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Hepatic steatosis due to altered lipid metabolism and accumulation of hepatic triglycerides is a hallmark of nonalcoholic fatty liver disease (NAFLD). Diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, catalyze the terminal reaction in triglyceride synthesis, making them attractive targets for pharmacologic intervention. There is a common misconception that these enzymes are related; however, despite their similar names, DGAT1 and DGAT2 differ significantly on multiple levels. As we look ahead to future clinical studies of DGAT2 inhibitors in patients with NAFLD and nonalcoholic steatohepatitis (NASH), we review key differences and include evidence to highlight and support DGAT2 inhibitor (DGAT2i) pharmacology.
Methods
Three Phase I, randomized, double-blind, placebo-controlled trials assessed the safety, tolerability, and pharmacokinetic properties of the DGAT2i ervogastat (PF-06865571) in healthy adult participants (Single Dose Study to Assess the Safety, Tolerability and Pharmacokinetics of PF-06865571 [study C2541001] and Study to Assess the Safety, Tolerability, and Pharmacokinetics of Multiple Doses of PF-06865571 in Healthy, Including Overweight and Obese, Adult Subjects [study C2541002]) or participants with NAFLD (2-Week Study in People With Nonalcoholic Fatty Liver Disease [study C2541005]). Data from 2 Phase I, randomized, double-blind, placebo-controlled trials of the DGAT1i PF-04620110 in healthy participants (A Single Dose Study of PF-04620110 in Overweight and Obese, Otherwise Healthy Volunteers [study B0961001] and A Multiple Dose Study of PF-04620110 in Overweight and Obese, Otherwise Healthy Volunteers [study B0961002]) were included for comparison. Safety outcomes were the primary end point in all studies, except in study C2541005, in which safety was the secondary end point, with relative change from baseline in whole liver fat at day 15 assessed as the primary end point. Safety data were analyzed across studies by total daily dose of ervogastat (5, 15, 50, 100, 150, 500, 600, 1000, and 1500 mg) or PF-04620110 (0.3, 1, 3, 5, 7, 10, 14, and 21 mg), with placebo data pooled separately across ervogastat and PF-04620110 studies.
Findings
Published data indicate that DGAT1 and DGAT2 differ in multiple dimensions, including gene family, subcellular localization, substrate preference, and specificity, with unrelated pharmacologic inhibition properties and differing safety profiles. Although initial nonclinical studies suggested a potentially attractive therapeutic profile with DGAT1 inhibition, genetic and pharmacologic data suggest otherwise, with common gastrointestinal adverse events, including nausea, vomiting, and diarrhea, limiting further clinical development. Conversely, DGAT2 inhibition, although initially not pursued as aggressively as a potential target for pharmacologic intervention, has consistent efficacy in nonclinical studies, with reduced triglyceride synthesis accompanied by reduced expression of genes essential for de novo lipogenesis. In addition, early clinical data indicate antisteatotic effects with DGAT2i ervogastat, in participants with NAFLD, accompanied by a well-tolerated safety profile.
Implications
Although pharmacologic DGAT1is are limited by an adverse safety profile, data support use of DGAT2i as an effective and well-tolerated therapeutic strategy for patients with NAFLD, NASH, and NASH with liver fibrosis. ClinicalTrials.gov identifiers: NCT03092232, NCT03230383, NCT03513588, NCT00799006, and NCT00959426.
Predicting NAFLD prevalence in the United States using National Health and Nutrition Examination Survey 2017–2018 transient elastography data and application of machine learning.
The final step in triglyceride synthesis is catalyzed by acyl-coenzyme A (CoA):diacylglycerol acyltransferase (DGAT) enzymes, combining fatty acyl-CoA with diacylglycerol to yield triacylglycerol.
DGAT1 and DGAT2 differ significantly. In this narrative review, we discuss key safety, pharmacokinetic (PK), and pharmacologic data relating to the clinical investigation of DGAT2 inhibitor (DGAT2i) ervogastat (PF-06865571, Pfizer Inc, New York, New York), to complement discussion of the differences between DGAT1 and DGAT2, and the clinical implications of different pharmacology. Data relating to the clinical investigation of the DGAT1i PF-04620110 are included for comparison.
