evaluation of the phase II trial is ongoing (NCT04171765).
      Aldafermin (NGM282) is a humanized FGF19 analog that significantly reduced the liver fat content in patients with NASH patients [
      • Harrison S.A.
      • Rinella M.E.
      • Abdelmalek M.F.
      • Trotter J.F.
      • Paredes A.H.
      • Arnold H.L.
      • et al.
      NGM282 for treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.
      ]. More recently, aldafermin improved liver fibrosis and NAFLD histological score in NASH patients. Treatment with aldafermin led to a relative reduction in liver fat content and improved biochemical parameters of liver functions [
      • Harrison S.A.
      • Rossi S.J.
      • Paredes A.H.
      • Trotter J.F.
      • Bashir M.R.
      • Guy C.D.
      • et al.
      NGM282 improves liver fibrosis and histology in 12 weeks in patients with nonalcoholic steatohepatitis.
      ]. A 24-week treatment with aldafermin significantly reduced liver fat content and improved fibrosis and NASH resolution in patients with NASH and stage 2 or 3 fibrosis [
      • Harrison S.A.
      • Neff G.
      • Guy C.D.
      • Bashir M.R.
      • Paredes A.H.
      • Frias J.P.
      • et al.
      CLINICAL-LIVER efficacy and safety of aldafermin, an engineered fgf19 analog, in a randomized, double-blind, placebo-controlled trial of patients with nonalcoholic steatohepatitis.
      ]. In the recent phase IIb (ALPINE 2/3) trial in NASH patients with stage 2/3 fibrosis, aldafermin failed to meet the primary endpoint (fibrosis improvement by >1 stage with no worsening of NASH). However, it significantly improved the secondary endpoints, including NASH resolution and reduction of liver fat content, ALT, AST, and fibrosis marker (Pro-C3). The phase III clinical development of aldafermin in F2-F3 NASH has now been halted [
      NGM bio reports topline results from 24-week phase 2b ALPINE 2/3 study of Aldafermin in NASH | NGM Biopharmaceuticals, Inc..
      ]. A phase II trial (ALPINE 4) with aldafermin is currently ongoing in subjects with compensated NASH cirrhosis
      Efruxifermin (AKR001) is an FcFGF21 analog with N- and C-terminal modification, which prevents its degradation and increases potency. In a phase I trial in individuals with T2DM, efruxifermin improved glycaemic control and insulin sensitivity, simultaneously improved markers of lipid metabolism (decreased plasma TG, LDL-C, and increased HDL-C) [
      • Kaufman A.
      • Abuqayyas L.
      • Denney W.S.
      • Tillman E.J.
      • Rolph T.
      AKR-001, an Fc-FGF21 analog, showed sustained pharmacodynamic effects on insulin sensitivity and lipid metabolism in type 2 diabetes patients.
      ]. In a recent phase II trial, efruxifermin met the primary endpoint and reduced the hepatic fat fraction in patients with NASH. The most common adverse effects were mild and transient gastrointestinal events [
      • Harrison S.A.
      • Ruane P.J.
      • Freilich B.L.
      • Neff G.
      • Patil R.
      • Behling C.A.
      • et al.
      Efruxifermin in non-alcoholic steatohepatitis: a randomized, double-blind, placebo-controlled, phase 2a trial.
      ].

      3.1.7 Glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic peptide (GIP) receptor analogs or agonists

      GLP-1 and GIP are the two primary incretin hormones secreted by the L-cells and K-cells of the small intestine, respectively [
      • Seino Y.
      • Fukushima M.
      • Yabe D.
      GIP and GLP-1, the two incretin hormones: similarities and differences.
      ]. GIP and GLP-1 bind to their specific G-protein coupled receptors, GIP receptor and the GLP-1 receptor, which initiates downstream signaling events in pancreatic β cells to stimulate glucose-dependent insulin secretion [
      • Seino Y.
      • Fukushima M.
      • Yabe D.
      GIP and GLP-1, the two incretin hormones: similarities and differences.
      ]. Substantial evidence from animal and human studies indicates that GLP-1 receptor agonists or mimetics have improved NAFLD, as reviewed by Petit et al. [
      • Petit J.M.
      • Vergès B.
      GLP-1 receptor agonists in NAFLD.
      ].
      Semaglutide, a GLP-1 receptor agonist, recently completed a phase II trial in patients with NASH and fibrosis, and the results show a significantly higher percentage of patients with NASH resolution without worsening of fibrosis [
      • Newsome P.N.
      • Buchholtz K.
      • Cusi K.
      • Linder M.
      • Okanoue T.
      • Ratziu V.
      • et al.
      A placebo-controlled trial of subcutaneous semaglutide in nonalcoholic steatohepatitis.
      ]. Semaglutide (0.4 mg) met the primary endpoint of NASH resolution with no worsening of fibrosis in 59% of patients with F2-F3 fibrosis compared with 17% in placebo. However, the confirmatory secondary endpoint of fibrosis improvement with no worsening of NASH was not met. Fibrosis was improved by one stage in all arms, with no difference between placebo (33%) and 0.4 mg semaglutide (43%) [
      • Newsome P.N.
      • Buchholtz K.
      • Cusi K.
      • Linder M.
      • Okanoue T.
      • Ratziu V.
      • et al.
      A placebo-controlled trial of subcutaneous semaglutide in nonalcoholic steatohepatitis.
      ].
      Liraglutide, a GLP-1 receptor agonist, also demonstrated efficacy in reducing liver fat content as well as levels of liver enzymes in patients with NASH [
      • Armstrong M.J.
      • Gaunt P.
      • Aithal G.P.
      • Barton D.
      • Hull D.
      • Parker R.
      • et al.
      Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis (LEAN): a multicentre, double-blind, randomised, placebo-controlled phase 2 study.
      ]. Considering the beneficial effects of GLP-1 receptor agonists, such as semaglutide, in maintaining glycemic control and weight loss, it represents an attractive potential therapeutic option in NASH [
      • Dichtel L.E.
      The GLP-1 receptor agonist semaglutide for the treatment of nonalcoholic steatohepatitis.
      ]. Moreover, the emerging evidence suggests that a GIP and GLP-1 receptor agonist coadministration results in additive effects and generates increased insulin response in healthy volunteers [
      • Gasbjerg L.S.
      • Helsted M.M.
      • Hartmann B.
      • Jensen M.H.
      • Gabe M.B.N.
      • Sparre-Ulrich A.H.
      • et al.
      Separate and combined glucometabolic effects of endogenous glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 in healthy individuals.
      ]. A dual GIP/GLP-1 receptor agonist tirzepatide (LY3298176) significantly improved liver functions and NASH-related biomarkers, such as reduced ALT and AST and increased adiponectin in T2DM patients as concluded in a post hoc analysis [
      • Hartman M.L.
      • Sanyal A.J.
      • Loomba R.
      • Wilson J.M.
      • Nikooienejad A.
      • Bray R.
      • et al.
      Effects of novel dual GIP and GLP-1 receptor agonist tirzepatide on biomarkers of nonalcoholic steatohepatitis in patients with type 2 diabetes.
      ]. The efficacy of tirzepatide is now being evaluated in a phase IIb trial SYNERGY-NASH (NCT04166773).
      HM15211, a long-acting triple (glucagon/GIP/GLP-1) agonist, has been reported to reduce the liver fat content, oxidative stress, and inflammatory markers in a mouse model of NASH and fibrosis [
      • Choi Jaehyuk
      • Kim Jung Kuk
      • Lee Seon Myeong
      • Kwon Hyunjoo
      • Lee Jongsoo
      • Sungmin Bae D.K.
      • et al.
      1830-P: therapeutic effect of a novel long-acting GLP-1/GIP/glucagon triple agonist (HM15211) in CDHFD-Induced NASH and fibrosis mice.
      ,
      • Choi In Young
      • Kim Jung Kuk
      • Lee Jong Suk
      • Park Eunjin
      • SYJ Young Hoon Kim
      • S. J.K.
      Effect of a novel long-acting GLP-1/GIP/glucagon triple agonist (HM15211) in a NASH and fibrosis animal model.
      ]. A phase II trial of HM15211 is currently ongoing in obese subjects with NAFLD (NCT03744182).
      MEDI0382 (cotadutide) is another GLP-1 and glucagon receptor dual agonist that improved metabolic and pathological components of NAFLD in two distinct mouse models of NASH by reducing steatosis, inflammation, and fibrosis [
      • Boland M.L.
      • Laker R.C.
      • Mather K.
      • Nawrocki A.
      • Oldham S.
      • Boland B.B.
      • et al.
      Resolution of NASH and hepatic fibrosis by the GLP-1R and GCGR dual-agonist cotadutide via modulating mitochondrial function and lipogenesis.
      ]. The safety and tolerability of MEDI0382 have been demonstrated in a phase IIa trial in healthy subjects who showed a linear pharmacokinetic profile. However, vomiting, nausea, dizziness, and increased heart rate were the most common reported adverse events [
      • Ambery P.
      • Parker V.E.
      • Stumvoll M.
      • Posch M.G.
      • Heise T.
      • Plum-Moerschel L.
      • et al.
      MEDI0382, a GLP-1 and glucagon receptor dual agonist, in obese or overweight patients with type 2 diabetes: a randomised, controlled, double-blind, ascending dose and phase 2a study.
      ]. MEDI0382 is currently in phase II clinical trial (NCT04019561).

