Advancements in Metabolic Associated Steatotic Liver Disease (MASLD) Research: Diagnostics, Small Molecule Developments and Future Directions

Abstract

Metabolic (dysfunction) associated steatotic liver disease (MASLD) formerly known as non-alcoholic fatty liver disease (NAFLD) is a growing global health concern with no approved pharmacological treatments. At the same time, there are no standard methods to definitively screen for the presence of MASLD because of its progressive nature and symptomatic commonality with other disorders. Recent advances in molecular understanding of MASLD pathophysiology have intensified research on development of new drug molecules, repurposing of existing drugs approved for other indications, and an educated use of dietary supplements for its treatment and prophylaxis. This review focused on depicting the latest advancements in MASLD research related to small molecule development for prophylaxis or treatment and diagnosis, with emphasis on mechanistic basis at the molecular level.

Graphical Abstract

Potential Mechanisms Involving in the Pathogenesis of MASLD and Targets for Pharmacotherapy.

1. Introduction

Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder that affects more than 25% of the global population, whether definitively diagnosed or not [1]. Because of high nutritional intake and lifestyle characteristics, the prevalence of NAFLD is as high as 30% in the United States, costing $103 billion annually in aggregate for costs related to therapy, liver transplantation, hospitalization, and medical management [2]. In 2020, an international expert panel introduced the term metabolic (dysfunction) associated steatotic liver disease (MASLD) to replace NAFLD. This renaming is aimed to create a more inclusive diagnostic criteria that consider metabolic dysfunction as the central component of this disease. The National Health and Nutrition Examination Survey (NHANES) III database study found that compared to the NAFLD criteria, the new MASLD definition is more appropriate for identifying patients with fatty liver at high risk of hepatic fibrosis [3]. The shift from NAFLD to MASLD is driven by the understanding that metabolic dysfunction, rather than just the absence of alcohol consumption, is a key factor in the disease. The MASLD criteria are believed to better identify patients with fatty liver who are at a high risk of developing hepatic fibrosis compared to the traditional NAFLD criteria [4]. MASLD is closely associated with a range of metabolic disorders, including obesity [5], esophagitis [6], atherosclerotic cardiovascular disease [4], and dyslipidemia. These metabolic factors contribute significantly to the overall pathogenesis of MASLD and its progression [7].

The pathogenesis of MASLD is complex and multifactorial, involving a combination of genetic, metabolism, gut microbiota, and lifestyle factors [8]. Insulin resistance, oxidative stress, inflammation, and dysregulated lipid metabolism are thought to play key roles in its progression [9]. Multiple stages of liver disease have been proposed to explain MASLD pathogenesis. Accordingly, the pathology initiated by lipid accumulation in hepatocytes, followed by ER/oxidative stress induced hepatocyte cell death and inflammation, is called lipotoxicity. These key drivers trigger immune cell dysregulation and hepatic stellate cell activation leading to fibrogenesis and fibrosis [7, 10]. There is evidence that saturated fat and fructose are more likely to stimulate hepatic lipid accumulation. Typically, the disease starts as isolated steatosis, but if the initial stimuli persist for a long time, it can advance to metabolic dysfunction-associated steatohepatitis (MASH). The progression involves hepatocyte ballooning, liver injury, inflammation, and varying degrees of fibrosis, which can ultimately lead to cirrhosis, liver failure, and hepatocellular carcinoma (HCC). The primary insult of lipid excess is exacerbated by contributions from other factors, such as obesity, alcohol intake, immune activation, genetic predisposition, dysbiosis, and diabetes. Albeit the onset of fatty liver disease is attributed to an imbalance in hepatic lipid homeostasis due to increased fatty acid uptake, increased de novo lipogenesis, inhibition of β-oxidation, and reduced lipid export, only a subset of MASLD patients develop MASH. It is hypothesized that progression to MASH is caused by “multiple parallel hits”, stating that a second trigger, such as insulin resistance, is necessary in addition to the initial fat deposition (Fig. 1). Insulin resistance activates inflammatory signaling and promotes the progression of “innocent” steatosis into MASH and liver fibrosis [11]. Abnormal lipid accumulation in hepatocytes can trigger oxidative stress, insulin resistance, inflammation, apoptosis, and fibrosis sequentially or simultaneously, leading to the progression of the disease. MASH patients carry a higher likelihood of progressing into irreversible liver damage and HCC. Interestingly, MASLD patients can be treated with Lenvatinib, a kinase inhibitor, to reduce the odds of progression to HCC [12].

