Resmetirom: An Orally Administered, Smallmolecule, Liver-directed, β-selective THR Agonist for the Treatment of Non-alcoholic Fatty Liver Disease and Non-alcoholic Steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of fatty liver disease, including non-alcoholic fatty liver (NAFL) and its more progressive form, non-alcoholic steatohepatitis (NASH). The prevalence of NAFLD/NASH along with type 2 diabetes and obesity is rising worldwide. In those who develop NASH, unlike those with bland steatosis (NAFL), lipotoxic lipids drive hepatocyte injury, inflammation and stellate cell activation leading to progressive accumulation of collagen or fibrosis, ultimately leading to cirrhosis and increased risk of hepatocellular carcinoma. Hypothyroidism is associated with NAFLD/NASH; specifically, intrahepatic hypothyroidism drives lipotoxicty in preclinical models. Agonists of thyroid hormone receptor (THR)-β, which is primarily found in the liver, can promote lipophagy, mitochondrial biogenesis and mitophagy, stimulating increased hepatic fatty acid β-oxidation, and thereby decreasing the burden of lipotoxic lipids, while promoting low-density lipoprotein (LDL) uptake and favourable effects on lipid profiles. A number of THR-β agonists are currently being investigated for NASH. This review focuses on resmetirom, an orally administered, once-daily, small-molecule, liver-directed, ß-selective THR agonist, as it is furthest along in development. Data from completed clincal studies outlined in this review demonstrate that resmetirom is effective in reducing hepatic fat content as measured by magnetic resonance imaging-derived proton density fat fraction, reduces liver enzymes, improves non-i nvasive markers of liver fibrogenesis and decreases liver stiffness, while eliciting a favourable cardiovascular profile with a reduction in serum lipids, including LDL cholesterol. Topline phase III biopsy data showed resolution of NASH and/or fibrosis improvement after 52 weeks of treatment, with more detailed peer-reviewed findings anticipated in order to certify these findings. Longer term clinical outcomes from both MAESTRO-NASH and MAESTRO-NASH OUTCOMES will be a pivotal juncture in the drug’s road towards being approved as a NASH therapeutic.

N on-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of fatty liver disease, including non-alcoholic fatty liver (NAFL) and its more progressive form, non-alcoholic steatohepatitis (NASH). The prevalence of NAFLD/NASH along with type 2 diabetes and obesity is rising worldwide. In those who develop NASH, unlike those with bland steatosis (NAFL), lipotoxic lipids drive hepatocyte injury, inflammation and stellate cell activation leading to progressive accumulation of collagen or fibrosis, ultimately leading to cirrhosis and increased risk of hepatocellular carcinoma. Hypothyroidism is associated with NAFLD/NASH; specifically, intrahepatic hypothyroidism drives lipotoxicty in preclinical models. Agonists of thyroid hormone receptor (THR)-β, which is primarily found in the liver, can promote lipophagy, mitochondrial biogenesis and mitophagy, stimulating increased hepatic fatty acid β-oxidation, and thereby decreasing the burden of lipotoxic lipids, while promoting low-density lipoprotein (LDL) uptake and favourable effects on lipid profiles. A number of THR-β agonists are currently being investigated for NASH. This review focuses on resmetirom, an orally administered, once-daily, small-molecule, liver-directed, ß-selective THR agonist, as it is furthest along in development. Data from completed clincal studies outlined in this review demonstrate that resmetirom is effective in reducing hepatic fat content as measured by magnetic resonance imaging-derived proton density fat fraction, reduces liver enzymes, improves non-invasive markers of liver fibrogenesis and decreases liver stiffness, while eliciting a favourable cardiovascular profile with a reduction in serum lipids, including LDL cholesterol. Topline phase III biopsy data showed resolution of NASH and/or fibrosis improvement after 52 weeks of treatment, with more detailed peer-reviewed findings anticipated in order to certify these findings. Longer term clinical outcomes from both MAESTRO-NASH and MAESTRO-NASH OUTCOMES will be a pivotal juncture in the drug's road towards being approved as a NASH therapeutic. touchREVIEWS in Endocrinology Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of fatty liver diseases, including non-alcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). 1 NAFLD is associated with metabolic disorders, including obesity, hypertension, dyslipidaemia, type 2 diabetes mellitus (T2DM), hypothyroidism and metabolic syndrome. 2 The diagnosis of NAFLD requires ≥5% histologic or imaging evidence of hepatic steatosis, with exclusion of secondary causes of hepatic steatosis, such as significant alcohol consumption, hereditary disorders and steatogenic medications. 1 NASH, the more severe and progressive form of the disease, is characterized by inflammation and hepatocyte injury (ballooning) with or without hepatic fibrosis. 1 Our understanding of the natural history of NAFLD and NASH continues to evolve. In a seminal paired liver biopsy study with a mean follow-up of 13.7 years, Ekstedt et al. conducted a biopsy study found that 16% of patients with NAFLD showed improvement in fibrosis, 43% remained stable, 41% showed fibrosis progression and 5% developed cirrhosisrelated complications, thus providing relevant insight into the natural history of NASH with no intervention. 3 NASH is a heterogenous disease, so multiple factors, including genetic determinants, environmental factors and comorbidities, interact to promote fibrosis progression in specific individuals. Ultimately, fibrosis is the most important predictor of clinical outcomes 4 and thus represents an important endpoint in clinical trials, while longer-term follow-up can be done to assess clinical outcomes.