      3.1.8 Thyroid hormone receptor (THR-β) selective agonist

      THR-β is highly expressed in hepatocytes and regulates hepatic lipoproteins, TG, and cholesterol metabolism. Several preclinical studies have demonstrated a protective role for THR-β or thyromimetics in NAFLD, which has been summarized by Sinha et al. [
      • Sinha R.A.
      • Bruinstroop E.
      • Singh B.K.
      • Yen P.M.
      Nonalcoholic fatty liver disease and hypercholesterolemia: roles of thyroid hormones, metabolites, and agonists.
      ]. MGL-3196 (resmetirom) is an orally administered liver-directed selective THR-β agonist which significantly reduced hepatic steatosis and decreased hepatocyte ballooning and inflammation in phase II clinical trial in NASH patients with stages 1-3 fibrosis [
      • Harrison S.A.
      • Bashir M.R.
      • Guy C.D.
      • Zhou R.
      • Moylan C.A.
      • Frias J.P.
      • et al.
      Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.
      ]. In 105 compensated NASH cirrhotic patients enrolled in the MAESTRO-NAFLD-1 trial (NCT04197479), resmetirom appeared to be safe. It reduced liver fat fraction, liver enzymes, fibrosis markers, LDL-C, and other atherogenic lipids [
      • Harrison S.A.
      • Alkhouri N.
      • Taub R.A.
      • Neff G.
      • Baum S.J.
      • Bashir M.
      Treatment with resmetirom in phase 3 maestronafld-1 nash study open label arm: effects on biomarkers and imaging.
      ]. Now it is being evaluated in phase III MAESTRO-NASH trial in non-cirrhotic NASH patients with stage 2 or 3 fibrosis for the primary endpoint to improve NASH with at least a 2-point reduction in the NASH activity score and no worsening of fibrosis (NCT03900429).
      VK2809 (MB07811) is a prodrug, which undergoes a first-pass hepatic metabolism to generate a negatively charged THR-β agonist – VK2809A [
      • Erion M.D.
      • Cable E.E.
      • Ito B.R.
      • Jiang H.
      • Fujitaki J.M.
      • Finn P.D.
      • et al.
      Targeting thyroid hormone receptor-β agonists to the liver reduces cholesterol and triglycerides and improves the therapeutic index.
      ]. VK2809 treatment of the glycogen storage disease in type Ia mice has been reported to reduce hepatic steatosis by enhancing fatty acid mitochondrial β-oxidation through stimulating hepatic autophagic flux, improving mitochondrial biogenesis, and increasing enzymes for lipid oxidation and FGF21 [
      • Zhou J.
      • Waskowicz L.R.
      • Lim A.
      • Liao X.H.
      • Lian B.
      • Masamune H.
      • et al.
      A liver-specific thyromimetic, VK2809, decreases hepatosteatosis in glycogen storage disease type Ia.
      ]. VK2809 is currently undergoing a phase II trial (NCT04173065)

      3.1.9 Fatty acid synthesis enzyme inhibitors

      Fatty acid biosynthesis involves several steps: glycolysis, lipogenesis, desaturation, elongation, and esterification, leading to TG accumulation in steatosis. Lipogenesis involves key enzymes, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). ACC carboxylates acetyl-CoA to malonyl-CoA, and FAS converts the malonyl-CoA to long-chain fatty acids. The desaturation and elongation steps require enzymes, including stearoyl-CoA desaturase (SCD-1), which catalyzes the synthesis of monounsaturated fatty acids. In the esterification step, TG is being synthesized by enzymes, including diacylglycerol acyltransferase (DGAT) [
      • Postic C.
      • Girard J.
      Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice.
      ].
      TVB-2640 is a highly potent selective and reversible FAS inhibitor currently undergoing a phase II trial (NCT03938246). TVB-2640 treatment in male subjects with metabolic abnormalities significantly reduced fructose-stimulated peak fractional de novo lipogenesis, improved liver function parameters, and decreased the liver fat content. However, treatment was associated with an adverse effect of alopecia in two subjects, which was reversed when TVB-2640 treatment was discontinued [
      • Syed-Abdul M.M.
      • Parks E.J.
      • Gaballah A.H.
      • Bingham K.
      • Hammoud G.M.
      • Kemble G.
      • et al.
      Fatty acid synthase inhibitor TVB-2640 reduces hepatic de novo lipogenesis in males with metabolic abnormalities.
      ].
      A potent ACC inhibitor PF-05221304 has been reported to improve hepatic steatosis, inflammation, and fibrosis in vitro in primary human hepatocyte cell cultures and in vivo in diet-induced NAFLD/NASH rats [
      • Ross T.T.
      • Crowley C.
      • Kelly K.L.
      • Rinaldi A.
      • Beebe D.A.
      • Lech M.P.
      • et al.
      Acetyl-CoA carboxylase inhibition improves multiple dimensions of NASH pathogenesis in model systems.
      ]. The safety and tolerability of PF-05221304 have been demonstrated in a phase I trial in healthy volunteers who were given PF-05221304 in different dosing regimens. Repeated PF-05221304 doses inhibited fructose-stimulated hepatic de novo lipogenesis in a dose-dependent manner, with near-complete inhibition seen at higher doses; however, the higher doses also elevated serum TG levels and reduced platelet count, which was not observed at lower doses [
      • Bergman A.
      • Carvajal-Gonzalez S.
      • Tarabar S.
      • Saxena A.R.
      • Esler W.P.
      • Amin N.B.
      Safety, tolerability, pharmacokinetics, and pharmacodynamics of a liver-targeting Acetyl-CoA carboxylase inhibitor (PF-05221304): a three-part randomized phase 1 study.
      ]. Therefore, the current phase II trial is being conducted in low doses 10 and 20 mg twice and four times daily, respectively, for six weeks in patients with NASH (NCT04399538).
      Another ACC inhibitor, firsocostat (GS-0976), significantly reduced hepatic de novo lipogenesis, steatosis, and liver stiffness, along with decreased serum ALT, AST, and fibrosis marker- tissue inhibitor of metalloprotease 1 (TIMP1) levels in a phase IIa clinical in NASH patients [
      • Lawitz E.J.
      • Coste A.
      • Poordad F.
      • Alkhouri N.
      • Loo N.
      • McColgan B.J.
      • et al.
      Acetyl-CoA carboxylase inhibitor GS-0976 for 12 weeks reduces hepatic de novo lipogenesis and steatosis in patients with nonalcoholic steatohepatitis.
      ]. In the subsequent phase IIb clinical study in NASH patients, firsocostat reduced steatosis and significantly reduced TIMP1, but no significant changes in liver stiffness and liver biochemistry parameter were observed, and the treatment was associated with elevated serum TG levels [
      • Loomba R.
      • Kayali Z.
      • Noureddin M.
      • Ruane P.
      • Lawitz E.J.
      • Bennett M.
      • et al.
      GS-0976 reduces hepatic steatosis and fibrosis markers in patients with nonalcoholic fatty liver disease.
      ].
      Aramchol is a synthetic lipid obtained by conjugating cholic acid and arachidic acid, which inhibits SCD-1. Administration of aramchol to diet-induced NASH in mice reduced NASH and fibrosis and maintained the redox balance by increasing cellular antioxidant capacity [
      • Iruarrizaga-Lejarreta M.
      • Varela-Rey M.
      • Fernández-Ramos D.
      • Martínez-Arranz I.
      • Delgado T.C.
      • Simon J.
      • et al.
      Role of aramchol in steatohepatitis and fibrosis in mice.
      ]. Aramchol significantly reduced hepatic fat content in the proof-of-concept phase II trial in NAFLD-NASH patients [
      • Safadi R.
      • Konikoff F.M.
      • Mahamid M.
      • Zelber-Sagi S.
      • Halpern M.
      • Gilat T.
      • et al.
      The fatty acid-bile acid conjugate aramchol reduces liver fat content in patients with nonalcoholic fatty liver disease.
      ]. In the phase IIb ARREST study in NASH patients who had overweight or obesity and had confirmed prediabetes or T2DM, aramchol 600 mg resolved NASH without worsening of fibrosis and fibrosis improvement by ≥1 stage without worsening NASH. It also improved liver function parameters (ALT, AST, and HbA1c). However primary end point of a reduction in liver fat did not meet the prespecified significance level [
      • Ratziu V.
      • de Guevara L.
      • Safadi R.
      • Poordad F.
      • Fuster F.
      • Flores-Figueroa J.
      • et al.
      Aramchol in patients with nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase 2b trial.
      ]. Aramchol is currently being investigated in phase III clinical trial (NCT04104321).