Figure 1.

Metabolic associated steatotic liver disease (MASLD) Spectrum

2. Challenges in the management and diagnosis of MASLD and MASH

The global prevalence and increasing incidence of fatty liver disease signify an urgent need for safe and effective options for prophylaxis and treatment of MASLD and MASH [13]. Currently, many different interventions, mostly experimental and non-specific, are used to manage MASLD (Table 1). However, barring isolated successes, the current standard of care for MASLD remains suboptimal. The two primary reasons for this bleak scenario are- difficulty in timely diagnosis of the disease and progressively increasing involvement of multiple mechanisms towards MASH development. The diagnostic handicap is mostly related to the manifestation of non-specific symptoms, which forces clinicians to diagnose MASLD by a process of elimination; other liver diseases are first ruled out through additional tests, before MASLD diagnosis is suggested and confirmed by biopsy. Moreover, MASLD is a chronic problem with a gradual progression characterized by long periods of asymptomatic presentation. When symptoms appear, they may include fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider-like blood vessels, jaundice, itching, fluid buildup, edema of the legs, and ascites.

Table 1.

Diagnosis of MASLD

Diagnostic method	Particulars	Ref.
Routine clinical parameters
NFS	Predicts probability of fibrosis based on composite score of age, BMI, albumin, AST/ALT ratio, hyperglycemia, and platelet count	[26]
FIB-4	Combination of age, platelet count, AST/ALT, it estimates the amount of scarring in the liver	[27]
AST-APRI	Based on platelet count and AST levels, predicts liver cirrhosis	[28]
Aminotransferase	AST/ALT > 1 indicates Cirrhosis	[29]
Non-invasive tests
NIS4	Effective way to rule in or rule out at-risk NASH	[18]
OWLiver	Detects MASH using metabolomic panel	[30]
PROC3	Detects fibrosis based on type III collagen	[31]
ELF	Identifies MASH with advanced fibrosis	[32]
Radiologic assessment
Conventional Ultrasound	Used in the diagnosis of advanced liver fibrosis and cirrhosis	[33]
Transient Elastography (TE)	Used in the diagnosis of liver stiffness and advanced liver fibrosis	[23]
Shear wave elastography	Estimates liver stiffness, and diagnoses early liver fibrosis with chronic liver disease	[34]
MR (elastography)	Used in the diagnosis of liver stiffness and liver fibrosis	[35]
MRI-PDFF	Used in the diagnosing liver steatosis	[36]

In the management of MASLD, it is crucial to identify patients at a higher risk of MASH and advanced fibrosis. Although liver biopsy remains a gold standard for determining steatohepatitis and fibrosis stage, it is invasive and costly. Moreover, biopsy is inherently afflicted with sampling errors and is not suitable for widespread screening. A non-invasive assessment of fibrosis severity is paramount in MASLD patient management, given that the stage of fibrosis directly impacts overall mortality. The non-invasive diagnostic methods currently employed for MASLD can be categorized into three groups:

1. Routine clinical parameters to calculate risk scores: Among the clinical tools, meta-analysis of histological data in viral hepatitis (METAVIR) scoring system, proposed by Bedossa et. al., is widely used for evaluating fibrosis severity [14]. Additionally, the MASH Clinical Research Network (CRN) scoring system is another highly validated option [15]. Even more scoring systems have been developed to distinguish fibrosis severity in MASLD patients, such as the fibrosis-4 (FIB-4) index score, and NAFLD fibrosis score (NFS). Among these, the FIB-4 score demonstrates predictive accuracy for advanced fibrosis in chronic hepatitis C virus (HCV) infection cases. In addition, aspartate aminotransferase (AST)-to-platelet ratio index (APRI) and alanine aminotransferase (ALT) levels are also commonly used. However, AST and ALT are inadequate for detecting the extent of hepatic fibrosis due to their moderate elevation in MASLD patients [16].