Methods
Clinical Investigation of the DGAT2i Ervogastat
Participants and Study Designs
Three Phase I, randomized, double-blind, placebo-controlled trials, Single Dose Study to Assess the Safety, Tolerability and Pharmacokinetics of PF-06865571 (study C2541001), Study to Assess the Safety, Tolerability, and Pharmacokinetics of Multiple Doses of PF-06865571 in Healthy, Including Overweight and Obese, Adult Subjects (study C2541002), and 2-Week Study in People With Nonalcoholic Fatty Liver Disease (study C2541005), assessed the safety, tolerability, and PK properties of the DGAT2i ervogastat (Table I). The effect of ervogastat on liver fat, as assessed by magnetic resonance imaging–proton density fat fraction (MRI-PDFF), versus placebo was also evaluated for the first time in participants with NAFLD enrolled in study C2541005.
Table IOverview of clinical study designs of 3 Phase I studies with the diacylglycerol acyltransferase 2 inhibitor ervogastat.
Three participants were randomized and received ervogastat in 2 studies (C2541001 and C2541002); however, they are counted only once (as part of repeated dose exposure) when summarizing unique participant experience.
Three participants were randomized and received ervogastat in 2 studies (C2541001 and C2541002); however, they are counted only once (as part of repeated dose exposure) when summarizing unique participant experience.
a Three participants were randomized and received ervogastat in 2 studies (C2541001 and C2541002); however, they are counted only once (as part of repeated dose exposure) when summarizing unique participant experience.
Studies C2541001 and C2541002 enrolled healthy participants (including obese and overweight participants in study C2541002) aged 18 to 55 years, with body mass indexes (BMIs) of 22.5 to 35.4 kg/m2 and total body weights >50 kg. Additionally, at screening, participants had a fasting LDL level ≤190 mg/dL (study C2541001) or >190 mg/dL (study C2541002) and a fasting triglyceride level ≤500 mg/dL (study C2541001) or >400 mg/dL (study C2541002) after an overnight fast of ≥10 hours. Participants were excluded if they had evidence or a history of any clinically relevant hematologic, renal, endocrine, pulmonary, gastrointestinal (GI), cardiovascular, hepatic, psychiatric, neurologic, or allergic disease, a history of regular alcohol consumption exceeding 7 or 14 drinks per week in females and 14 or 21 drinks per week in males (studies C2541001 and C2541002, respectively), supine blood pressure ≥140 mm Hg (systolic) or ≥90 mm Hg (diastolic) 6 months before screening, and aspartate aminotransferase or alanine aminotransferase levels greater than upper limit of normal (≥1.25 × upper limit of normal) at screening (study C2541002). Participants were also excluded if they had a history of HIV, hepatitis B, or hepatitis C or tested positive for HIV, hepatitis B surface antigen or core antibody, or hepatitis C antibody.
Study C2541005 enrolled participants with NAFLD aged 18 to 65 years with BMIs ≥28 kg/m2 and total body weights >50 kg at screening. Participants had to meet the following inclusion criteria: controlled attenuation parameter ≥260 dB/m via transient elastography at screens 1 and 2, and liver fat ≥6% assessed by MRI-PDFF at screen 3. Participants were excluded from this study if they had an estimated glomerular filtration rate <60 mL/min/1.73 m2 or evidence or diagnosis of other forms of chronic liver disease, including, but not limited to, alcoholic liver disease, HIV, or hepatitis B or C determined by the presence of antibodies, surface antigen or core antibody, or by antibody and RNA, respectively, at screen 1. Participants with type 1 diabetes mellitus, type 2 diabetes mellitus that was pharmacologically managed, and those with a recent history of congestive heart failure, unstable angina, myocardial infarction, stroke, or transient ischemic attack in the 6 months before screen 1 were also excluded.
Details of the study design and dosing are outlined in Table I. Briefly, study C2541001 was a single-ascending oral dose, 4-period, crossover study with placebo substitution. In each period, approximately 6 participants were planned to receive ervogastat and 2 participants to receive placebo. Each participant received placebo plus up to 3 of 7 single-ascending doses of ervogastat (5, 15, 50, 150, 500 1000, and 1500 mg), with random insertion of placebo and a ≥14-day washout interval between dosing.