      3.1.10 Glucocorticoid receptor antagonist

      Glucocorticoid (GC) hormones have been implicated across all stages in the NAFLD pathogenesis. They substantially affect metabolic pathways in the liver and adipose tissue, including hepatic energy homeostasis, lipogenesis, uptake, and efflux of FFAs or lipoproteins [
      • Woods C.P.
      • Hazlehurst J.M.
      • Tomlinson J.W.
      Glucocorticoids and non-alcoholic fatty liver disease.
      ]. Miricorilant (CORT118335), an antagonist of GC receptor, has been reported to prevent and reverse hepatic lipid accumulation in high-fat diet (HFD)-fed mice [
      • Koorneef L.L.
      • van den Heuvel J.K.
      • Kroon J.
      • Boon M.R.
      • ’t Hoen PAC, Hettne KM, et al.
      Selective glucocorticoid receptor modulation prevents and reverses non-alcoholic fatty liver disease in male mice.
      ]. In a phase II trial, miricorilant has been reported to reduce the liver fat content. However, surprisingly, ALT and AST levels were elevated in four of the first five patients who received miricorilant for four weeks. The ongoing phase II trial (NCT03823703) is, therefore, now suspended, pending investigation of the abnormal laboratory values in NASH patients [
      Corcept Therapeutics Observes Large Reductions of Liver Fat and Transient Liver Enzyme Elevations in Phase 2 Trial of Miricorilant As a Potential Treatment for Patients with Nonalcoholic Steatohepatitis (NASH) BioSpace 2021.
      ].

      3.1.11 Growth Hormone Releasing Hormone (GHRH) analog

      Growth hormone (GH) has emerged as an important potential target for NAFLD. Several case reports and clinical studies suggest that the GH deficiency state in adults is associated with an increased prevalence of NAFLD and NASH [
      • Takahashi Y.
      • Iida K.
      • Takahashi K.
      • Yoshioka S.
      • Fukuoka H.
      • Takeno R.
      • et al.
      Growth hormone reverses nonalcoholic steatohepatitis in a patient with adult growth hormone deficiency.
      ,
      • Takahashi Y.
      Essential roles of growth hormone (GH) and insulin-like growth factor-I (IGF-I) in the liver.
      ]. Consistently, GH replacement therapy significantly improved NAFLD, evidenced by improved liver function parameters, including ALT, AST, and GGT, improved histological changes in the liver, and reduced fibrotic markers [
      • Nishizawa H.
      • Iguchi G.
      • Murawaki A.
      • Fukuoka H.
      • Hayashi Y.
      • Kaji H.
      • et al.
      Nonalcoholic fatty liver disease in adult hypopituitary patients with GH deficiency and the impact of GH replacement therapy.
      ]. Tesamorelin is a GHRH analog approved for treating human immunodeficiency virus (HIV)-associated lipodystrophy. In a recent clinical trial in HIV patients with NAFLD, tesamorelin reduced liver fat content and prevented liver inflammation and fibrosis progression [
      • Stanley T.L.
      • Fourman L.T.
      • Feldpausch M.N.
      • Purdy J.
      • Zheng I.
      • Pan C.S.
      • et al.
      Effects of tesamorelin on non-alcoholic fatty liver disease in HIV: a randomised, double-blind, multicentre trial.
      ]. A prospective phase II trial with tesamorelin in targeted NAFLD patients is underway (NCT03375788).

      3.1.12 Cholecystokinin receptor (CCKR) antagonist

      Cholecystokinin is a hormone secreted by the I-cells of the small intestine in response to fat, protein, and nutrients [
      • Liddle R.A.
      Cholecystokinin cells.
      ]. Proglumide is a cholecystokinin receptor (CCKR) antagonist which non-selectively binds to both the cholecystokinin receptors (CCK1R and CCK2R) [
      • Miederer S.E.
      • Lindstaedt H.
      • Kutz K.
      • Mayershofer R.
      Efficient treatment of gastric ulcer with proglumide (Milid®) in outpatients (double blind trial).
      ]. Proglumide prevented diet-induced NASH and HCC in mice and reversed biochemical and histologic abnormalities, including steatosis, fibrosis, and inflammation [
      • Tucker R.D.
      • Ciofoaia V.
      • Nadella S.
      • Gay M.D.
      • Cao H.
      • Huber M.
      • et al.
      A cholecystokinin receptor antagonist halts nonalcoholic steatohepatitis and prevents hepatocellular carcinoma.
      ]. It is currently undergoing a phase I trial (NCT04152473).