2. Non-invasive tests (NITs): Although there are no approved diagnostic biomarkers of MASLD, intense efforts are on to identify and validate the diagnostic panels based on the disease markers in circulation. A consortium of National Institutes of Health, industry partners, and academic researchers is working on a project called non-invasive biomarkers for metabolic liver disease (NIMBLE). Its first report was recently released [17]. The study evaluated the diagnostic performance of five biomarker panels (NIS4, OWLiver, PROC3, ELF and FibroMeter VCTE). NIS4 is a blood-based test, where at-risk NASH is defined by NAFLD activity score ≥4 and fibrosis stage ≥2 based on four biomarkers, namely miR-34a-5p, alpha-2 macroglobulin, YKL-40, and glycated hemoglobin [18]. OWLiver employs lipidomic analysis of fasting blood samples by ultra high-resolution liquid chromatography coupled with mass spectrometry (UHPLC-MS) to measure a panel of lipid biomarkers reflecting liver fat content, inflammation, and fibrosis. The PROC3 test grades fibrosis based on the levels pro-peptide of type lll collagen in blood [19]. ELF (enhanced liver fibrosis) test predicts severity of liver fibrosis by quantifying three analytes in blood, namely hyaluronic acid, amino-terminal propeptide of type III procollagen, and tissue inhibitor of matrix metalloproteinase 1 [20]. The utility of these NITs in screening for NASH is still under investigation, but these tests can be used for first-line risk stratification and exclude severe disease.

3. Radiologic assessment of liver morphology: Hepatic fibrosis is characterized by increased deposition of extracellular matrix, which makes the liver tissue stiffer than normal. This elasticity difference is commonly detected by ultrasonography (US). US is a low-cost, reliable, highly accessible, and accurate technique for detection of moderate-severe disease [21]. European guidelines recommend US as the first-choice imaging modality to identify risk of MASLD in adult population. The major drawback of conventional US is its low sensitivity for early-stage disease [22]. Thus, US-elastography has been adopted to assess liver stiffness. Transient elastography (TE or FibroScan), strain elastography (SE), and 2D-shear wave elastography (2D-SWE) are modifications of US-based elastography [23]. These are highly effective techniques, but they may fail in obese patients or those with narrow intercostal space. In addition, US-based methods cannot technically be performed in patients with ascites because the US push pulse is prevented by the ascites from reaching the liver parenchyma [23]. Besides US, magnetic resonance elastography (MRE) has been recently evaluated to stage liver fibrosis. MRE has higher repeatability and reproducibility and thus provides the more reliable liver stiffness measurement [24]. Magnetic resonance imaging proton density fat fraction (MRI-PDFF) is a cutting-edge imaging technique that plays a crucial role in the field of MAFLD. The fundamental principle underlying MRI-PDFF is based on the distinct magnetic properties of water and fat protons in the human body. Under strong magnetic field and radiofrequency pulses, these protons emit signals that can be detected and processed to create detailed images [25].

3. Current therapies for MASLD

3.1. Lifestyle Changes:

Diet and exercise are recommended as the first-line therapy for MASLD [37]. Weight loss, even modest amounts, has been shown to improve liver function and reduce liver fat content [38]. Studies have shown that a diet low in saturated fats and simple sugars, and high in fiber, fruits, and vegetables can improve liver enzymes and reduce liver fat content [39]. Studies also suggest that aerobic exercises help in processing fats and carbohydrates into usable energy in the form ATP. Aerobic metabolism is a slower process, this process generates sustained energy required for day-to-day functional needs. Aerobic condition is very efficient, and can breakdown fats, carbohydrates to glycogens or ketone bodies, a process called ketosis. Unlike aerobic metabolism, anaerobic metabolism is an immediate energy generator that uses mostly carbohydrates, but not fat or protein. However, some studies showed that both aerobic and resistance exercise reduce hepatic steatosis in MASLD [40, 41].

3.2. Bariatric Surgery:

Gastric bypass and sleeve gastrectomy have been shown to significantly improve MASLD, particularly in patients with obesity and type 2 diabetes [1]. Bariatric surgery has been associated with improvements in steatosis, hepatic fibrosis, hyperglycemia, and decrease all-cause morbidity and mortality [42].

