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Safety and efficacy of combination therapy with semaglutide, cilofexor and firsocostat in patients with non-alcoholic steatohepatitis: A randomised, open-label phase II trial
Combining therapies with complementary mechanisms may be beneficial in patients with NASH.
•
In a phase II trial in 108 patients with NASH, semaglutide alone or in combination with cilofexor and/or firsocostat was well tolerated.
•
Combinations resulted in greater improvements in liver steatosis, liver biochemistry, and non-invasive tests of fibrosis.
Background & Aims
Non-alcoholic steatohepatitis (NASH) is associated with increased risk of liver-related and cardiovascular morbidity and mortality. Given the complex pathophysiology of NASH, combining therapies with complementary mechanisms may be beneficial. This trial evaluated the safety and efficacy of semaglutide, a glucagon-like peptide-1 receptor agonist, alone and in combination with the farnesoid X receptor agonist cilofexor and/or the acetyl-coenzyme A carboxylase inhibitor firsocostat in patients with NASH.
Methods
This was a phase II, open-label, proof-of-concept trial in which patients with NASH (F2–F3 on biopsy, or MRI-proton density fat fraction [MRI-PDFF] ≥10% and liver stiffness by transient elastography ≥7 kPa) were randomised to 24 weeks’ treatment with semaglutide 2.4 mg once weekly as monotherapy or combined with once-daily cilofexor (30 or 100 mg) and/or once-daily firsocostat 20 mg. The primary endpoint was safety. All efficacy endpoints were exploratory.
Results
A total of 108 patients were randomised to semaglutide (n = 21), semaglutide plus cilofexor 30 mg (n = 22), semaglutide plus cilofexor 100 mg (n = 22), semaglutide plus firsocostat (n = 22) or semaglutide, cilofexor 30 mg and firsocostat (n = 21). Treatments were well tolerated – the incidence of adverse events was similar across groups (73–90%) and most events were gastrointestinal in nature. Despite similar weight loss (7–10%), compared with semaglutide monotherapy, combinations resulted in greater improvements in liver steatosis measured by MRI-PDFF (least-squares mean of absolute changes: −9.8 to −11.0% vs. −8.0%), liver biochemistry, and non-invasive tests of fibrosis.
Conclusions
In patients with mild-to-moderate fibrosis due to NASH, semaglutide with firsocostat and/or cilofexor was generally well tolerated. In exploratory efficacy analyses, treatment resulted in additional improvements in liver steatosis and biochemistry vs. semaglutide alone. Given this was a small-scale open-label trial, double-blind placebo-controlled trials with adequate patient numbers are warranted to assess the efficacy and safety of these combinations in NASH.
Clinical Trial registration number
NCT03987074.
Lay summary
Non-alcoholic fatty liver disease and its more severe form, non-alcoholic steatohepatitis (NASH), are serious liver conditions that worsen over time if untreated. The reasons people develop NASH are complex and combining therapies that target different aspects of the disease may be more helpful than using single treatments. This trial showed that the use of 3 different types of drugs, namely semaglutide, cilofexor and firsocostat, in combination was safe and may offer additional benefits over treatment with semaglutide alone.
Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent causes of chronic liver disease worldwide, and its more severe form, non-alcoholic steatohepatitis (NASH), is associated with hepatic fibrosis, cirrhosis, end-stage liver disease and hepatocellular carcinoma.
After alcohol-related liver disease, NASH is the second most frequent indication for liver transplantation in the US and is the leading indication in women.
NASH is also closely associated with metabolic disorders, especially type 2 diabetes. Cardiovascular disease along with liver-related complications are the leading causes of death in patients with NASH.
The pathophysiology of NASH is complex and includes metabolic abnormalities (e.g., insulin resistance), lipotoxicity, fibrogenesis and inflammatory processes.
Increased understanding of these multifactorial pathways has resulted in a range of therapeutic targets being investigated for NASH. Cilofexor is a non-steroidal agonist of farnesoid X receptor (FXR) that inhibits lipogenesis, gluconeogenesis and bile acid synthesis, while firsocostat, an acetyl-coenzyme A carboxylase (ACC) inhibitor, reduces hepatic de novo lipogenesis. In phase II trials, both therapies as single agents were well tolerated and decreased hepatic steatosis in patients with NASH.
In patients with NASH and advanced fibrosis (stage F3 or F4), treatment with a combination of cilofexor and firsocostat for 48 weeks was well tolerated and led to significantly greater improvements in necroinflammatory activity and some fibrosis-related endpoints than the individual agents, supporting the safety and potential additional benefits of a combination approach vs. monotherapy.
Semaglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist that improves glycaemic control and reduces body weight and markers of inflammation in patients with type 2 diabetes and obesity.
Efficacy and safety of once-weekly semaglutide monotherapy versus placebo in patients with type 2 diabetes (SUSTAIN 1): a double-blind, randomised, placebo-controlled, parallel-group, multinational, multicentre phase 3a trial.
Semaglutide has also been shown to reduce the incidence of major adverse cardiovascular events in patients with type 2 diabetes and high cardiovascular risk.
In patients with NASH and F2 or F3 fibrosis, semaglutide resulted in significantly more patients achieving NASH resolution without worsening of fibrosis than placebo after 72 weeks of treatment in a phase IIb trial.
Although statistically significant differences in fibrosis improvement assessed by histology were not observed, semaglutide-treated patients had reduced rates of fibrosis progression and improvements in several non-invasive tests (NITs) of fibrosis vs. placebo, including liver fibrosis measured by transient elastography (TE) and enhanced liver fibrosis (ELF) test score. Semaglutide was also associated with improvements in metabolic parameters including body weight, glycated haemoglobin (HbA1c) and lipids.