      3.1.13 Sodium-glucose co-transporter type 2 (SGLT2) inhibitor

      SGLT2 is almost exclusively expressed in the kidney and reabsorbs >90% of the glucose filtered at the glomerulus [
      • Wright E.M.
      • DDFL Loo
      • Hirayama B.A.
      Biology of human sodium glucose transporters.
      ]. Pharmacological inhibition of SGLT2 reduces hyperglycemia by promoting the urinary excretion of glucose (glycosuria) via inhibition of SGLT channels in proximal convoluted tubules in kidneys [
      • Tahrani A.A.
      • Barnett A.H.
      • Bailey C.J.
      SGLT inhibitors in management of diabetes.
      ]. Dapagliflozin monotherapy has been reported to reduce hepatocyte injury biomarkers and FGF21 in T2DM and NAFLD patients [
      • Eriksson J.W.
      • Lundkvist P.
      • Jansson P.-A.
      • Johansson L.
      • Kvarnström M.
      • Moris L.
      • et al.
      Effects of dapagliflozin and n-3 carboxylic acids on non-alcoholic fatty liver disease in people with type 2 diabetes: a double-blind randomised placebo-controlled study.
      ]. A recent study evaluated the effect of SGLT2 inhibitors in patients with T2DM and NAFLD, where SGLT2 inhibition by dapagliflozin improved liver steatosis and attenuated liver fibrosis only in patients with significant liver fibrosis [
      • Shimizu M.
      • Suzuki K.
      • Kato K.
      • Jojima T.
      • Iijima T.
      • Murohisa T.
      • et al.
      Evaluation of the effects of dapagliflozin, a sodium-glucose co-transporter-2 inhibitor, on hepatic steatosis and fibrosis using transient elastography in patients with type 2 diabetes and non-alcoholic fatty liver disease.
      ]. Dapagliflozin improved liver function parameters, decreased the serum level of hepatocytes-secreted soluble dipeptidyl peptidase-4 (DPP4), which is responsible for adipose tissue inflammation and insulin resistance in patients with T2DM and NAFLD [
      • Aso Y.
      • Kato K.
      • Sakurai S.
      • Kishi H.
      • Shimizu M.
      • Jojima T.
      • et al.
      Impact of dapagliflozin, an SGLT2 inhibitor, on serum levels of soluble dipeptidyl peptidase-4 in patients with type 2 diabetes and non-alcoholic fatty liver disease.
      ]. Dapagliflozin has also been demonstrated to reduce liver fat fraction and visceral adipose tissue volume in obese patients with T2DM. Although glycemic control was improved, no effect on tissue-level insulin sensitivity was observed [
      • Latva-Rasku A.
      • Honka M.-J.
      • Kullberg J.
      • Mononen N.
      • Lehtimäki T.
      • Saltevo J.
      • et al.
      The SGLT2 Inhibitor dapagliflozin reduces liver fat but does not affect tissue insulin sensitivity: a randomized, double-blind, placebo-controlled study with 8-week treatment in Type 2 diabetes patients.
      ]. In another study, dapagliflozin and pioglitazone have been shown to exert equivalent beneficial effects in NAFLD patients [
      • Kinoshita T.
      • Shimoda M.
      • Nakashima K.
      • Fushimi Y.
      • Hirata Y.
      • Tanabe A.
      • et al.
      Comparison of the effects of three kinds of glucose-lowering drugs on non-alcoholic fatty liver disease in patients with type 2 diabetes: A randomized, open-label, three-arm, active control study.
      ]. A phase III trial with dapagliflozin evaluating the efficacy and safety in NASH is underway (NCT03723252).
      Empagliflozin has been reported to improve hepatic steatosis and fibrosis in patients with NAFLD and T2DM and reduce body weight and abdominal fat [
      • Chehrehgosha H.
      • Sohrabi M.R.
      • Ismail-Beigi F.
      • Malek M.
      • Babaei M.R.
      • Zamani F.
      • et al.
      Empagliflozin improves liver steatosis and fibrosis in patients with non-alcoholic fatty liver disease and type 2 diabetes: a randomized, double-blind, placebo-controlled clinical trial.
      ]. The proof of concept trial with empagliflozin showed a decrease in hepatic fat content, circulating uric acid, and increased adiponectin levels despite no effects on tissue-specific insulin sensitivity in T2DM patients [
      • S. K.
      • S. G.
      • K. S.
      • C. H.
      • J. M.
      • H. K.
      • et al.
      Empagliflozin effectively lowers liver fat content in well-controlled type 2 diabetes: a randomized, double-blind, phase 4, placebo-controlled trial.
      ]. Moreover, empagliflozin has been shown to improve steatosis and fibrosis and significantly reduce ALT and AST levels in NAFLD patients without T2DM [
      • Taheri H.
      • Malek M.
      • Ismail-Beigi F.
      • Zamani F.
      • Sohrabi M.
      • Reza babaei M.
      • et al.
      Effect of empagliflozin on liver steatosis and fibrosis in patients with non-alcoholic fatty liver disease without diabetes: a randomized, double-blind, placebo-controlled trial.
      ].
      Canagliflozin also reduced intrahepatic TG and improved hepatic insulin sensitivity, insulin secretion, and clearance in patients with T2DM [
      • Cusi K.
      • Bril F.
      • Barb D.
      • Polidori D.
      • Sha S.
      • Ghosh A.
      • et al.
      Effect of canagliflozin treatment on hepatic triglyceride content and glucose metabolism in patients with type 2 diabetes.
      ]. It also reduced body mass, fat mass, hepatic fat content, and improved liver enzymes and glycemic control in T2DM patients with NAFLD [
      • Inoue M.
      • Hayashi A.
      • Taguchi T.
      • Arai R.
      • Sasaki S.
      • Takano K.
      • et al.
      Effects of canagliflozin on body composition and hepatic fat content in type 2 diabetes patients with non-alcoholic fatty liver disease.
      ].
      Licogliflozin is another novel SGLT1/2 inhibitor whose efficacy, safety, and tolerability as a monotherapy and combination with tropifexor are being evaluated in adult patients with NASH and liver fibrosis in the (ELIVATE) phase II trial (NCT04065841). In a phase IIa study with NASH patients, licogliflozin decreased liver function markers such as serum ALT, AST, GGT, and reduced body weight, insulin resistance, HbA1c, hepatic fat content, and markers of liver fibrosis. The most common adverse effect was diarrhea [
      • Harrison Stephen A.
      • Sicard Eric
      • Manghi Frederico Perez
      • Smith William B.
      • Alpenidze Diana
      • Aizenberg Diego
      • et al.
      LIK066 (Licogliflozin), an SGLT1/2 inhibitor, improves markers of hepatic and metabolic health in patients with nonalcoholic fatty liver disease: full study analysis of a 12-week, randomized, placebo-controlled, phase 2a study.
      ].

      3.1.14 Fatty acid derivative

      Icosabutate is a structurally engineered liver-targeted eicosapentaenoic acid derivative. It selectively improved insulin sensitivity, hepatic steatosis, inflammation, lipotoxicity, oxidative stress, and fibrosis in a mice model of T2DM or hepatic inflammation or metabolic dysregulation [
      • Hoek A.M.
      • Pieterman E.J.
      • Hoorn J.W.
      • Iruarrizaga-Lejarreta M.
      • Alonso C.
      • Verschuren L.
      • et al.
      Icosabutate exerts beneficial effects upon insulin sensitivity, hepatic inflammation, lipotoxicity, and fibrosis in mice.
      ]. The interim analysis of the ongoing phase IIb ICONA trial treatment of NASH patients with icosabutate for 16 weeks showed a decrease in the biomarkers of liver injury, inflammation, and fibrogenesis, along with improvements in glycemic control and atherogenic lipids. There were no changes in weight or body mass index (BMI) and liver fat content. Treatment was well tolerated with no safety concerns [
      • Harrison Stephen
      • Gunn Nadege T.
      • Sheikh Muhammad Y.
      • Rudraraju Madhavi
      • Anita Kohli D.
      • Fraser A.
      • et al.
      Icosabutate, a novel structurally engineered fatty acid receptor agonist, significantly reduces relevant biomarkers of NASH and fibrosis in 16 weeks: results of an interim analysis of the phase 2b ICONA trial.
      ].

      3.2 Oxidative stress targeted therapies

      Redox imbalance is highly relevant for NAFLD pathogenesis, and preclinical and epidemiological studies have also shown a positive correlation between NAFLD and oxidative stress [
      • Spahis S.
      • Delvin E.
      • Borys J.M.
      • Levy E.
      Oxidative stress as a critical factor in nonalcoholic fatty liver disease pathogenesis.
      ]. Therefore, various preclinical and clinical studies target oxidative stress through micronutrients or antioxidants to maintain redox homeostasis. In the PIVENS clinical trial, daily administration of vitamin E (a potent antioxidant) for 96 weeks improved the histological features of NASH, such as hepatic steatosis, lobular inflammation, and hepatocellular ballooning, in approximately 43% of non-diabetic patients; no significant improvement in fibrosis was found [
      • Sanyal A.J.
      • Chalasani N.
      • Kowdley K.V.
      • McCullough A.
      • Diehl A.M.
      • Bass N.M.
      • et al.
      Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis.
      ]. Moreover, the combination of vitamin E with pioglitazone in a proof-of-concept trial was found to improve the histological features of NASH, compared to placebo. However, improvement in fibrosis was not observed in any group, and vitamin E alone was not effective against the histological improvements [
      • Bril F.
      • Biernacki D.M.
      • Kalavalapalli S.
      • Lomonaco R.
      • Subbarayan S.K.
      • Lai J.
      • et al.
      Role of Vitamin E for Nonalcoholic Steatohepatitis in Patients With Type 2 diabeTes: a Randomized Controlled Trial.
      ]. A meta-analysis using data from randomized controlled trials concluded that high-dosage (≥400 IU/d) of vitamin E supplements might increase all-cause mortality [
      • Miller E.R.
      • Pastor-Barriuso R.
      • Dalal D.
      • Riemersma R.A.
      • Appel L.J.
      • Guallar E.
      Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality.
      ]. Moreover, in the SELECT trial, vitamin E at the dose of 400 IU/day increased the risk of prostate cancer among healthy men [
      • Klein E.A.
      • Thompson I.M.
      • Tangen C.M.
      • Crowley J.J.
      • Lucia M.S.
      • Goodman P.J.
      • et al.
      Vitamin E and the risk of prostate cancer: the Selenium and vitamin E Cancer Prevention Trial (SELECT).
      ].
      Idebenone is a synthetic analog of coenzyme Q10, an important antioxidant that participates in the mitochondrial electron transport chain [
      • Bergamasco B.
      • Scarzella L.
      • La Commare P.
      Idebenone, a new drug in the treatment of cognitive impairment in patients with dementia of the Alzheimer type.
      ]. The antioxidant potential to scavenge the free radicals of idebenone and coenzyme Q10 has been well documented [
      • Mordente A.
      • Martorana G.E.
      • Minotti G.
      • Giardina B.
      Antioxidant properties of 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)- 1,4-benzoquinone (idebenone).
      ]. However, it has recently been demonstrated that idebenone and coenzyme Q10 have partial agonistic activity for both PPARα and PPARγ and reverse hepatic steatosis in db/db mice model of T2DM [
      • Tiefenbach J.
      • Magomedova L.
      • Liu J.
      • Reunov A.A.
      • Tsai R.
      • Eappen N.S.
      • et al.
      Idebenone and coenzyme Q10 are novel PPARα/γ ligands, with potential for treatment of fatty liver diseases.
      ]. The safety and efficacy study of idebenone is being evaluated in a phase I/IIa clinical trial in adults aged 18+ with NASH and stage 1-3 fibrosis (NCT04669158).