3.3. Liver Transplantation:

In advanced cases of MASLD with cirrhosis, liver transplantation may be necessary [43]. However, it is noteworthy that long-term outcomes following liver transplantation for MASLD have presented unique challenges and, in some cases, shown to be less favorable compared to transplants performed for other indications [44]. One of the primary concerns is the recurrence of MASLD in the transplanted liver. Studies have indicated that up to 30-40% of patients may experience graft steatosis, which refers to the accumulation of fat in the transplanted liver [45, 46].

4. Emerging therapies for MASLD

4.1. Drug repurposing:

Although no medications are approved specifically for MASLD, ongoing clinical trials have identified several drugs for potential repurposing as treatment options (Table 2). It is a cost-effective approach to rapidly transition existing drugs into the clinic for new indications. Notably, certain antidiabetic drugs improve MASLD pathology and can be used in carefully selected individuals with MASH and comorbid conditions of diabetes and obesity. As per American Association for the Study of Liver Diseases (AASLD) guidelines pioglitazone, a potent PPARγ agonist is recommended for patients with biopsy proven MASH and after evaluating risks and benefits [47]. Pioglitazone improves steatosis activity and insulin sensitivity albeit its role in the resolution of hepatic fibrosis is unclear [48]. Other antidiabetic drugs including GLP-1 receptor agonists liraglutide, semaglutide, tirzepatide, and SGLT2 inhibitors such as dapagliflozin, canagliflozin have also been shown to improve steatosis and insulin sensitivity but have no proven benefits in reversing fibrosis [49-51]. Finally, vitamin E may be considered for its antioxidant properties in adults only in biopsy proven MASH. However, it is not recommended for diabetic MASLD or cirrhosis [48]. Many other existing drugs listed in Table 2 have been trialed with varying degree of success, Clinically speaking, these repurposed medications have shown efficacy in reducing liver fat and improving liver function, but long-term safety and efficacy data are needed to definitively conclude [52-54].

Table 2.

Repurposing of existing drugs for MASLD treatment

Drug Target	Drug Name	Repurposing to MASLD	Status	Refs
SGLT2 inhibition	Empagliflozin	BMI, HOMA-IR, CAP score	Phase 4	[55]
	Dapagliflozin	Hepatic lipid content, Stiffness	Phase 4	[56]
	Ertugliflozin	Hepatic Transaminase	Phase 4	[57]
GLP1R Agonist	Semaglutide	ALT	Phase 2	[49]
	Exenatide	Hepatic TG	Phase 4	[58]
PPAR agonist	Pioglitazone	ALT, AST	Phase 3	[48]
PDE inhibitor	Pentoxifylline	Fatty acid β oxidation	Phase 3	[59]
AMPK activator	Metformin	Attenuate de novo lipogenesis, fatty acid β oxidation, insulin resistance, improve liver enzymes	Phase 3	[60]
Angiotensin II	Ademetionine	ALT, AST, ALP	Phase 3	[61]
Angiotensin II receptor antagonist	Losartan	Liver function	Phase 2	[62]
DPP-4 Inhibitor	Sitagliptin	Fibrosis score, liver enzymes	Phase 2	[63]
Leptin receptor agonist	Metreleptin	Hepatic injury	Phase 2	[64]
GABA analog	Vigabatrin	HOMA-IR	Phase 2	[65]

4.2. Fibroblast growth factor 21 (FGF21) analogs:

FGF21 is a hormone that regulates glucose and lipid metabolism. Several FGF21 analogs are currently being developed as potential therapies for MASLD. These analogs have been shown to improve insulin sensitivity, reduce liver fat content, and improve liver function in animal models [66]. Clinical trials in humans are ongoing to evaluate the safety and efficacy of FGF21 analogs in the treatment of MASLD. Interestingly, circulating levels of FGF21 can also serve as biomarker for the presence of MASLD (10.1016/j.metabol.2019.153994). FGF21 binds to β-Klotho co-receptor, and the FGF21-β-Klotho complex interacts specifically with the receptors (FGFR1c/2c/3c), enabling downstream signaling via mitogen-activated protein kinase (MAPK) and AKT signaling [67].