As increasingly applied in other diseases, such as type 2 diabetes, combination approaches to the treatment of NASH may offer greater benefits than monotherapies, given the heterogeneity of the patient population and the potential to simultaneously target multiple relevant pathways.
Semaglutide, cilofexor and firsocostat have different and potentially complementary mechanisms of action, including metabolic and anti-inflammatory effects in the case of semaglutide and direct hepatic effects for cilofexor and firsocostat. Key questions related to the safety and tolerability of combination approaches include the potential for semaglutide to mitigate lipid changes associated with ACC inhibition and FXR agonism, to mitigate pruritus associated with the latter, and the compatibility of these compounds’ mechanisms of action, including their impact on gastrointestinal tolerability. In this phase II randomised trial, we evaluated the safety and tolerability of combination treatments including semaglutide with cilofexor and/or firsocostat in patients with mild-to-moderate fibrosis due to NASH. In addition, several exploratory efficacy endpoints, including NITs of hepatic steatosis and fibrosis, markers of liver function and metabolic parameters, were assessed.
Patients and methods
This phase II, randomised, open-label, proof-of-concept trial (NCT03987074) evaluated the safety and tolerability of subcutaneous semaglutide administered as monotherapy or in combination with cilofexor and/or firsocostat in patients with mild-to-moderate fibrosis due to NASH, as defined by biopsy or clinical and non-invasive criteria. The trial was conducted at 18 centres in the USA. The trial protocol was approved by the institutional review board at each site, and the trial was undertaken in accordance with ICH Good Clinical Practice guidelines and the Declaration of Helsinki. The trial protocol is provided in the supplementary materials.
Participants
Patients aged 18–75 years were eligible for inclusion if they had a historical liver biopsy within 6 months of screening consistent with NASH (defined as the presence of steatosis, inflammation and ballooning) with stage 2 or 3 fibrosis according to the NASH Clinical Research Network classification (or equivalent), or if they met all 4 criteria of: a clinical diagnosis of NAFLD, MRI-proton density fat fraction (MRI-PDFF) with ≥10% steatosis, liver stiffness by TE (FibroScan®, Echosens, Paris, France) ≥7 kPa (consistent with fibrosis) and FibroTest® <0.75 (to exclude cirrhosis). Other inclusion criteria included alanine aminotransferase (ALT) ≤5x the upper limit of normal (43 U/L in females and 48 U/L in males), HbA1c ≤9.5%, body weight >60 kg and BMI >23 kg/m2. Key exclusion criteria included a historical liver biopsy consistent with cirrhosis, history of decompensated liver disease, liver transplantation or hepatocellular carcinoma, other causes of liver disease, excessive alcohol consumption (>21 oz/week for men or 14 oz/week for women), unstable cardiovascular disease or weight loss >5% within 6 months prior to screening. There were no criteria based on lipid levels; however, treatment with GLP-1 receptor agonists within 90 days prior to screening was exclusionary, and patients on a vitamin E dose of ≥800 IU/day at baseline were required to be on a stable dose for at least 180 days prior to screening. Full eligibility criteria are listed in Table S1. All participants provided written, informed consent before any trial-related activities.
Procedures
Patients were randomised (1:1:1:1:1) to 1 of 5 groups to receive open-label treatment with semaglutide monotherapy, semaglutide plus cilofexor 30 mg (SEMA + CILO 30), semaglutide plus cilofexor 100 mg (SEMA + CILO 100), semaglutide plus firsocostat 20 mg (SEMA + FIR) or semaglutide plus cilofexor 30 mg plus firsocostat 20 mg (SEMA + CILO + FIR). An interactive mobile/web response system (IXRS/IWRS) was used for centralised randomisation and treatment assignment. Randomisation was stratified by the presence or absence of type 2 diabetes.
The trial consisted of a 2-week screening period, a 2-week pre-treatment period, a 24-week treatment period and a 7-week follow-up period (Fig. S1).
Semaglutide was administered by subcutaneous injection once weekly with a prefilled pen-injector and was initiated at a starting dose of 0.24 mg, which was increased at 4-weekly intervals (to 0.5 mg, 1.0 mg and 1.7 mg) until the recommended target dose of 2.4 mg was reached (from week 17 onwards). Patients who could not tolerate the planned dose-escalation schedule were encouraged to extend any single-dose step for a maximum of 1 additional week, and to re-attempt dose escalation to 2.4 mg at least once. Cilofexor and firsocostat were given orally once daily with or without food.
Imaging assessments (MRI-PDFF, liver stiffness by 2-D magnetic resonance elastography [MRE] and TE) were conducted at screening (or within 4 weeks prior to screening), and at weeks 12 and 24. TE was performed by experienced operators, and MRI-PDFF and MRE images were analysed by a central reader as previously described.
Serum samples were collected at screening, baseline and every 4 weeks through to week 24 for clinical laboratory values; blood biomarkers were assessed at screening, baseline and weeks 12 and 24.
Outcomes
The primary endpoint was the safety and tolerability of study regimens, including semaglutide alone and in combination with semaglutide, cilofexor, and/or firsocostat in patients with NASH. Safety assessments included adverse events, clinical laboratory assessments, vital signs, electrocardiograms and physical examination. Clinical and laboratory adverse events were coded using the Medical Dictionary for Regulatory Activities version 23.0.