      3.3 Inflammation targeted therapies

      Hepatic inflammation is one of the main pathogenic factors of NASH, which is induced by the proinflammatory state created by lipotoxic lipids-mediated hepatocyte injury [
      • Wang H.
      • Mehal W.
      • Nagy L.E.
      • Rotman Y.
      Immunological mechanisms and therapeutic targets of fatty liver diseases.
      ]. JKB-122 is a toll-like receptor 4 (TLR4) antagonist with hepatoprotective effects. In concanavalin A-induced hepatitis in male mice, JKB-122 reduced liver lesions (necrosis) and hepatic injury, also improved liver function enzymes ALT, AST alone or in combination with prednisolone by inhibiting pro-inflammatory cytokines including IFN-γ, IL-4, IL-6, IL-17A, and TNF-α [
      • Hsu M.C.
      • Liu S.H.
      • Wang C.W.
      • Hu N.Y.
      • Wu E.S.C.
      • Shih Y.C.
      • et al.
      JKB-122 is effective, alone or in combination with prednisolone in Con A-induced hepatitis.
      ]. Presently, it is being evaluated in a phase II trial in NASH and fibrosis patients (NCT04255069).
      CRV431, a cyclosporin A analog, is a pan-cyclophilin inhibitor reported to reduce fibrosis and the number and size of liver tumors in a carbon tetrachloride-induced mouse model of NASH or chronic liver disease [
      • Kuo J.
      • Bobardt M.
      • Chatterji U.
      • Mayo P.R.
      • Trepanier D.J.
      • Foster R.T.
      • et al.
      A pan-cyclophilin inhibitor, CRV431, decreases fibrosis and tumor development in chronic liver disease models.
      ]. Cyclophilins are a group of proteins involved in various processes such as inflammation and pathologies such as diabetes and liver disease including NASH, and fibrosis [
      • Naoumov N.V.
      Cyclophilin inhibition as potential therapy for liver diseases.
      ]. CRV431 is now being evaluated in a phase II trial (NCT04480710).
      HPN-01 is a novel potent and selective inhibitor of IκB kinases (IKK) developed for NAFLD treatment, which is now being evaluated for safety parameters and pharmacokinetic properties in healthy patients (NCT04481594).
      CC-90001 is a selective c-Jun N-terminal kinase (JNK) inhibitor, the efficacy, and safety of which in subjects with NASH and stage 3 or 4 liver fibrosis is being evaluated in phase II clinical trial (NCT04048876).

      3.4 Apoptosis-targeted therapies

      Cell death, including apoptosis, is critical for the progression of NAFLD and NASH, and inhibitors of apoptosis have been shown to improve NAFLD and NASH in preclinical and clinical settings [
      • Kanda T.
      • Matsuoka S.
      • Yamazaki M.
      • Shibata T.
      • Nirei K.
      • Takahashi H.
      • et al.
      Apoptosis and non-alcoholic fatty liver diseases.
      ]. Emricasan is a pan-caspase inhibitor that has been shown to decrease liver injury and fibrosis by inhibiting apoptosis and inflammatory responses in preclinical models of NASH [
      • Barreyro F.J.
      • Holod S.
      • Finocchietto P.V.
      • Camino A.M.
      • Aquino J.B.
      • Avagnina A.
      • et al.
      The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis.
      ]. However, it failed to achieve the primary efficacy endpoint in several clinical studies, including phase IIb trials in non-cirrhotic NASH patients with fibrosis (ENCORE-NF), in compensated or decompensated cirrhosis and severe portal hypertension (ENCORE-PH) and for liver function for those with decompensated NASH–cirrhosis (ENCORE-LF) [
      • Harrison S.A.
      • Goodman Z.
      • Jabbar A.
      • Vemulapalli R.
      • Younes Z.H.
      • Freilich B.
      • et al.
      A randomized, placebo-controlled trial of emricasan in patients with NASH and F1-F3 fibrosis.
      ,
      • Garcia-Tsao G.
      • Bosch J.
      • Kayali Z.
      • Harrison S.A.
      • Abdelmalek M.F.
      • Lawitz E.
      • et al.
      Randomized placebo-controlled trial of emricasan for non-alcoholic steatohepatitis-related cirrhosis with severe portal hypertension.
      ]
      Selonsertib is an inhibitor of apoptosis signal-regulating kinase 1 (ASK1), with potential anti-inflammatory and antifibrotic properties. Two randomized, double-blind, placebo-controlled, phase III trials with selonsertib (18 mg and 6 mg once a day for 48 weeks) in patients with NASH and bridging fibrosis F3 (STELLAR-3 trial) or compensated cirrhosis (STELLAR-4 trial) were performed. Neither of these trials met the primary endpoint, and no significant effects on liver biochemistry, fibrosis, or cirrhosis were noted [
      • Harrison S.A.
      • Wong V.W.S.
      • Okanoue T.
      • Bzowej N.
      • Vuppalanchi R.
      • Younes Z.
      • et al.
      Selonsertib for patients with bridging fibrosis or compensated cirrhosis due to NASH: results from randomized phase III STELLAR trials.
      ]. Both the monotherapy trials (NCT03053050, NCT03053063) were later terminated due to the lack of efficacy in a 48-week analysis.
      Moreover, an open-label phase II trial evaluating the ASK1 inhibitor selonsertib, alone or in combination with simtuzumab (a monoclonal antibody against lysyl oxidase-2) in NASH patients with liver fibrosis (stage 2/3) demonstrated a decrease in fibrosis associated with a reduction in liver stiffness, collagen, and lobular inflammation as well as improvements in serum biomarkers of apoptosis and necrosis in patients treated with selonsertib for 24 weeks. However, the addition of simtuzumab had no observable benefits [
      • Loomba R.
      • Lawitz E.
      • Mantry P.S.
      • Jayakumar S.
      • Caldwell S.H.
      • Arnold H.
      • et al.
      The ASK1 inhibitor selonsertib in patients with nonalcoholic steatohepatitis: a randomized, phase 2 trial.
      ]. Simtuzumab also did not benefit the patients with bridging fibrosis nor with compensated cirrhosis [
      • Harrison S.A.
      • Abdelmalek M.F.
      • Caldwell S.
      • Shiffman M.L.
      • Diehl A.M.
      • Ghalib R.
      • et al.
      Simtuzumab is ineffective for patients with bridging fibrosis or compensated cirrhosis caused by nonalcoholic steatohepatitis.
      ]

      3.5 Fibrosis targeted therapies

      3.5.1 C-C motif chemokine receptor type 2 and 5 (CCR2/CCR5 chemokine) antagonists

      Inhibition of CCR2/5 by cenicriviroc has shown potent anti-inflammatory and anti-fibrotic effects and significantly reduced NALFD in vivo in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced NASH [
      • Lefebvre E.
      • Moyle G.
      • Reshef R.
      • Richman L.P.
      • Thompson M.
      • Hong F.
      • et al.
      Antifibrotic effects of the dual CCR2/CCR5 antagonist cenicriviroc in animal models of liver and kidney fibrosis.
      ]. However, in a phase IIb study in adults with NASH and liver fibrosis (CENTAUR Study), cenicriviroc showed a significant antifibrotic benefit at year 1 but failed to meet the primary endpoint of at least 2-point improvement in NASH without worsening of fibrosis [
      • Friedman S.L.
      • Ratziu V.
      • Harrison S.A.
      • Abdelmalek M.F.
      • Aithal G.P.
      • Caballeria J.
      • et al.
      A randomized, placebo-controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis.
      ]. Moreover, continued long-term (two years) treatment was not associated with further improvement in fibrosis beyond the effect observed at 1 year [
      • Ratziu V.
      • Sanyal A.
      • Harrison S.A.
      • Wong V.W.
      • Francque S.
      • Goodman Z.
      • et al.
      Cenicriviroc treatment for adults with nonalcoholic steatohepatitis and fibrosis: final analysis of the phase 2b CENTAUR study.
      ]. The phase III trial (AURORA) was recently terminated due to the lack of efficacy outcomes in part I of the AURORA study. Leronlimab or PRO 140 is a humanized monoclonal antibody antagonist of CCR5, currently undergoing a phase II clinical trial (NCT04521114) in NASH patients.