LY2405319 is glycosylated FGF21, with better thermal stability and reduced tendency to aggregate, but it still needs daily administration as it is cleaved in circulation. PF-05231023, is FGF21 covalently linked to Fab regions of IgG1κ mAb scaffold, requiring weekly administration. Pegbelfermin is a PEGylated FGF21, but it remains susceptible to cleavage by FAP and thus, needs frequent dosing. Efruxifermin is yet another FGF21 analog studied in a clinical trial of biopsy-confirmed NASH patients. It is an Fc-FGF21 fusion protein with a 3-3.5-day half-life which is substantially longer than that of other FGF21 analogs described above. For complete review of FGF21 and its analogs, readers can access two excellent recent reviews [67] [68].

4.3. Glucagon-like peptide-1 (GLP-1) receptor agonists:

GLP-1 receptor agonists are a class of medications used to treat type 2 diabetes. These medications have been shown to improve glucose control, reduce bodyweight, and improve liver function in patients with MASLD [69]. Several GLP-1 receptor agonists, including liraglutide and semaglutide, have shown promising results in clinical trials for the treatment of MASLD [70].

4.4. Gut Microbiota:

The gut microbiota plays a crucial role in the pathogenesis of MASLD. Gut dysbiosis, characterized by an imbalance in the composition of the gut microbiota, has been linked to the development and progression of MASLD [71]. Several gut microbiota-targeted therapies are currently being developed as potential treatments for MASLD. These include prebiotics, probiotics, and fecal microbiota transplantation [72].

Probiotics are live bacteria or yeasts that restore a healthy intestinal microbiome and improve the gut barrier integrity. The commonly used probiotics include Bifidobacterium and Lactobacilli. Prebiotics are non-digestible food, such as inulin, raw oats, and pectin that promote the growth and survival of beneficial bacteria. Synbiotic treatments combine the probiotic and prebiotic in a single product. The gut microbiota can also be altered using antibiotics [73]. For example, co-administration of polymyxin B and neomycin was found to prevent hepatic lipid accumulation in mice on a high-fructose diet [74]. vancomycin, metronidazole ampicillin, rifaximin, and solithromycin are other antibiotics that have been used in preclinical and clinical studies [75-77]. Among these, probiotic and synbiotic treatment appear to be a low-risk and low-cost supplement to the standard of care, whereas the use of antibiotics to modulate MASLD pathology is still experimental and more focused clinical trials are needed to decide on their utility in MASLD treatment. Finally, fecal microbiota transplantation involves transferring of processed microbial community from healthy individuals to the gastrointestinal tract of the patients. This procedure is in trials, and it is too early to judge the promise of microbiota transplantation for MASLD.

5. Small Molecule Developments

Development of small molecules as therapeutic candidates for treatment of MASLD is beginning to show signs of success. Defined as any organic compound with low molecular weight (< 1,000 Da), small molecule drugs exhibit distinct advantages as therapeutics. For example, most can be administered orally, and they reach intracellular targets relatively unhindered. Moreover, small molecules can be designed to target specific pathways involved in disease pathogenesis. Thus, target-specific small molecules have the potential to be more effective than the non-specific and broad-spectrum therapies. Several small molecules are in clinical development for MASLD, and they have shown promising results in early-stage trials (Figure 2). However, there are currently no FDA-approved small molecule drugs for the treatment of MASLD.

Figure 2.

Potential Mechanisms Involving in the Pathogenesis of MASLD and Targets for Pharmacotherapy. Drugs with agonist actions are labeled in blue and with antagonist action are labeled in red. Abbreviations: ACC, acetyl-CoA carboxylase; AP1, activator protein 1; ASK1, apoptosis signal-regulating kinase1; CCR2/5, C-C chemokine receptor type 2/5; DGAT, diacylglycerol O-acyltransferase; FASN, Fatty acid synthase; FXR, Farnesoid X receptor; JNK, Jun N-terminal kinases; SCD1, stearoyl-CoA desaturase 1; SREBP1, sterol regulatory element binding protein 1; TCA, tricarboxylic acid cycle; TRβ, thyroid receptor β; VLDL, very-low-density lipoprotein.