All efficacy endpoints were exploratory and included changes from baseline at week 24 in liver steatosis as measured by MRI-PDFF and controlled attenuation parameter (CAP; FibroScan). Proportions of patients with ≥5% absolute and ≥30% relative reductions in MRI-PDFF at week 24 were also assessed. In addition to these protocol-defined analyses, post hoc assessments included proportions of patients with ≥50% relative reduction in MRI-PDFF and MRI-PDFF normalisation (defined as <5%) at week 24. Changes in liver biochemistry (ALT, aspartate aminotransferase [AST], alkaline phosphatase, gamma-glutamyltransferase, total and direct bilirubin), platelets, albumin, international normalised ratio, model for end-stage liver disease score, NITs of fibrosis including ELF test score (and proportion of patients with a ≥0.5-unit reduction
), FibroSure/FibroTest score and markers of inflammation and apoptosis (C-reactive protein [CRP], CK-18 M30) were evaluated. Changes in liver stiffness were assessed by TE and MRE, including the proportion of patients with a ≥25% relative reduction in liver stiffness by TE. Changes in FibroScan-AST (FAST) score, which combines liver stiffness measured by TE, steatosis by CAP and serum AST for the non-invasive identification of patients with NASH and ≥F2 fibrosis, were also assessed post hoc.
FibroScan-AST (FAST) score for the non-invasive identification of patients with non-alcoholic steatohepatitis with significant activity and fibrosis: a prospective derivation and global validation study.
Finally, metabolic parameters (body weight, BMI, HbA1c, fasting plasma glucose, fasting insulin, homeostatic model assessment of insulin resistance), blood pressure, estimated glomerular filtration rate, serum creatinine and patient-reported health-related quality-of-life outcomes (Chronic Liver Disease Questionnaire-NAFLD, EuroQol-5 D questionnaire and visual analogue scale) were assessed.
Statistical analyses
Due to the exploratory nature of this trial, sample size was informed by clinical experience with prior proof-of-concept studies, including rates of adverse events and safety-related laboratory changes, rather than a formal power calculation. In a previous phase II study, in which a similar patient population was treated with CILO 30 mg once daily for 24 weeks, pruritus of any severity occurred in 7 of 56 treated patients (12.5%).
As firsocostat has not been observed to mitigate pruritus incidence in prior trials, approximately 40–45 patients randomised to a treatment arm including CILO 30 mg was deemed to be sufficient to support comparisons with existing data. Safety and efficacy analyses included all randomised patients who took at least 1 dose of any study drug; descriptive statistics are provided.
All protocol-defined efficacy analyses were exploratory in nature. Comparative analyses between combination regimens and semaglutide monotherapy were conducted post hoc. For evaluation of changes from baseline to week 24, analysis of covariance (ANCOVA) with adjustment for baseline value and diabetes status was used, and least-squares (LS) means, LS mean differences, associated 95% CIs and p values were calculated. Proportions of binary responders were compared by Fisher’s exact test.
Due to the COVID-19 pandemic, week 24 imaging (MRI-PDFF, MRE and/or TE) was delayed for a small number of patients. Accordingly, sensitivity analyses were conducted for these parameters excluding patients in whom imaging data were collected more than 30 days after the last dose of study drug(s).
Given the exploratory nature of the analyses, nominal p values are reported. All comparisons were made at a significance level of 0.05. All analyses were based on observed data and were performed using SAS version 9.4 (SAS; Cary, NC).
Results
Between 29 July 2019 and 9 December 2019, 209 patients were screened and 108 were randomised to 24 weeks of treatment with semaglutide (n = 21), SEMA + CILO 30 (n = 22), SEMA + CILO 100 (n = 22), SEMA + FIR (n = 22) or SEMA + FIR + CILO 30 (n = 21). All 108 patients who were randomised received at least 1 dose of any study drug. Ninety-two patients (85%) completed study drug treatment and 96 (89%) completed the study (Fig. 1). The majority of patients in each group had treatment adherence rates of 90–100%. Overall, 89 patients (82%) achieved the semaglutide target dose of 2.4 mg with no meaningful differences between semaglutide alone and combination groups.
Across treatment groups, most patients were female (68.5%), white (85.2%) and had type 2 diabetes (54.6%); the median (Q1–Q3) age was 54 years (48−61). At baseline, NITs were consistent with mild-to-moderate fibrosis (median ELF 9.4 [8.9−9.9] and liver stiffness by TE 9.3 kPa [7.7−12.0]) and moderate-to-severe steatosis (median MRI-PDFF 17.9% [12.0−24.3]). Baseline demographic and clinical characteristics were similar between the groups (Table 1), although median body weight, MRI-PDFF and liver stiffness by TE were higher, and serum ALT and AST were lower in the combination groups compared with the semaglutide group. Overall, 37%, 10% and 18% of patients were on a statin, fibrate or fish oil, respectively, at baseline, and rates were similar across groups.
Table 1Baseline demographics and clinical characteristics.
Most patients experienced at least 1 adverse event, with the incidence similar across treatment groups (73% to 90% of patients; Table 2). Most adverse events were grade 1 or 2 in severity; the incidence of grade ≥2 events was also similar across groups (41% to 48%). The most commonly reported adverse events were gastrointestinal, including nausea, diarrhoea, constipation and decreased appetite. Rates of nausea and vomiting were similar in the semaglutide group (43% and 10%, respectively) and the combination groups (14–36% and 5–9%), with the exception of the SEMA + FIR + CILO group (67% and 29%); however, this did not result in increased treatment discontinuations. Pruritus was observed in 5 patients, all on CILO (1 [4.5%] in the SEMA + CILO 30 group, 2 [9.1%] in the SEMA + CILO 100 group and 2 [9.5%] in the SEMA + FIR + CILO group). All pruritus events were mild and none led to treatment discontinuation. Ten hypoglycaemic episodes were reported in 5 patients (4.6%); all were grade 1 or 2 in severity and none were assessed as serious. Dose modification of semaglutide was required in 1 patient with type 2 diabetes treated with metformin.