      3.5.2 Galectin-3 inhibitor

      Galectin-3 plays a key role in developing hepatic and other organ fibrosis [
      • Henderson N.C.
      • Mackinnon A.C.
      • Farnworth S.L.
      • Poirier F.
      • Russo F.P.
      • Iredale J.P.
      • et al.
      Galectin-3 regulates myofibroblast activation and hepatic fibrosis.
      ]. GB1211 is an orally administered galectin-3 inhibitor and is currently under safety, tolerability, and pharmacokinetics evaluations in healthy adults and subjects with NASH and hepatic fibrosis (NCT03809052). Another galectin-3 inhibitor, belapectin (GR-MD-02), has demonstrated a good safety profile, but treatment was not associated with improvement/reduction in hepatic venous pressure gradient (HVPG) or fibrosis compared to placebo. However, in a subgroup analysis of patients without esophageal varices, 2 mg/kg belapectin reduced HVPG and development of varices in a phase IIb study of 162 patients with NASH, cirrhosis, and portal hypertension [
      • Chalasani N.
      • Abdelmalek M.F.
      • Garcia-Tsao G.
      • Vuppalanchi R.
      • Alkhouri N.
      • Rinella M.
      • et al.
      Effects of Belapectin, an inhibitor of galectin-3, in patients with nonalcoholic steatohepatitis with cirrhosis and portal hypertension.
      ].

      3.5.3 Angiogenesis inhibitor

      ALS-L1023 is a herbal extract from Melissa officinalis (lemon-balm) leaves with antiangiogenic and matrix metalloproteinase inhibitory effects [
      • Park B.Y.
      • Lee H.
      • Woo S.
      • Yoon M.
      • Kim J.
      • Hong Y.
      • et al.
      Reduction of adipose tissue mass by the angiogenesis inhibitor ALS-L1023 from Melissa officinalis.
      ]. ALS-L1023 ameliorated obesity and insulin resistance in obese female mice [
      • Lee D.
      • Shin Y.
      • Jang J.
      • Park Y.
      • Ahn J.
      • Jeong S.
      • et al.
      The herbal extract ALS-L1023 from Melissa officinalis alleviates visceral obesity and insulin resistance in obese female C57BL/6J mice.
      ] and inhibited obesity, hepatic steatosis, and fibro-inflammation in ovariectomized mice [
      • Kim J.
      • Lee H.
      • Lim J.
      • Lee H.
      • Yoon S.
      • Shin S.S.
      • et al.
      The lemon balm extract ALS-L1023 inhibits obesity and nonalcoholic fatty liver disease in female ovariectomized mice.
      ]. It also inhibited HFD-induced NAFLD in mice by modulating gene expression involved in lipid metabolism, inflammation, fibrosis, antioxidation, and apoptosis [
      • Kim J.
      • Lee H.
      • Lim J.
      • Oh J.
      • Shin S.
      • Yoon M.
      The angiogenesis inhibitor ALS-L1023 from lemon-balm leaves attenuates high-fat diet-induced nonalcoholic fatty liver disease through regulating the visceral adipose-tissue function.
      ]. It is now being evaluated in a phase II trial in NASH patients to evaluate the efficacy against liver fibrosis and steatosis (NCT04342793).

      4. Multidrug combination therapy

      With the failure of several monotherapies in clinical trials (Table 3), rational combination therapy seems to be a way forward for effective management for NAFLD. For instance, FXR agonist (cilofexor) and ACC inhibitor (firsocostat) represent one such potential combination. The combination therapy of cilofexor with firsocostat for 48 weeks improved the NASH activity score; and significantly improved liver function parameters including ALT, AST, bilirubin, bile acid, insulin, and cytokeratin-18 in NASH patients with either bridging fibrosis (F3) or compensated cirrhosis (F4) [
      • Loomba R.
      • Noureddin M.
      • Kowdley K.V.
      • Kohli A.
      • Sheikh A.
      • Neff G.
      • et al.
      Combination therapies including cilofexor and firsocostat for bridging fibrosis and cirrhosis attributable to NASH.
      ]. Several other combinations therapies are also being evaluated at various phases of the clinical trial. For instance, FXR agonist tropifexor, with leukotriene A4 hydrolase inhibitor (LYS006), is in a phase II trial (NCT04147195). A non-bile FXR agonist (MET409) is also undergoing a phase IIa combination trial with SGLT2 inhibitor (empagliflozin) in patients with T2DM and NASH (NCT04702490).
      Table 3Major clinical trial failure of investigational drugs for NAFLD-NASH during the advanced development stage (phase II phase III).
      Drug candidatePrimary MOAReason for failureCT identifier
      ElafibranorPPARα/δ agonistDid not meet the predefined primary surrogate efficacy endpoint.NCT02704403 (RESOLVE-IT)
      SelonsertibASK1 inhibitorTerminated early due to lack of efficacy based on the results of the week 48 analysis.NCT03053050 (STELLAR-3)

      NCT03053063 (STELLAR-4)
      CenicrivirocCCR2/CCR5 inhibitorTerminated early due to lack of efficacy based on the results of part I of the AURORA study.NCT03028740 (AURORA)
      SeladelparPPARβ/δ agonistFailed to meet the primary efficacy endpoint.NCT03551522
      EmricasanCaspase inhibitorDid not meet the primary efficacy endpoints.NCT02686762 (ENCORE-NF)

      NCT02960204 (ENCORE-PH)

      NCT03205345 (ENCORE-LF)
      SimtuzumabAntibody against LOX2No benefit to patients with bridging fibrosis or compensated cirrhosis due to NASH.NCT01672866
      MSDC-0602KMPC inhibitorDid not demonstrate statistically significant effects on primary and secondary liver histology endpoints.NCT02784444 (EMMINENCE)
      AldaferminFGF19 analogDid not meet the primary efficacy endpoint.NCT03912532 (ALPINE 2/3)
      Abbreviations: PPAR, peroxisome proliferator-activated receptor; ASK1, apoptosis signal-regulating kinase 1; CCR,2/5 C-C chemokine receptor type2/5; LOX2, lysyl oxidase-like 2; MPC, mitochondrial pyruvate carrier; FGF19, fibroblast growth factor 19.
      Another rational combination therapy includes metabolic enzymes DGAT2 and ACC inhibition with PF-06865571 and PF-05221304, respectively. These are being investigated as a combination therapy in a phase II study in adults with NAFLD (NCT03776175). Saroglitazar and vitamin E combination is being evaluated in a phase III trial (NCT04193982). An ileal bile acid transporter (IBAT) inhibitor, elobixibat, and bile acid sequestrants, cholestyramine, is also undergoing a phase II clinical trial (NCT04235205).