The global prevalence of NAFLD is estimated to be 25%, with the highest prevalence in the Middle East and South America and the lowest in Africa. 5 It is thought to affect 55.5% of individuals with T2DM 6 and 70-80% of patients with obesity worldwide. 7 The global prevalence of NASH is about 5%, and among those with T2DM, the prevalence is estimated at 37.3% 6 and 33.5% among individuals who are overweight or obese. 8 In USANorth America, the prevalence of NAFLD is estimated to be about 24.13% in the adult population, while NASH is estimated to affect 5-6% of adults. 5,9,10 Based on data from the Scientific Registry of Transplant Recipients (2002−2019), NASH was the leading cause of liver transplantation in women and the second most common indication overall in waitlisted transplant candidates without hepatocellular carcinoma in the USA. 11 Markov modelling suggests that by 2030, approximately 100.9 million people will have NAFLD, and 27.0 million people will have NASH with varying stages of fibrosis. 9 The latter represents a 63% increase in NASH cases from 2015. Of those with NASH, 14 million are projected to have a fibrosis score of F2 or higher (i.e. moderate-to-high fibrosis), which may represent the eligible population for treatment should a drug be approved for therapy. 9 Despite the increasing prevalence of NASH with the increase in obesity, 10 no treatment has been approved by the European Medicines Agency or the US Food and Drug Administration (FDA).

Lipooxiuiy as a key nriver in ce pacogenesis of non-aouocooiu seaocepaiis
The pathogenesis of hepatic steatosis is extremely complex but is thought to be conceptionally driven by the excess delivery of free fatty acids to the liver, coupled with increased de novo lipogenesis fuelled with excess carbohydrates, particularly fructose. 12,13 The two major fates of fatty acids in hepatocytes are mitochondrial β-oxidation and re-esterification to form triglycerides. 13 Triglycerides can be exported into the blood as very-low-density lipoproteins or stored in lipid droplets. 14 Lipid-droplet triglyceride undergoes regulated lipolysis to release fatty acids back into the hepatocyte free fatty acid pool. 14 When the disposal of fatty acids through β-oxidation or the formation of triglyceride is overwhelmed, fatty acids can contribute to the formation of lipotoxic species that lead to endoplasmic reticulum stress, oxidant stress and inflammasome activation. 14 Insulin resistance and hyperinsulinaemia further fuel lipid accumulation by not only diverting glucose from glycogen synthesis to de novo lipogenesis in the liver but also impacting peripheral lipolytic pathways. 13 When the hepatocytes' ability to handle this stress is overwhelmed, hepatocyte injury and apoptosis result in the recruitment and activation of inflammatory cells and the activation of hepatic stellate cells, which are the predominant cellular source of collagen/fibrosis in response to any chronic liver injury. 15 The activation of these repair pathways moves diagnosis from simple steatosis (NAFL) to the the more active progressive form of NAFLD, steatohepatitis (NASH). 15 In its quiescent state, the hepatic stellate cell is vitamin rich and produces predominantly type IV collagen. With injury, it undergoes phenotypic changes, including increased proliferation and contractility and a shift towards producing type I and III collagens, which are characteristic of the cirrhotic liver. 16 Progressive fibrosis is often defined histologically on a scale ranging from F0 to F4, as shown in Table 1. 15 As fibrosis is the most important predictor of clinical outcomes, 17,18 improvement in fibrosis has become the most important surrogate endpoint in NASH clinical trials while awaiting longer-term follow-up for clinical outcomes.