Table 2Adverse events, laboratory abnormalities and changes in serum lipids.
Adverse event leading to discontinuation of any study drug
3 (14)
1 (5)
1 (5)
2 (9)
1 (5)
Death
0
0
0
0
0
Most common adverse events (>10% of individuals in any treatment group)
Nausea
9 (43)
8 (36)
6 (27)
3 (14)
14 (67)
Diarrhoea
5 (24)
5 (23)
1 (5)
4 (18)
3 (14)
Constipation
2 (10)
5 (23)
3 (14)
2 (9)
5 (24)
Decreased appetite
4 (19)
6 (27)
0
4 (18)
3 (14)
Vomiting
2 (10)
2 (9)
1 (5)
2 (9)
6 (29)
Abdominal pain
1 (5)
1 (5)
1 (5)
4 (18)
2 (10)
Fatigue
2 (10)
3 (14)
1 (5)
1 (5)
2 (10)
Dizziness
2 (10)
3 (14)
2 (9)
1 (5)
0
Abdominal pain upper
3 (14)
1 (5)
0
1 (5)
2 (10)
Arthralgia
5 (24)
0
2 (9)
0
0
Headache
1 (5)
1 (5)
1 (5)
3 (14)
1 (5)
Hypoglycaemia
0
4 (18)∗
0
2 (9)
0
Gastroenteritis
0
0
3 (14)
1 (5)
1 (5)
Gastroesophageal reflux disease
0
1 (5)
0
4 (18)
0
Early satiety
1 (5)
0
0
3 (14)
0
Abdominal distension
0
0
0
3 (14)
0
Adverse event of interest
Pruritus
0
1 (5)
2 (9)
0
2 (10)
Laboratory abnormalities
Grade 3
0
0
0
1 (5)†
0
Grade 4
0
0
2 (9)‡
0
0
Lipid changes, least-squares mean change from baseline to week 24 (95% CI)
Total cholesterol, mg/dl
–15 (–29, –1)
–9 (–21, 3)
15 (2, 29)§
–7 (–20, 6)
3 (–10, 16)
LDL cholesterol, mg/dl
–9 (–22, 4)
0 (–11, 12)
23 (11, 36)§
–5 (–17, 7)
7 (–6, 21)
HDL cholesterol, mg/dl
–1 (–3, 2)
–2 (–4, 0)
–4 (–7, –1)
–5 (–8, –3)§
–5 (–8, –2)§
VLDL cholesterol, mg/dl
–7 (–12, –2)
–5 (–9, –1)
–5 (–9, 0)
4 (0, 8)§
1 (–4, 5)§
Triglycerides, mg/dl
–28 (–60, 4)
–43 (–72, –14)
–16 (–47, 15)
15 (–16, 46)
26 (–7, 58)§
Data are n (%) except for lipid changes.
∗One patient was reported to have experienced hypoglycaemia but not a hypoglycaemic episode (defined as plasma glucose levels of ≤70 mg/dl). †Grade 3 hypertriglyceridaemia at week 4 (577 mg/dl) in a patient with grade 2 elevation at baseline (487 mg/dl). ‡Grade 4 creatine phosphokinase elevations in 2 patients, neither attributed to study drug. §p < 0.05 vs. semaglutide alone.
Two patients had serious adverse events, 1 in the semaglutide group (grade 3 diarrhoea and vomiting) and 1 in the SEMA + CILO 100 group (grade 3 pancreatitis); in both patients, study drug was discontinued. Overall, 8 patients (7.4%) discontinued any study drug and 15 patients (13.9%) required dose modification or interruption of any study drug due to adverse events, mostly gastrointestinal in nature. Drug discontinuation due to adverse events was not increased in the combination groups vs. the semaglutide monotherapy group. No deaths occurred during the trial.
Changes in serum lipids between baseline and week 24 are summarised in Table 2, and triglycerides and LDL cholesterol are shown in Fig. S2. Semaglutide resulted in improvements in triglycerides, and total, LDL and VLDL cholesterol. While LDL cholesterol increased at week 24 in the SEMA + CILO 100 group, no change was observed in patients treated with CILO 30 mg. Increases in triglycerides were observed in FIR-containing groups, including 1 patient with grade 3 hypertriglyceridaemia (defined as >500 to 1,000 mg/dl) in the SEMA + FIR group. In this patient, triglycerides increased from 487 mg/dl (grade 2) at baseline to 577 mg/dl (grade 3) at week 4, after which the patient remained on study drug with no further grade 3 or 4 triglyceride elevations. Otherwise, grade ≥3 laboratory abnormalities were reported in 2 patients in the SEMA + CILO 100 group; both were grade 4 increases in blood creatine phosphokinase considered unrelated to study treatment. No evidence of drug-related hepatotoxicity was observed. At week 24, median increases in heart rate of 1 to 10 bpm were observed across treatment groups, with no abnormal or clinically significant electrocardiogram findings or other clinically relevant changes in vital signs.