Study C2541002 was a sequential, multiple-ascending oral dose study. Participants were randomly assigned to 1 of 5 cohorts of ervogastat (30, 100, 240, 400, and 600 mg) or matching placebo in a 4:1 ratio and were administered the study drug q8h with breakfast or snacks for 14 consecutive days during inpatient stay. After discharge, participants returned for an on-site follow-up visit 7 to 10 days, and received a follow-up telephone call 28 to 35 days, after the last dose of study drug.
Study C2541005 was a 3-arm, placebo-controlled, parallel-group study. Participants were randomly assigned to 1 of 2 ervogastat dosing regimens (50 or 300 mg q12h) or placebo in a 1:1:1 ratio and were administered the study drug during a 14-day inpatient stay. Participants were discharged from the clinical research unit 2 days after dosing was completed on day 14 and returned for a follow-up visit 7 to 10 days after the last dose of the study drug. Participants also received a follow-up telephone call 28 to 35 days after the last dose of the study drug.
The primary end point in studies C2541001 and C2541002 was safety, with plasma PK analysis and evaluation of pharmacology as secondary and tertiary end points, respectively. The primary end point in study C2541005 was the relative change from baseline in whole liver fat at day 15, as assessed by MRI-PDFF, and safety was assessed as a secondary end point. All studies were conducted in accordance with the International Conference on Harmonisation Guideline for Good Clinical Practice and the ethical principles of the Declaration of Helsinki and were registered on ClinicalTrials.gov (NCT03092232, NCT03230383 and NCT03513588). All participants provided written informed consent.
Safety Assessments
The frequency and severity of treatment-emergent AEs (TEAEs) were reported from the first dose of study drug to the last follow-up in all 3 studies. Safety data are presented by ascending ervogastat total daily dose, with placebo data pooled across all 3 studies. TEAEs are reported by preferred term using Medical Dictionary for Regulatory Activities version 23.1 coding.
Pharmacokinetics
Blood samples for analysis of ervogastat plasma concentrations were collected at the following time points in each study: day 1 before dosing and 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 36, and 48 hours after dosing (study C2541001); days 2, 4, 8, and 10 before dosing, days 15 and 16 before dosing (24, 30, 36, and 48 hours after the last morning dose on day 14), and before dosing and at 1, 2, 3, 4, 6, 8, and 12 hours after dosing on days 1, 7, and 14 (study C2541002); and before dosing on days 4, 8, and 15, before dosing on day 14, and 1, 2, 3, 4, 6, 8, and 12 hours after dosing (study C2541005). Geometric mean values for Cmax, AUC0–τ, AUC0–∞, and Tmax were calculated using standard noncompartmental methods.
Assessment of Pharmacology
Serum triglycerides were evaluated as tertiary end points in healthy participants and in those with NAFLD to assess pharmacology or target engagement. In study C2541002, blood samples were collected from healthy participants after a ≥10-hour fast at screening, at follow-up visits 21 to 24 days after the last dose of study drug, and after a ≥7.25-hour fast before dosing at 0 hours on days 2, 4, 8, 10, and 15. The percent change from baseline in fasting serum triglycerides at day 14 was calculated, along with the percent change from baseline in AUC0–18 for postprandial triglycerides at day 14. In study C2541005, blood samples for serum triglycerides were collected before dosing at day 15. Serial blood samples were also collected before the first dose on day 1 and at 2, 4, 6, 8, 10, 12, 14, 16, and 18 hours after the first dose on days –1, 1, and 14. The percent change from baseline in fasting serum triglycerides at day 14 was calculated, along with the AUC0–24 for postprandial serum triglycerides.
The relative change from baseline in liver fat at day 15 was assessed by the MRI-PDFF in participants with NAFLD. The MRI-PDFF assessment was performed at screening, baseline (day –2) and day 15. Images were acquired by trained personnel, and image analysis was performed at a sponsor-identified central imaging vendor. Where possible, analysis was performed by the same reader, who was blinded to individual participants’ clinical data and treatment assignment.