In the USA, approximately 20% of patients with NAFLD have disease progression to NASH, and up to 20% of patients with NASH may develop cirrhosis within their lifetime ( Figure 1). 19 While the most common cause of death in patients with NASH is cardiovascular disease, 20 the risk of liver-related mortality increases exponentially with fibrosis stage increase, 17 and liver fibrosis is the most important predictor of liverrelated mortality in patients with NAFLD. 18 Patients with advanced fibrosis are at an increased risk of morbidity and mortality due to the disease progressing to liver failure, cirrhosis and hepatocellular carcinoma. 15,17,18 In addition to obesity and T2DM, other conditions associated with NAFLD include dyslipidaemia, polycystic ovary syndrome, metabolic syndrome, obstructive sleep apnoea and endocrine disorders including hypogonadism, hypopituitarism and hypothyroidism. 1 This review will focus on the role of thyroid hormone signalling in NASH pathogenesis and the potential role of thyroid hormone receptor (THR)-β agonists as a treatment strategy. Tcyroin cormone-meniaen reguoaion of oipin meabooism ann miouconnriao auiviy in ce uonex of non-aouocooiu fay oiver nisease/nonaouocooiu seaocepaiis Low serum thyroid hormone levels are associated with non-alcoholic fatty liver disease The hypothalamus produces thyroid-releasing hormone, which stimulates the production of thyroid-stimulating hormone (TSH) from the pituitary gland. 21 TSH promotes the secretion of prohormone thyroxine (T4) and triiodothyronine (T3) from the thyroid gland. T3 binds to the THR-α (mainly in the heart and bone) and -β (mainly in the liver) to regulate gene transcription. Population studies have shown associations between NAFLD and overt hypothyroidism, subclinical hypothyroidism and low thyroid hormone in the normal range. [22][23][24][25] Patients with higher baseline TSH levels and no evidence of liver disease were more likely to develop NAFLD. 26 In addition, the prevalence of NAFLD was found to significantly increase as serum TSH level increased, even after adjusting for age, gender and smoking status. 23  Overall, a 12% decrease in intrahepatic lipid content was observed, with a 23% reduction in those over the age of 50 years. However, in patients with subclinical hypothyroidism or euthyroidism, the beneficial effects of longer treatment with thyroid hormone would need to be weighed against long-term clinical adverse effects, such as atrial tachycardia, arrhythmias and loss of bone density through the effect of THR-α. As THR-β is the predominant isoform in the liver, specific targeting may be a better approach.
The effect of thyroid hormones on autophagy, mitophagy, mitochondrial biogenesis and β-oxidation Autophagy is a normal physiologic metabolic process wherein a cell consumes its own redundant or damaged organelles to fuel regeneration of newer healthier organelles. T3 induces autophagy in the hepatoma cell line HepG2 in a dose-dependent manner and specifically promotes lipophagy, resulting in the delivery of additional internal free fatty acids to the mitochondria for β-oxidation. 29 In addition, T3 promotes mitochondrial oxidation due to a combination of mitophagy and mitochondrial biogenesis. 30 Taken together, these results show that thyroid hormones can regulate lipid homeostasis via autophagy and help to explain how thyroid hormones increase oxidative metabolism through effects on mitochondrial turnover and activity.