Exploratory efficacy endpoints
Hepatic steatosis by MRI-PDFF and CAP
Changes from baseline to week 24 in hepatic steatosis, as measured by MRI-PDFF, are shown in Fig. 2A. Compared with the semaglutide group (LS mean change: −8.0%), greater absolute reductions were observed in the combination treatment groups (LS mean change range: −9.8 to −11.0%). This difference was statistically significant between the semaglutide and SEMA + FIR groups (p = 0.0353). Similar results were observed in a sensitivity analysis that excluded patients with imaging data collected at least 30 days after the last dose of study drug, although the difference between semaglutide and the SEMA + FIR + CILO group (LS mean change: −8.6% vs. −12.6%) was also significant (p = 0.0078) in this analysis (Fig. S3).
Fig. 2Change from baseline to week 24 in hepatic steatosis assessed by MRI-PDFF.
(A) Absolute change from baseline at week 24. (B) Proportion of patients with a ≥30% and ≥50% relative reduction in MRI-PDFF. (C) Proportion of patients with normalisation of liver fat (<5%) and ≥5% absolute reduction in MRI-PDFF. LSmean change based on ANCOVA models adjusted for baseline and diabetes status. ANCOVA, analysis of covariance; LSmean, least-squares mean; MRI-PDFF, magnetic resonance imaging proton density fat fraction.
Greater median relative reductions in MRI-PDFF at week 24 were observed in the combination groups (−55.7 to −59.4%) vs. the semaglutide group (−46.2%) (Fig. S4). More patients in the combination groups achieved relative reductions in MRI-PDFF of ≥30% and ≥50% compared with the semaglutide group (Fig. 2B). A ≥5% absolute reduction in liver fat was achieved by 64.7% of patients in the semaglutide group vs. 76.5% to 94.4% in the combination groups (Fig. 2C). Normalisation of liver fat by MRI-PDFF to <5% in patients with ≥5% liver fat at baseline was achieved by 38.1% to 41.2% of patients treated with combinations vs. 29.4% with semaglutide (Fig. 2C).
Improvements in hepatic steatosis were also observed when assessed by CAP, with median reductions of 52 to 80 dB/m for the combination groups compared to 21 dB/m with semaglutide monotherapy (Fig. S5). These reductions were statistically significant for the SEMA + FIR (p = 0.0034) and SEMA + CILO 30 (p = 0.0379) groups vs. the semaglutide group.
Liver biochemistry
Across treatment groups, reductions from baseline in serum ALT and AST were observed (Fig. 3A,B). Compared with semaglutide monotherapy, all combination groups had significantly greater improvements in ALT; the SEMA + FIR and SEMA + FIR + CILO groups also had significantly greater reductions in AST. Normalisation of ALT in patients with elevated levels at baseline was reported in 50% (6/12) of patients in the semaglutide group and between 86% and 100% in the combination groups (Fig. 3C). Changes in other liver biochemistry parameters are summarised in Table 3 and Table S2.
Fig. 3Changes from baseline to week 24 in liver enzymes.
(A) Absolute change from baseline to week 24 in alanine aminotransferase. ∗p <0.05 vs. semaglutide 2.4 mg alone. LSmean change based on ANCOVA models adjusted for baseline and diabetes status. (B) Absolute change from baseline to week 24 in aspartate aminotransferase. ∗p <0.05 vs. semaglutide 2.4 mg alone. LSmean change based on ANCOVA models adjusted for baseline and diabetes status. (C) Proportion of patients with normalisation of alanine aminotransferase at week 24 in patients with elevated levels at baseline. ANCOVA, analysis of covariance; LSmean, least-squares mean.
Biomarkers of inflammation and hepatocellular apoptosis
C-reactive protein, mg/dl
n = 16 –0.23 (–0.42, –0.04)
n = 20 –0.13 (–0.30, 0.04)
n = 17 0.05 (–0.13, 0.24)∗
n = 18 –0.01 (–0.19, 0.17)
n = 17 –0.19 (–0.38, –0.01)
CK-18 M30, U/L
n = 16 –179 (–252, –107)
n = 20 –259 (–323, –194)
n = 17 –213 (–284, –143)
n = 18 –312 (–381, –243)∗
n = 17 –247 (–318, –176)
Metabolic parameters
Body weight, kg
n = 15 –7.0 (–9.9, –4.0)
n = 20 –11.0 (–13.5, –8.4)∗
n = 17 –9.9 (–12.6, –7.1)
n = 18 –8.3 (–11.0, –5.6)
n = 18 –7.7 (–10.4, –4.9)
Body weight, %†
−7.6 (−9.2, −4.2)
−8.6 (−13.3, −6.0)
−9.6 (−14.7, −6.3)
−7.6 (−11.7, −4.1)
−7.0 (−10.3, −3.7)
Patients with ≥10 weight loss, n (%)
3 (20)
9 (45)
8 (47)
5 (28)
5 (28)
Body mass index, kg/m2
n = 15 –2.6 (–3.7, –1.6)
n = 20 –4.0 (–4.9, –3.2)∗
n = 17 –3.7 (–4.7, –2.7)
n = 18 –3.0 (–4.0, –2.1)
n = 18 –2.5 (–3.5, –1.5)
Fasting plasma glucose, mg/dl
n = 16 –31 (–40, –23)
n = 20 –22 (–30, –14)
n = 17 –18 (–26, –10)∗
n = 18 –32 (–40, –23)
n = 16 –23 (–31, –14)
HbA1c, %
n = 16 –1.0 (–1.2, –0.7)
n = 19 –1.1 (–1.3, –0.9)
n = 17 –1.0 (–1.2, –0.8)
n = 18 –1.2 (–1.4, –1.0)
n = 17 –1.0 (–1.2, –0.8)
In patient with type 2 diabetes
n = 7 –1.4 (–1.8, –1.0)
n = 10 –1.7 (–2.0, –1.3)
n = 8 –1.2 (–1.6, –0.8)
n = 12 –1.7 (–2.0, –1.4)
n = 10 –1.4 (–1.8, –1.1)
Data are least-squares mean (95% CI) unless otherwise stated.