      5. Novel and innovative therapies/approaches

      ION839, previously known as IONIS-AZ6-2.5-LRx and AZD2693, is a ligand-conjugated investigational antisense drug designed to inhibit the production of patatin-like phospholipase domain-containing 3 (PNPLA3) protein. PNPLA3 is a protein that is found on the surface of intracellular lipid droplets. Silencing PNPLA3 with antisense oligonucleotides in mice has been shown to ameliorate NASH and fibrosis [
      • Lindén D.
      • Ahnmark A.
      • Pingitore P.
      • Ciociola E.
      • Ahlstedt I.
      • Andréasson A.C.
      • et al.
      Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice.
      ].
      AMG 609 is a small interfering RNA (siRNA) that selectively targets a variant allele of PNPLA3 I148M, which is being investigated as a possible way of NASH treatment. RNA interference (RNAi) is a biological process where RNA molecules are used to inhibit gene expression. ARO-HSD and ALN-HSD are two RNA interference (RNAi) therapeutics targeting hydroxysteroid 17-beta dehydrogenase 13 protein (HSD17B13). HSD17B13 is known to be involved in the metabolism of hormones, fatty acids, and bile acids. However, it has recently been identified as a lipid droplet-associated protein with hepatic retinol dehydrogenase activity [
      • Ma Y.
      • Belyaeva O.V.
      • Brown P.M.
      • Fujita K.
      • Valles K.
      • Karki S.
      • et al.
      17-Beta hydroxysteroid dehydrogenase 13 Is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.
      ]. HSD17B13 has been reported to accelerate the progression of NAFLD to chronic liver diseases such as NASH, fibrosis, and cirrhosis, whereas a loss-of-function variant in HSD17B13 (a protein-truncating variant (rs72613567:TA) reduced the risk of chronic liver disease, including progression of NAFLD [
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • Xin Y.
      • Schurmann C.
      • Stevis P.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.
      ,
      • Pirola C.J.
      • Garaycoechea M.
      • Flichman D.
      • Arrese M.
      • Martino J.S.
      • Gazzi C.
      • et al.
      Splice variant rs72613567 prevents worst histologic outcomes in patients with nonalcoholic fatty liver disease.
      ]. Both (ARO-HSD and ALN-HSD) are at the initial phases of evaluation (phase I). Other novel therapies include modulation of the gut microbiome by administering probiotics/synbiotics, which also represents a promising therapeutic target for NAFLD-NASH [
      • Sharpton S.R.
      • Maraj B.
      • Harding-Theobald E.
      • Vittinghoff E.
      • Terrault N.A.
      Gut microbiome-targeted therapies in nonalcoholic fatty liver disease: a systematic review, meta-analysis, and meta-regression.
      ].
      Precision medicine based upon the individuals’ genetic background, molecular pathogenic factors, and environmental factors has also been suggested to provide more targeted treatment for NAFLD-NASH. Such a personalized approach could maximize the therapeutic benefits and minimize the adverse effects [
      • Lonardo A.
      • Arab J.P.
      • Arrese M.
      Perspectives on precision medicine approaches to NAFLD diagnosis and management.
      ]. The systems biology approach may assist in precision drug development programs by identifying drug candidates based upon the disease-associated genes or proteins [
      • Sookoian S.
      • Pirola C.J.
      Precision medicine in nonalcoholic fatty liver disease: new therapeutic insights from genetics and systems biology.
      ]. At present, the cost of this approach is the key limiting factor, but it can be expected that the concept will become more common and affordable with time.

      6. Regulatory perspective for NAFLD-NASH drug development and approval

      Regulatory agencies, including US FDA and European Medicine Agency (EMA), have made several efforts to expedite NAFLD-NASH drug development and approvals. Several guidance documents on clinical development methodologies published in recent years provide a comprehensive recommendation to sponsors for the clinical development of NAFLD-NASH drugs [
      ,
      ,
      ]. The US FDA and EMA consider that the therapies that will delay the progression, halt, or reverse NASH and NAFLD will address an unmet medical need. Therefore, the potential therapies for NAFLD-NASH thus qualify for US FDA's expedited programs such as accelerated approval, fast track designation, breakthrough therapy designation, and priority review to allow expedited drug development and accelerated evaluation of marketing authorization application [
      Fast track, breakthrough therapy, accelerated approval.
      ]. Lanifibranor, EDP-305, MET642, and HM15211 have been given fast track designation, and conditional market approval will be provided after evaluation.
      Similarly, EMA supports the drug development process through the PRIority MEdicine (PRIME) scheme [
      ]. The PRIME scheme allows sponsors to have an early and proactive regulatory dialogue with the EMA, which enables them to optimize clinical trial design and generate robust data to support accelerated assessment of marketing authorization applications for therapies targeting an unmet medical need. Efruxifermin has already been granted PRIME designation by EMA to support the drug development and accelerated approval.

      7. New Approach Methodologies (NAM) and adverse outcome pathways for NAFLD-NASH research

      The failure of potential drugs at clinical trials in NAFLD-NASH suggests that the current test systems are not capable of recapitulating human disease pathology and/or complex pathological features of NAFLD or NASH. Therefore, NAM in the preclinical drug development phase is gaining importance and regulatory acceptance as it can increase the predictivity of clinical outcomes [
      • Avila A.M.
      • Bebenek I.
      • Bonzo J.A.
      • Bourcier T.
      • Davis Bruno K.L.
      • Carlson D.B.
      • et al.
      An FDA/CDER Perspective on Nonclinical Testing Strategies: Classical Toxicology Approaches and New Approach Methodologies (NAMs).
      ]. The use of human biology-based in vitro test systems and in silico predictions in hazard identification and characterization during preclinical stages of drug development holds great promise [
      • Dirven H.
      • Vist G.E.
      • Bandhakavi S.
      • Mehta J.
      • Fitch S.E.
      • Pound P.
      • et al.
      Performance of preclinical models in predicting drug-induced liver injury in humans: a systematic review.
      ]. For NAFLD-NASH research, in vitro monolayer cell culture to complex three-dimensional (3D) spheroids/organoids and model organisms have been developed to mimic the human physiological and pathological conditions. 3D co-cultures, e.g., spheroids/organoids with hepatocytes, Kupffer cells, and/or stellate cells, are the closest to mimic the improved hepatocyte metabolic activity and an enhanced liver phenotype function that can also be employed for repeat dose studies [
      • Müller F.A.
      • Sturla S.J.
      Human in vitro models of nonalcoholic fatty liver disease.
      ]. The complex in vivo phenotype of liver disease such as NASH can now be modeled in vitro using differentiated human pluripotent stem cells [
      • Bin Ramli M.N.
      • Lim Y.S.
      • Koe C.T.
      • Demircioglu D.
      • Tng W.
      • KAU Gonzales
      • et al.
      Human pluripotent stem cell-derived organoids as models of liver disease.
      ]. However, replacing animal testing with NAMs requires a solid and comprehensive knowledge of the biological mechanisms linking the effects of drugs in cells or tissues with expected effects on the organism.
      The mechanistic information from the Organization for Economic Co-operation and Development (OECD) endorsed Adverse Outcome Pathway (AOP), which links the early events (molecular initiating events) to the disease outcomes through casually linked key events facilitates the understanding of not only toxicological but also pathophysiological pathways responsible for the occurrence and progression of human diseases [
      • Bal-Price A.
      • Bette Meek M.E.
      Adverse outcome pathways: application to enhance mechanistic understanding of neurotoxicity.
      ]. AOPs and/or AOP networks linking several key events could potentially help to understand the interaction between several associated (interlinked) pathways and contribute to a holistic and deeper understanding of the disease pathophysiology, including the complex pathogenesis of NAFLD and NASH. In particular, several AOPs for drug-induced liver injury, including hepatic steatosis, have already been described [
      • Vinken M.
      Adverse outcome pathways and drug-induced liver injury testing.
      ] and can be utilized for mechanistic understanding. These AOPs can contribute to the mechanistic understanding of disease pathophysiology, which is crucial for identifying potential targets, developing relevant diagnostic biomarkers and novel/advanced therapeutics.
      Extrapolation of preclinical outcomes to clinical relevance is always a challenging task where utilization of the AOP framework could facilitate the development of clinically relevant human liver disease models to understand the pathophysiology of disease progression and therapeutic testing. Pre-clinical data derived from computational models, high throughput screening, and novel human-relevant in vitro cellular models can be integrated through the AOP framework with data from clinical and population studies to better understand the disease pathophysiology. In addition, the powerful multi-omics technologies can also be utilized to integrate complex disease pathology into the mechanistic framework of AOP. At the same time, challenges associated with the integration of mechanisms or pathways to the AOP framework, such as the identification of molecular initiating events in the case of NAFLD driven by genetic factors, cannot be neglected.