Intrahepatic hypothyroidism as a driver of nonalcoholic steatohepatitis
In a mouse NASH model where mice were fed a Western diet with fructose for 16 weeks, increased steatosis, inflammation and fibrosis were associated with a statistically significant decrease in intrahepatic T4 and T3. 31 In addition, in a dietary mouse model of NASH, administration of thyroid hormones decreased hepatic triglyceride content (3.19 ± 0.68 mM/g liver versus 8.04 ± 0.42 mM/g liver) and hydroxyproline (1.44 ± 0.07 mg/g liver versus 2.58 ± 0.30 mg/g liver) compared with mice with untreated NASH. 32 Moreover, thyroid hormones restored autophagy and mitochondrial biogenesis to increase β-oxidation of fatty acids and reduced lipotoxicity, oxidative stress, hepatic inflammation and fibrosis. 32 In the liver, deiodinase type 1 (DIO1) converts the prohormone T4 to the bioactive hormone T3. Interestingly, in a model of early NASH, DIO1 levels and activity are increased suggesting that there is a compensatory increase to handle excess lipids. 31 However, low DIO1 levels and activity have been observed in humans and rodents with advanced NASH, 33 and DIO1 knockdown leads to increased intrahepatic lipid content. 31 At the cellular level, in both rodent and human healthy livers, hepatocytes strongly expressed DIO1 and stromal cells weakly expressed DIO3. 33 Very little DIO1 was detected in other non-parenchymal cells of the liver. During injury, hepatocyte expression of DIO1 decreased, whereas stromal expression of DIO3 increased, particularly in myofibroblasts. 33 In patients, this was also reflected by increased serum reverse T3 (rT3).  Moreover, the decreases in the free T3 to rT3 and free T4 to rT3 ratios distinguished advanced from mild fibrosis, even in individuals with similar serum levels of TSH and free T4. 33 Therefore, it is thought that with chronic liver injury, intrahepatic thyroid hormone signalling may be impaired; this impairment would decrease conversion of T4 by DIO1 to active T3 and increase conversion of T4 by DIO3 to inactive rT3, leading to the accumulation of lipotoxic species, stimulating a cycle of repetitive liver injury. 33 Taken together, these findings suggest that intrahepatic hypothyroidism may be a driver of NASH pathogenesis ( Figure 2).

Specific role of thyroid hormone receptor-β in hepatic lipid metabolism
Hepatocytes highly express THR-β, which is responsible for regulating metabolic pathways that are impaired in NAFLD and NASH. 34 Most hepatic fat is derived from free fatty acids released by adipocytes. In NASH, β-oxidation of hepatic lipids is decreased, resulting in lipotoxicity. 35 Animal studies have shown that the activation of THR-β plays an important role in reducing triglycerides and cholesterol. 33,36 In cultured In one study, patients with THR-β mutations had increased liver fat (as assessed by controlled attenuation parameters using transient elastography) compared with their unaffected family members, while controlling for body mass index. 37 All participants belonged to the same family, lived on the same small island and were, therefore, exposed to similar environmental conditions. No difference in insulin resistance was observed between the two groups. Individuals with NASH display low THR-β activity, which exacerbates mitochondrial dysfunction and lipotoxicity. 35 Given the role played by THR-β signalling in liver metabolism, a strong rationale for developing THR-β-selective thyromimetics exists.
Consequently, new molecules with a very high THR-β affinity and hepatic selectivity have been developed to treat lipid-associated hepatic disorders, in particular NAFLD.
Thyroid hormone receptor-β agonists for the treatment of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis For the treatment of NASH, a THR-β agonist ideally needs to achieve three goals: 1) reduced hepatic steatosis, inflammation and fibrosis; 2) liver specificity with no effect on the hypothalamus-pituitary-thyroid axis, which regulates serum thyroid levels; 3) high THR-β selectivity to limit off-target THR-α effects on the bone/cartilage and heart.
Sobetirome (GC-1) and eprotirome (KB2115) were the first THR-β agonists shown to reduce intrahepatic lipid content in preclinical models. 38,39 Despite its favourable lipid-lowering effects, eprotirome development was halted in phase III because it caused cartilage damage in dogs following chronic treatment. 40 In addition, liver toxicity was noted when patients with familial hypercholesterolaemia were treated after only 6 weeks. 34

WSummary of preuoiniuao naa for resmeirom
Resmetirom is an investigational liver-directed agonist of THR that is 28 times more selective for THR-β over THR-α. 52 In vitro studies have demonstrated resmetirom's preferential use of the organic anion transporting polypeptides 1B1 receptor for hepatocyte uptake and stronger activation of THR-β. 53,54 Moreover, oxygen consumption rate studies found that both T3 and resmetirom increased basal and maximal respiration, accompanied by an increase in the production of adenosine triphosphate, reflecting higher oxidation of substrates. 53 While it is known that increased hepatic THR-β agonism increases the level of hepatic DIO1, 55,56 additional in vitro and in vivo data suggest that resmetirom upregulates the expression of DIO1, supporting its role not only as a thyromimetic but also in potentially increasing the conversion of T4 to T3. 54 Although not definitive, the low serum T4 detected in patients treated with resmetirom indirectly suggests this mechanism may be relevant. 53 Given its specificity for THR-β and its impact on lipophagy, mitophagy, mitogenesis and β-oxidation within hepatocytes, resmetirom is an appealing agent for treating NASH. ≥4 and hepatic fat fraction of ≥10% at baseline when assessed by MRI-PDFF. 47 The NAS comprises three components: steatosis (0-3), ballooning (0-2) and inflammation (0-3). The maximum NAS is 8, and patients had to have a score ≥4 with the requirement of ballooning for inclusion.