ANCOVA, analysis of covariance; CK, cytokeratin; ELF, enhanced liver fibrosis; HbA1c, glycated haemoglobin; MRE, magnetic resonance elastography.
∗p <0.05 vs. semaglutide alone. †Data for % weight loss are medians (Q1, Q3). Least-squares means, 95% CIs and p values were estimated from the ANCOVA models of the change from baseline at week 24 adjusted for baseline value and baseline diabetes status.
Reductions in liver stiffness by TE were similar across treatment groups; LS mean changes ranged from −2.29 to −3.74 kPa (Fig. 4A). The largest reductions from baseline to week 24 were observed in the 2 FIR-containing groups (SEMA + FIR, −3.50 kPa; SEMA + FIR + CILO, −3.74 kPa). The proportions of patients with a ≥25% relative reduction in liver stiffness by TE from baseline to week 24 were higher in the combination groups (50% to 60%) compared to the semaglutide group (36%) (Fig. 4B). Liver stiffness by MRE did not change between baseline and week 24 and no significant differences were observed between treatment groups (Table 3).
Fig. 4Changes from baseline to week 24 in liver stiffness assessed by transient elastography (FibroScan).
(A) Absolute change from baseline to week 24 in liver stiffness by transient elastography. LSmean change based on ANCOVA models adjusted for baseline and diabetes status. (B) Proportions of patients with a ≥25% relative reduction in liver stiffness by transient elastography. Percentages calculated among patients with elevated alanine aminotransferase at baseline who had non-missing post-baseline values (n/n in bars). Total numbers of patients with elevated alanine aminotransferase levels overall are shown under the bars (N). ANCOVA, analysis of covariance; LSmean, least-squares mean.
At week 24, statistically significant reductions from baseline in ELF score were observed in all groups (from 0.42 to 0.59 units); no significant differences between groups were observed (Table 3). Changes in individual ELF components are reported in Table S2. In a post hoc analysis, all combinations except SEMA + CILO 100 led to significantly greater improvements in FAST score compared with semaglutide (Fig. S6). Reductions in FibroSure/FibroTest from baseline to week 24 were also observed; however, there were no significant differences between the semaglutide group and the combination therapies (Table 3; Table S2).
Other efficacy endpoints
Reductions from baseline in serum levels of CK-18 M30, a biomarker of hepatocyte apoptosis, were observed in all treatment groups at week 24; these reductions were significantly greater in the SEMA + FIR group vs. the semaglutide group (p = 0.0102). With the exception of the SEMA + CILO 100-treated patients, CRP declined in all treatment groups.
Relative reductions from baseline to week 24 in body weight were similar across groups (−7.6 with semaglutide, −7.0 to −9.6% with combinations) (Table 3). Proportions of patients with ≥10% weight loss at week 24 ranged from 20% with semaglutide to 28−47% with combinations. Decreases in HbA1c at week 24 were similar across the treatment groups (−1.0 to −1.2%) (Table 3). No consistent pattern of improvement in health-related quality of life across treatment groups was observed for any of the patient-reported outcomes (Table S3).
Discussion
In this randomised trial of patients with NASH and mild-to-moderate fibrosis, treatment with semaglutide in combination with cilofexor and/or firsocostat was well tolerated and, in exploratory analyses, associated with additional improvements in hepatic steatosis (as measured by MRI-PDFF and CAP), liver stiffness (as measured by TE), FAST score and serum ALT and AST, compared with semaglutide alone.
The tolerability of combinations including semaglutide, cilofexor and/or firsocostat was similar to that of semaglutide monotherapy. Most adverse events were mild-to-moderate in severity, treatment discontinuation due to adverse events was infrequent and only 2 patients had serious adverse events. The overall rates of adverse events observed in this trial (73–90%) are consistent with those observed in prior studies of patients with mild-to-moderate fibrosis due to NASH (71–89%).
the most frequent adverse events were gastrointestinal, in particular, nausea, diarrhoea, constipation and decreased appetite. The incidence of gastrointestinal events was similar in the semaglutide and combination groups, with the exception of higher rates of nausea and vomiting in the SEMA + FIR + CILO group, but this did not translate into a higher rate of discontinuations. The incidence of pruritus, an adverse event associated with both NASH and FXR agonist therapy,
was low, occurring in just 5 of 65 cilofexor-treated patients (7.7%). Importantly, all pruritus events were mild and none led to discontinuation of treatment.
Treatment with both FXR agonists and ACC inhibitors has been associated with increases in serum lipids, including LDL cholesterol and triglycerides, respectively.
Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial.
Previous studies have shown that ACC inhibitor-related hypertriglyceridaemia is greatest in patients with pre-existing dyslipidaemia and can be mitigated via the use of fibrates and/or fish oil.
Indeed, in the current trial, total cholesterol, LDL cholesterol and triglycerides decreased with semaglutide monotherapy. These benefits were generally reduced with the addition of cilofexor and/or firsocostat to semaglutide. Conversely, firsocostat-related hypertriglyceridaemia was relatively mitigated by the addition of semaglutide compared with data from prior studies.
Importantly, the trial duration was short (24 weeks) and the treatment period at the target 2.4 mg dose of semaglutide was limited; hence, the utility of longer durations of semaglutide to fully mitigate these lipid abnormalities requires additional study.