      8. Summary and conclusion

      NAFLD is a major growing concern. Despite remarkable progress in understanding the pathogenesis of NAFLD, no approved medication has been made available yet. The current recommendation includes pioglitazone and vitamin E for clinical use in NAFLD/NASH. However, based on the positive outcomes of the recent phase II trial, GLP-1 receptor agonists such as semaglutide may also be an attractive potential therapeutic option for NASH. Among several investigational therapies being evaluated in clinical trials, potential drugs, including dapagliflozin, aramchol, resmetirom, semaglutide, and lanifibranor, have already entered phase III clinical trials. Studies indicate their potential beneficial effects in clinical settings, as summarized in Table 4. However, further updates from ongoing clinical trials are much awaited.
      Table 4Summary of major clinical trial outcomes of potential monotherapy drugs currently undergoing phase III clinical trial for NAFLD NASH.
      Drug candidatePrimary MOASubjectsMain outcomeReferencesCurrent phase III CT identifier
      LanifibranorPan-PPAR agonistNASH

      (24-weeks)
      • Decreased steatosis activity score with no worsening of fibrosis.
      • Resolved NASH with no worsening of fibrosis and improved fibrosis with no worsening of NASH.
      • Resolved NASH and improved fibrosis
      • Decreased NAFLD activity score without worsening of fibrosis.
      • Decreased TG and liver enzymes (ALT, AST, and GGT), increased HDL-C.
      [
      • InventivaPharma
      PRESS RELEASEInventiva’s lanifibranor meets the primary and key secondary endpoints in the Phase IIb NATIVE clinical trial in non-alcoholic steatohepatitis (NASH).
      ]
      (NATiV3)

      NCT04849728
      Obeticholic acidFXR agonistNASH

      (72 weeks)
      • Improved liver histology (2-point or greater improvement in NAFLD activity score) without worsening of fibrosis.
      • Reduced serum ALT, AST levels.
      [
      • Neuschwander-Tetri B.A.
      • Loomba R.
      • Sanyal A.J.
      • Lavine J.E.
      • Van Natta M.L.
      • Abdelmalek M.F.
      • et al.
      Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial.
      ]
      (REGENERATE)

      NCT02548351

      NCT03439254 (REVERSE
      NASH

      (18 months)
      • Improved fibrosis by at least one stage and other markers of hepatocellular injury (ALT and AST, GGT) in month-18 interim analysis of ongoing phase III study.
      • A post-hoc analysis showed that obeticholic acid treatment resulted in NASH resolution with no worsening of fibrosis.
      [
      • Younossi Z.M.
      • Ratziu V.
      • Loomba R.
      • Rinella M.
      • Anstee Q.M.
      • Goodman Z.
      • et al.
      Articles obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial.
      ]
      Semaglutide

      (72 weeks)
      GLP-1 receptor agonistNASH and liver fibrosis
      • Achieved NASH resolution with no worsening of fibrosis in patients with stage F2 or F3 fibrosis in 59% patients, (17% in the control).
      [
      • Newsome P.N.
      • Buchholtz K.
      • Cusi K.
      • Linder M.
      • Okanoue T.
      • Ratziu V.
      • et al.
      A placebo-controlled trial of subcutaneous semaglutide in nonalcoholic steatohepatitis.
      ]
      NCT04822181
      Resmetirom

      (36-week)
      THR β agonistNASH
      • Reduced relative and absolute hepatic fat fraction after 12 weeks and 36 weeks of treatment.
      • Reduced mean ALT, AST, and GGT levels.
      • Decreased non-invasive fibrosis markers- PRO-C3 atherogenic lipids and lipoproteins.
      • Higher percentage of patients with NASH resolution (at least a 1-point reduction in ballooning or inflammation on week 36 biopsy) in resmetirom group (46%) compared with placebo (19%).
      [
      • Harrison S.A.
      • Bashir M.R.
      • Guy C.D.
      • Zhou R.
      • Moylan C.A.
      • Frias J.P.
      • et al.
      Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.
      ]
      (MAESTRO)

      NCT03900429
      AramcholSCD1 inhibitorNAFLD

      (12 Weeks)
      • Reduced liver fat content.
      [
      • Safadi R.
      • Konikoff F.M.
      • Mahamid M.
      • Zelber-Sagi S.
      • Halpern M.
      • Gilat T.
      • et al.
      The fatty acid-bile acid conjugate aramchol reduces liver fat content in patients with nonalcoholic fatty liver disease.
      ]
      (ARMOR)

      NCT04104321
      NASH, T2DM

      (52 Weeks)
      • Resolved NASH without worsening of fibrosis (16.7% aramchol versus 5% placebo group) and reduced fibrosis without worsening of NASH in 29.5% aramchol versus 17.5% placebo group.
      • Improved liver biochemistry parameters (ALT, AST and HbA1c).
      [
      • Ratziu V.
      • de Guevara L.
      • Safadi R.
      • Poordad F.
      • Fuster F.
      • Flores-Figueroa J.
      • et al.
      Aramchol in patients with nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase 2b trial.
      ]
      DapagliflozinSGLT2 inhibitorT2DM, NAFLD

      (24 weeks)
      • Reduced liver stiffness and CAP (an indicator of hepatic steatosis).
      • Decreased HbA1c, HOMA-IR, AST, ALT, and GGT, VAT, SCT, HMW adiponectin, serum ferritin and body weight.
      [
      • Shimizu M.
      • Suzuki K.
      • Kato K.
      • Jojima T.
      • Iijima T.
      • Murohisa T.
      • et al.
      Evaluation of the effects of dapagliflozin, a sodium-glucose co-transporter-2 inhibitor, on hepatic steatosis and fibrosis using transient elastography in patients with type 2 diabetes and non-alcoholic fatty liver disease.
      ]
      (DEAN)

      NCT03723252
      Abbreviations: SGLT2, sodium-glucose cotransporter type 2; T2DM, type 2 diabetes mellitus; THR-β, thyroid receptor β; GLP-1R, glucagon-like peptide-1 receptor; PPAR, peroxisome proliferator-activated receptor; LXR, liver X receptor; MOA, mechanism of action; SCT, subcutaneous adipose tissue; VAT, visceral adipose tissue; CAP, controlled attenuation parameter; HMW, high-molecular-weight
      The pathogenesis of NAFLD is complex involving the interplay of several factors, and many medications have failed in one or another aspect in providing beneficial effects in NAFLD. For instance, investigational monotherapy drugs such as elafibranor, selonsertib, cenicriviroc, emricasan, simtuzumab, seladelpar, and more recently MSDC-0602K and aldafermin. These drug candidates failed to meet the predefined efficacy endpoint in the advanced stages of the trial. Therefore, targeting multiple targets or mechanisms by combination therapy could provide therapeutic benefits and mitigate the adverse effects. Several new investigational drugs have also been developed for NAFLD-NASH, but more focus has been given to repurposing or repositioning drugs to explore the possibility of using existing approved therapies to reduce the overall risk, time, and developmental cost. Currently, most pharmacotherapies focus on improving metabolic deregulations. Indeed, the liver is primarily involved in metabolic activities, including lipid and glucose regulation, nutrient storage, and detoxification activities. However, it is also a site of critical immune functions as well. Understanding the immune pathogenesis of NAFLD will help to target immune checkpoints that can eventually help in developing new treatment therapies for NAFLD-NASH. Failure of several investigational drugs despite adequate preclinical data also suggests the need to revisit the preclinical and clinical considerations in NAFLD research. More advanced, close-to-human models and testing strategies should be developed to realistically and mechanistically mimic or recapitulate human NAFLD pathology in preclinical settings. Using NAMs and the AOP concept in preclinical research or early development phases could potentially expedite the drug development process.

      Funding

      This work was supported by the Research Infrastructure RECETOX RI [No LM2018121] financed by the Ministry of Education, Youth and Sports , and Operational Programme Research, Development, and Innovation -project CETOCOEN EXCELLENCE [No CZ.02.1.01/0.0/0.0/17_043/0009632 ], and the Czech Science Foundation [No 19-19143S ].

      CRediT authorship contribution statement

      Chander K. Negi: Conceptualization, Data curation, Writing – original draft, Validation. Pavel Babica: Conceptualization, Data curation, Writing – review & editing, Validation. Lola Bajard: Writing – review & editing, Validation. Julie Bienertova-Vasku: Writing – review & editing, Validation. Giovanni Tarantino: Writing – review & editing, Validation.

      Declaration of competing interest

      None.

      Acknowledgments

      The authors apologize to all colleagues whose work could not be cited due to space limitations.
      Figures created with BioRender.com.

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