5.1. Thyroid hormone receptor beta (THRβ) agonists:

THRβ is predominant in the liver, kidneys, pituitary gland, and brain and it is responsible for thyroid hormones effects on lipid metabolism [78], whereas THRα is highly expressed in heart, responsible for most of the cardiovascular effects [79]. Based on these links, in the last few decades considerable effort has been put towards developing TRβ targeted treatments for MASLD. However, compounds Sobetirome (GC-1) and Eprotirome (KB2115) targeting THRβ failed to reach the therapeutic market due modest selectivity towards THRβ and onset of unwanted side-effects [78].

Further efforts to identify THRβ-specific agonists led to the discovery of compound MGL-3196 (Resmetirom). Resmetirom is an orally active agonist of THRβ that is around 28 times more selective than endogenous triiodothyronine (T3). Resmetirom has a specific uptake into the liver and poor tissue penetration outside the liver. Several clinical trials have been conducted to evaluate the efficacy and safety of resmetirom in MASLD/MASH patients [80]. In a Phase 3 MAESTRO-MASLD-1 study, the primary safety endpoint and various key secondary endpoints, including reductions in LDL-C, apolipoprotein B, triglycerides, and liver fat measured by MRI-PDFF, were met (NCT03900429). Importantly, resmetirom has received breakthrough therapy designation from FDA for the treatment of patients with MASH with liver fibrosis.

Another THRβ compound which has shown encouraging results for MASLD is VK2809, originally developed as MB07811 by Metabasis Therapeutics. It is an orally available liver-selective agonist prodrug that successfully completed Phase 2a clinical trials for the treatment of MASLD and elevated LDL-C. It is a new generation Hep-Directed prodrug with a cyclic phosphate group, which distributes into hepatocytes and is converted to its active metabolite VK2809A (3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy methyl phosphonic acid) [81]. Phase 1 and 2 safety trials showed safety and tolerance. Viking Therapeutics is initiating a Phase 2b clinical trial of VK2509, in patient with biopsy-confirmed MASH (NCT4173065).

5.2. Farnesoid X Receptor (FXR) Agonists:

FXR agonists are another promising drug class for MASLD treatment [82]. FXR is highly expressed in tissues that are involved in regulating bile acid metabolism. Thus, it has been considered for the treatment of cholestatic disorders, including primary biliary cirrhosis (PBC). FXR is also important for the regulation of the lipid and glucose metabolism. The activation of FXR displays beneficial effects on various metabolic diseases, including fatty liver diseases, type 2 diabetes, dyslipidemia, and obesity. Evidence is also accumulating to suggest that FXR agonism is favorable to liver regeneration and hepatocarcinogenesis. It also contributes to the protection of atherosclerosis and renal diseases, indicating the systemic effects of FXR activation. FXR agonists have been shown to improve liver histology, reduce inflammation, and promote lipid oxidation in preclinical models of MASLD [82, 83].

Among companies targeting FXR for MASLD, steroidal obeticholic acid (OCA) from Intercept Pharmaceuticals is currently leading the race. It is an analog of the chenodeoxycholic acid (CDCA), a bile acid [84]. Given its positive interim 18-month analyses from the regenerate trial and a detailed safety and tolerability profile for long-term OCA use, the FDA has recently allowed an NDA for the use of OCA to treat pre-cirrhotic liver fibrosis with (NCT02548351). However, in another trial OCA failed to show superiority compared to placebo in improving liver scarring in patients with compensated MASH-related cirrhosis (NCT03439254). Thus, the FDA issued a complete response letter on June 22, 2023, indicating that OCA cannot be approved in its present form. It is noteworthy that a combination treatment of OCA (5-10 mg) with bezafibrate showed normalization in PBC biomarkers in a phase 2 study [85].

Cilofexor (GS-9674) is a potent, selective non-steroidal FXR agonist that primarily functions to activate FXR in the intestine and does not undergo enterohepatic circulation [86]. In a phase 2 study, patients with non-cirrhotic MASH given a 24-week therapy of cilofexor showed significant improvement in hepatic steatosis, liver biochemistry, and bile acids [87]. Cilofexor improved cholestatic liver enzymes and non-invasive markers of liver fibrosis in patients with primary sclerosing cholangitis [88]. Similarly, cilofexor improved insulin sensitivity and liver enzymes in patients with non-diabetic MASLD [88]. Cilofexor reduced hepatic fat content and serum γGT in patients with MASH; pruritus was noted as a minor side effect [87, 89]. Patients treated with a combination of cilofexor and firsocostat (acetyl-CoA carboxylase inhibitor) in a Phase 2b (NCT03449446) achieved improvements in multiple response measures of fibrosis and liver function compared with placebo.