Based on exploratory efficacy analyses, combinations including cilofexor and/or firsocostat resulted in greater reductions in hepatic steatosis assessed by MRI-PDFF and CAP in comparison with semaglutide monotherapy. These beneficial effects were observed as early as 12 weeks after treatment initiation. Given that the 2.4 mg target dose of semaglutide was not reached until week 17 onwards, further reductions in hepatic steatosis may be expected with longer-term treatment, as was observed with semaglutide monotherapy between 24 and 48 weeks in a previous placebo-controlled trial in 67 patients with NAFLD.
Randomised clinical trial: semaglutide versus placebo reduced liver steatosis but not liver stiffness in subjects with non-alcoholic fatty liver disease assessed by magnetic resonance imaging.
In addition, more patients treated with combinations achieved reductions of ≥30% and ≥50% in MRI-PDFF compared with semaglutide. Eighty-six percent of patients treated with combinations had a ≥30% improvement in MRI-PDFF and 40% had normalisation of liver fat (<5%) after 24 weeks of treatment. These improvements are among the highest reported in a NASH trial and are of a magnitude that has been associated with an increased likelihood of histologic response, including NASH resolution and fibrosis improvement.
In addition, all combinations were associated with improvements in liver biochemistry that were similar or greater than those observed with semaglutide monotherapy. Significantly greater reductions in ALT were observed in all combination groups, and normalisation of ALT occurred in 86% to 100% of patients across these groups. Approximately 60% to 70% of patients had a ≥17 U/L reduction in ALT at week 24 (Table S4), a threshold that has been associated with histological improvement.
Moreover, reductions from baseline in CK-18 M30, a biomarker of hepatocellular apoptosis, as well as CRP, a biomarker of inflammation, were observed. These data are in keeping with prior observations regarding the beneficial effects of semaglutide monotherapy on necroinflammatory activity in NASH,
and suggest potential additive benefits of combinations including cilofexor and/or firsocostat.
While necroinflammatory activity is the key driver of fibrosis progression in NASH, fibrosis is the primary determinant of liver-related morbidity and mortality.
In this regard, beneficial effects on multiple NITs of fibrosis were observed with semaglutide alone and in combination with cilofexor and firsocostat. For example, ELF improvements were similar across all groups and ranged from 0.42 to 0.59 units. Similarly, improvements in liver stiffness by TE were observed in all groups, with a trend towards greater reductions in FIR-treated patients. Moreover, a higher proportion of patients in combination groups had a ≥25% relative reduction in liver stiffness by TE (Fig. 4B). While the trial lacked liver biopsy for histologic confirmation of anti-fibrotic effects, changes in ELF and liver stiffness by TE of these magnitudes have been associated with reduced rates of disease progression in patients with advanced fibrosis due to NASH.
Obeticholic acid improves experimental non-invasive markers of non-alcoholic steatohepatitis and advanced fibrosis: a secondary analysis of the phase 3 regenerate study.
in the combination groups vs. the semaglutide monotherapy group. Based on the ability of FAST to identify patients with NASH, with fibrosis (≥F2) and active necroinflammatory activity (NAS ≥4), these findings add support to the potential of these combination therapies.
Treatment with semaglutide, alone and in combination with cilofexor and/or firsocostat, also led to improvements in metabolic parameters, including body weight and glycaemic control. Body weight declined in all groups, with percentage weight loss of 7% to 9.6% across groups. This magnitude of weight loss has been associated with histological improvement in NASH.
Similar weight loss across the groups indicates that the greater reductions in liver fat, FAST, ALT and AST with combinations are not mediated by additional body weight reduction with cilofexor and/or firsocostat, and support the complementarity of FXR agonism and ACC inhibition to GLP-1 receptor agonism with semaglutide. The changes in body weight observed in this trial are consistent with previous observations regarding semaglutide in type 2 diabetes and obesity.
Efficacy and safety of once-weekly semaglutide monotherapy versus placebo in patients with type 2 diabetes (SUSTAIN 1): a double-blind, randomised, placebo-controlled, parallel-group, multinational, multicentre phase 3a trial.
In the phase II trial of semaglutide in NASH, a mean weight loss of 13% was observed after 72 weeks of therapy with the highest dose of semaglutide (0.4 mg once daily),
which is similar to the 2.4 mg weekly target dose utilised in the current trial. Based on these data, additional weight loss would be expected with longer-term semaglutide therapy, especially given that the target dose of semaglutide was not reached until week 17 of the trial. As expected, all groups experienced improvements in glycaemic parameters, including fasting plasma glucose and HbA1c, the latter ranging from –1.2 to –1.7% among patients with type 2 diabetes. Considering the increased cardiovascular risk characteristic of NASH, the impact of these improvements on cardiovascular events warrants evaluation in long-term studies.
This study has several limitations. First, the trial enrolled small numbers of patients across 5 treatment groups, was open-label, and lacked a placebo-controlled group. As such, comparisons of safety and efficacy across groups should be interpreted cautiously. Additionally, group sizes and baseline imbalances in select clinical parameters, potentially related to the potential to meet eligibility based on either historical liver biopsy or screening non-invasive parameters, yielded limited power for comparisons of efficacy. Second, patients were not required to follow specific dietary or lifestyle habits, nor were these monitored; thus, any contribution of lifestyle changes to efficacy assessments is unknown. Third, since the trial included patients with NASH and mild-to-moderate fibrosis, the relevance of these findings to patients with advanced fibrosis requires additional study. Fourth, the trial lacked histological assessment, although improvements in non-invasive parameters in this trial have been associated with a histologic response in prior studies.