Patients were randomly assigned to receive either resmetirom 80 mg or placebo orally once a day. Serial hepatic fat measurements were obtained at Weeks 12 and 36, and a second liver biopsy was obtained at Week 36 ( Figure 4; the 36-week main study). 47 The primary goal of this study was to determine whether resmetirom could effectively reduce the burden of lipotoxic lipids that may be driving liver injury. Therefore, the primary  Table 3). 47 Furthermore, resmetirom seemed to produce statistically significant reductions in a number of secondary and exploratory endpoints, including liver enzymes, multiple lipoproteins and atherogenic lipids (     Change in absolute NAS was not significantly different between groups but did show a difference in those with high exposure, high SHBG and Week 12 MRI-PDFF response ( Table 5). 47 The authors suggested that thyroid hormone-responsive protein SHBG may serve as a biomarker for monitoring compliance and optimizing dosing. There was no difference in patients who achieved NASH resolution with no worsening of fibrosis, except in a subgroup analysis of those with <9.5% weight loss and MRI-PDFF response, defined as >30% reduction of MRI-PDFF at Week 12.
Overall, this study met its primary endpoint of MRI-PDFF reduction at Week 12. Moreover, resmetirom responders with a 30% MRI-PDFF reduction at Week 12 had higher rates of NASH resolution (37%) at the Week 36 liver biopsy compared with non-responders (4%) along with a reduction in liver enzymes and fibrosis biomarkers, suggesting that early MRI-PDFF response may predict future NASH resolution and anti-fibrotic effect. In patients treated with resmetirom, a higher incidence of mild diarrhoea and nausea was reported; however, most adverse events were mild, selflimiting and balanced between the two groups studied. There were no reports of adverse events related to THR-α activity, such as changes in bone mineral density based on dual-energy X-ray absorptiometry (DEXA) scan, thyroid axis suppression or cardiovascular adverse events. 47 At the end of the main 36-week, phase II study, a 36-week, activetreatment, open-label extension (OLE) study was conducted in 31 patients with persistently mild to markedly elevated liver enzymes ( Figure 4). 58 All OLE study endpoints were exploratory. p<0.0001) and mean relative reduction of -52.3% (standard error 4.4%, p<0.0001). Interestingly, the controlled attenuation parameter score, which is commonly used to assess steatosis with FibroScan ® (Echosens, Paris, France), did not correlate with changes in MRI-PDFF, suggesting that it may not be a reliable assessment of steatosis reduction in response to treatment. Other key outcomes, including liver enzymes, lipid profiles and markers of fibrogenesis, were favourably reduced and are shown in Table 6. 58 While no repeat liver biopsy was conducted at the end of the OLE study, several markers of fibrogenesis were assessed. Of particular interest were PRO-C3 and C3M levels, which had not been initially analysed in the main 36-week study. PRO-C3 reflects the production of the pro-peptide (N-terminal pro-collagen) of type III collagen and is thought to be a biomarker of new collagen production by activated stellate cells or fibrogenesis. On the other hand, C3M is the matrix metallopeptidase 9-mediated degradation product of type III collagen and, thus, reflects fibrinolysis. Therefore, the PRO-C3/C3M ratio is thought to reflect net fibrosis production. 59 In the post hoc analysis from the main study, the   The MAESTRO-NASH-OUTCOMES trial ( ClinicalTrials. gov identifier NCT05500222) will be enrolling patients with early Child-Pugh class A cirrhosis (score of 5-6) and, through a rigorous adjudication process, define clinical events with a composite clinical endpoint that includes hepatic decompensation events, Model for End-stage Liver Disease score ≥15, liver transplant and all-cause mortality. 68 Ultimately, it is improvement in clinical outcomes that will move both the FDA and payors.
The path to NASH therapeutics reaching the market has been challenging. Early optimism was tempered by numerous failed trials. However, optimism has been renewed with the promising data collected for resmetirom. Its safety profile, coupled with potential efficacy that will be determined in upcoming data from phase III trials, positions it as a leader in a crowded field of candidate therapeutics. q