EDP-305 is another selective and potent FXR agonist that exhibits anti-fibrotic and anti-inflammatory gene expressions and favorable effects on lipid metabolism in hepatocytes. In a phase 2b trial (NCT04378010), EDP-305 was found to improve fibrosis without worsening the steatohepatitis and/or resolution of steatohepatitis [90].

5.3. Chemokine Antagonists:

Hepatic stellate cells (HSCs) and Kupffer cells (KCs) play a crucial role in the progression of MASLD. During liver injury, HSCs and KCs secrete large amounts of chemokines, including CCR2, CCR5, and IL-1β. These cytokines promote the development of liver fibrosis by inducing the accumulation of large amounts of extracellular matrix protein (Figure 2) [91]. Cenicriviroc, is a novel orally administered and potent dual CCR2 and CCR5 antagonist. Currently, there are several ongoing clinical trials evaluating the safety and efficacy of cenicriviroc in MASH and liver fibrosis. The CENTAUR Phase 2b trial [92, 93] (NCT02217475) showed promising results, with cenicriviroc demonstrating a significant improvement in liver fibrosis without worsening MASH after 1yr of treatment. Following these results, Allergan initiated the AURORA Phase 3 trial (NCT03028740) to evaluate its efficacy and safety. Recently, Allergan announced that the trial did not meet its primary endpoint of fibrosis improvement without worsening MASH after 1-y of treatment [94]. However, the trial did meet a key secondary endpoint of MASH resolution without worsening fibrosis. Despite the mixed results from the AURORA trial, cenicriviroc continues to be evaluated in other ongoing clinical trials, including the ATLAS phase 3 trial (NCT04338958) and the TANDEM Phase 2b trial (NCT03722562).

5.4. Apoptosis Signal Regulating Kinase 1 (ASK1):

ASK1 is a mediator in mitogen-activated protein kinase signaling and is involved in the pathogenesis of MASLD and its progression to MASH [95]. In animal models of MASLD/MASH, ASK1 activation leads to inflammation, fibrosis, and hepatocyte death (Figure 2) [96]. Selonsertib is an investigational small molecule inhibitor of ASK1 that is being developed by Gilead Sciences for the treatment of MASLD/MASH. In a phase 2 trail (NCT 01672866), oral selonsertib was tested in patients with MASH and liver fibrosis. It was found that selonsertib treatment significantly reduced liver fibrosis as measured by magnetic resonance elastography (MRE) compared to placebo. Unfortunately, the phase 3 study did not meet its primary endpoint of a statistically significant improvement in fibrosis without worsening MASH compared to placebo. Thus, Gilead discontinued development of selonsertib in June 2019 [97].

5.5. Stearoyl-CoA Desaturase:

SCD1 is the key determinant of triglycerides biosynthesis pathway [98]. Aramchol is an oral, liver-targeted, fatty acid-bile acid conjugate that partially inhibits hepatic SCD1 protein expression, resulting in a reduction of liver triglycerides. In phase 2a trial (NCT01094158), aramchol significantly reduced liver fat content compared to placebo [99, 100]. A follow up phase 3 ARMOR trial also showed that aramchol improves fibrosis in MASH [101].

5.6. Acetyl-CoA Carboxylase (ACC) inhibitors:

ACC1 and ACC2 are essential enzymes for synthesis of malonyl-CoA (M-CoA) from acetyl-CoA. ACC1 is predominantly expressed in lipogenic tissues such as liver and adipose. It regulates lipogenesis flux in the liver. In MASLD conditions, ACC1 is upregulated in the liver [102]. Firsocostat is an allosteric dual ACC inhibitor that is being developed for MASLD treatment. Results from preclinical and clinical studies support potential benefits of firsocostat in MASH, including improvements in steatosis, necro-inflammatory activity, and fibrosis [103]. In a phase 2 ATLAS study in patients with advanced fibrosis due to MASH, firsocostat monotherapy and a combination regimen (firsocostat + cilofexor) improved fibrosis without worsening MASH after 48 weeks of treatment (NCT04971785) [104].