Finally, the trial duration was short (24 weeks) and included a 16-week dose-escalation period for semaglutide. As NASH will likely require chronic therapy, longer duration trials are required to evaluate long-term safety.
In the previous ATLAS study, the combination of cilofexor and firsocostat was safe and well tolerated, and led to greater improvements in liver histology, liver stiffness, and the ELF score in patients with F3–F4 fibrosis.
In addition to established cardiovascular benefits in patients with type 2 diabetes, semaglutide as a monotherapy led to significantly greater rates of NASH resolution and improvements in NITs of fibrosis in a prior phase II trial in patients with NASH and F1–F3 fibrosis.
In patients with advanced fibrosis due to NASH, both hepatic and cardiovascular outcomes are relevant, supporting the rationale for further evaluation of combinations including semaglutide, which, in addition to the improvements seen in liver histology, has a known cardiovascular benefit.
In this phase II trial, semaglutide in combination with cilofexor and/or firsocostat was well tolerated in patients with mild-to-moderate fibrosis due to NASH. Combination treatments resulted in greater improvements in hepatic steatosis, liver biochemistry and several metabolic and hepatic biomarkers, including NITs of fibrosis, than achieved with semaglutide alone. Based on these data, a 72-week, phase IIb, randomised controlled trial that includes histologic endpoints is planned to further evaluate the efficacy and safety of combination treatments including semaglutide, cilofexor and firsocostat in patients with NASH.
This trial was funded by Gilead Sciences Inc., CA, USA, and Novo Nordisk A/S, Denmark.
Authors’ contributions
YZ, AB, LD, KB, MK, CB, RPM: data collection, data interpretation and manuscript development. NA, RSH, KH, ZK, TH, AK, RH, RL, MN: study conduct, data collection, data interpretation and manuscript development. All authors had access to study data, approved the final version of the manuscript, agreed to submission for publication, and vouch for data accuracy and fidelity to the protocol. NA and RSH have verified the data in the manuscript.
Data availability statement
Gilead shares anonymised Individual Patient Data (IPD) upon request or as required by law and/or regulation with qualified external researchers. Approval of such requests is at Gilead’s discretion and is dependent on the nature of the request, the merit of the research proposed, the availability of the data, and the intended use of the data. Data requests should be sent to [email protected]
Role of the funding source
The funders (Gilead Sciences, Inc., and Novo Nordisk A/S) designed the trial, monitored trial sites and collected and analysed data. The manuscript was drafted by a medical writer under the guidance of the authors. All authors had full access to all the data, actively contributed to all drafts of the manuscript and made the decision to submit the manuscript for publication.
Conflict of interest
NA: advisory board/review panel member for Echosens, Gilead, Intercept, Perspectum, Pfizer and Zydus; grant/research support from Akero, Allergan, Bristol Myers Squibb, Genentech, Gilead, Intercept, Madrigal, NGM Bio, Novo Nordisk, Pfizer, Viking and Zydus; speaker for AbbVie, Alexion, Echosens, Gilead and Intercept. TH: advisory board/review panel member and/or grant/research support from AbbVie, Bristol Myers Squibb, Gilead, Mallinckrodt, Merck and Organovo; speaker for AbbVie, Allergan, Amarex/Cytodyn, AstraZeneca, BeiGene, Boehringer Ingelheim, Bristol Myers Squibb, CARA, Cymabay, DURECT Corporation, Enanta, Galectin, GenFit, Gilead, Grifols, Intercept, Mallinckrodt, Merck, Mirum, Novartis, Novo Nordisk, Pfizer and Salix. AK: grant/research support from Gilead; speaker for Gilead, Intercept and Novartis. RSH, YZ, ANB, RPM: employees and shareholders of Gilead Sciences, Inc., LHD, KB, MSK, CB: employees and shareholders of Novo Nordisk A/S. RL: consultant for 89bio, Eli Lilly, Gilead, Glympse Bio, Ionis, Merck, Metacrine, NGM Bio, Novo Nordisk, Pfizer, Sagimet and Viking; grant/research support from AstraZeneca, Eli Lilly, Gilead, Intercept, Inventiva, Janssen, Madrigal, NGM Bio, Novartis and Novo Nordisk. MN: advisory committees or review panel member for Abbott, EchoSens, Intercept and OWL; grant/research support from Conatus, Galectin, Galmed, Genfit, Gilead, Shire and Zydus; speaker for Echosens, and holds stock in Anaeots. RH, HK and ZK: no interests to declare.
Please refer to the accompanying ICMJE disclosure forms for further details.
Acknowledgements
We thank the patients, investigators, trial site staff and all Gilead and Novo Nordisk employees involved in the trial. We also thank Andy Bond of Spirit Medical Communications Group Ltd, for medical writing and editorial assistance (funded by Gilead Sciences Inc. and Novo Nordisk A/S).
Supplementary data
The following are the supplementary data to this article:
Efficacy and safety of once-weekly semaglutide monotherapy versus placebo in patients with type 2 diabetes (SUSTAIN 1): a double-blind, randomised, placebo-controlled, parallel-group, multinational, multicentre phase 3a trial.
FibroScan-AST (FAST) score for the non-invasive identification of patients with non-alcoholic steatohepatitis with significant activity and fibrosis: a prospective derivation and global validation study.
Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial.
Randomised clinical trial: semaglutide versus placebo reduced liver steatosis but not liver stiffness in subjects with non-alcoholic fatty liver disease assessed by magnetic resonance imaging.
Obeticholic acid improves experimental non-invasive markers of non-alcoholic steatohepatitis and advanced fibrosis: a secondary analysis of the phase 3 regenerate study.
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