Clesacostat is a second-generation reversible liver-directed ACC inhibitor designed to have asymmetric distribution to the liver, which enables potent inhibition of hepatic ACC to normalize de novo lipogenesis in patients with MASH. Clesacostat was well tolerated at all single and multiple oral doses [105]. In a phase 2b clinical trial in patients with MASH and liver fibrosis, clesacostat showed improvement in fibrosis (NCT03248882). Recently, clesacostat obtained fast-track designation from the FDA for its investigational combination therapy with ervogastat. This combination could potentially offer benefits in overcoming both inflammation and fibrosis associated with MASLD.

6. Future Directions

Several pathways have been targeted with mixed outcomes, and there are various mechanisms related to lipid metabolism, inflammation and oxidative stress that can be further explored to potentially reverse the MASLD condition. There are many fascinating fields of research that are being explored. Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) could be a potential target for the development of new interventions against MASLD. MAP4K4 is a serine/threonine kinase. The molecular target of MAP4K4 is TGFβ-activated kinase 1 (TAK1, MAP3K7) that is a key activator of MKK4/7 which in turn activates c-Jun N-terminal kinase (JNK) and p38. Apart from MAPK/JNK pathway, NF-κB, Notch, and JAK-STAT pathways are downstream effectors of MAP4K4. A preclinical study in a high-fat diet fed mouse model indicated improved insulin sensitivity in liver and adipose tissues in response to tamoxifen-inducible MAPK4K4 deletion [106]. Similarly, a novel pharmacological inhibitor of MAP4K4 activity, PF-6260933, showed improved glucose tolerance in ob/ob mouse model of type 2 diabetes mellitus [107]. Recent study using human liver biopsies showed that the expression of MAP4K4 is positively correlated with MASLD severity. Moreover, inhibition of MAP4K4 in human hepatocytes initiated metabolic reprograming and protected against steatosis [108]. However, additional in vivo studies are warranted to assess potential of MAP4K4 inhibitors as small molecule therapeutics against MASLD.

Another potential target for small-molecule drug development is the AMP-activated protein kinase (AMPK) pathway. AMPK is a cellular energy sensor that regulates the lipid and glucose metabolism, inflammation, and autophagy. AMPK activation has been shown to improve liver histology, reduce inflammation, and promote lipid oxidation in preclinical models of MASLD. Several AMPK activators, including metformin and AICAR, have already been studied in clinical trials for the treatment of MASLD, but a more specific and potent small molecule will be an attractive advancement.

Conclusion

The field of MASLD research has witnessed significant advancements in recent years, leading to a deeper understanding of the mechanisms involved in the disease’s pathogenesis, diagnostic approaches, and therapeutic strategies. Improved imaging techniques and non-invasive biomarkers have enhanced screening, early detection, and risk stratification. Furthermore, studies exploring the intricate interplay between genetic factors, gut microbiota, and metabolic dysregulation have shed light on novel targets for therapeutic intervention. The huge interest in further drug discovery for MASLD is evidenced by the 1,225 clinical trials for MASLD currently listed in Clinical Trials.gov (see related links: Search Results ∣ Beta ClinicalTrials.gov). While lifestyle modifications remain a cornerstone in its management, emerging pharmacological agents show promise in treating advanced stages of MASLD and disease progression. By controlling dietary habits through timely interventions and the use of effective therapeutics, the liver’s remarkable regeneration capacity can offer the possibility of reversing steatohepatitis.

Abbreviations:

MASLD

Metabolic (dysfunction) associated steatotic liver disease

MASLD

Non-alcoholic fatty liver disease

MASH

non-alcoholic steatohepatitis

HCC

hepatocellular carcinoma

AST/ALT

aspartate transaminase/alanine transaminase

PPARγ

Peroxisome Proliferator-Activated Receptor gamma

SGLT2

Sodium-glucose cotransporter-2 (SGLT2)

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.