Clinical Investigation of the DGAT1i PF-04620110
Two Phase I, randomized, double-blind, placebo-controlled trials (A Single Dose Study of PF-04620110 in Overweight and Obese, Otherwise Healthy Volunteers and A Multiple Dose Study of PF-04620110 in Overweight and Obese, Otherwise Healthy Volunteers) assessed the safety, tolerability, PK, and pharmacology of the DGAT1i PF-04620110 (Supplemental Table I). These studies were included for comparison with results of the DGAT2i C2541001, C2541002, and C2541005 studies. Full details of the DGAT1i study design and data analysis are included in the Supplemental Methods.
Statistical Analysis
In all studies, assessment of safety and pharmacology included all participants who received at least 1 dose of the randomized study drug. The AUCs for serum triglycerides at the prespecified intervals were calculated using the linear trapezoidal rule in any participant, provided at least the first, last, and 75% of the total number of planned samples within the given interval were available for analysis.
In study C2541002, placebo-adjusted treatment group means were calculated for fasting serum triglycerides using the mixed model for repeated measures (MMRM) of natural log-transformed changes from baseline with treatment, time, and treatment × time interaction as fixed effects. Natural log-transformed baseline value was included as a covariate. Results from the model were exponentiated to express as geometric means with associated CIs. Baseline was defined as the measurement taken on day –1. Percent changes from the placebo group in AUC0–18 for serum triglycerides were calculated using ANCOVA on log-transformed change from baseline with treatment as a fixed effect and log-transformed baseline value as a covariate.
In study C2541005, percent change from baseline in fasting triglycerides was analyzed using MMRM with treatment, study day, baseline diabetic status, and treatment × study day interaction as fixed effects and baseline value and baseline whole liver PDFF as covariates. No adjustments for multiple comparisons were made. Baseline was defined as the closest measurement before the first dose on day 1. The relative changes in AUC0–24 for serum triglycerides from the placebo group were calculated using ANCOVA on log-transformed relative change from baseline with treatment and baseline diabetic status as factors and natural log-transformed baseline value and whole liver PDFF value as covariates. Liver fat assessed by MRI-PDFF at day 15 was analyzed using ANCOVA on natural log-transformed relative change from baseline with treatment as a fixed effect and natural log-transformed baseline liver fat by MRI-PDFF as a covariate. Results from the model were exponentiated to express as geometric means with associated CIs.
Results and discussion
DGAT1 and DGAT2 Differ Genetically and Structurally and Have Unique Patterns of Tissue Expression
A molecular overview of DGAT1 and DGAT2 is presented in Figure 1. The genes encoding human and murine DGAT1 and DGAT2 were cloned in 1998 and 2001, respectively.
Topological orientation of acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) and identification of a putative active site histidine and the role of the n terminus in dimer/tetramer formation.
Recent analysis of the human DGAT1 protein structure using cryoelectron microscopy revealed a dimer, with the N-terminal region on the cytosolic side of the endoplasmic reticulum membrane and the C-terminal region on the luminal side.
Topological orientation of acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) and identification of a putative active site histidine and the role of the n terminus in dimer/tetramer formation.
Murine diacylglycerol acyltransferase-2 (DGAT2) can catalyze triacylglycerol synthesis and promote lipid droplet formation independent of its localization to the endoplasmic reticulum.
The endoplasmic reticulum enzyme DGAT2 is found in mitochondria-associated membranes and has a mitochondrial targeting signal that promotes its association with mitochondria.
Specifically, DGAT1 appears to show a preference for exogenous fatty acids for reesterification of diacylglycerides from lipase-mediated hydrolysis of triglycerides, whereas evidence suggests that DGAT2 has a specific role in incorporating de novo synthesized endogenous fatty acids, in addition to exogenous fatty acids, for triacylglycerol synthesis.
Diacylglycerol acyltransferase 2 acts upstream of diacylglycerol acyltransferase 1 and utilizes nascent diglycerides and de novo synthesized fatty acids in HepG2 cells.
DGAT2 is also associated with lipid droplets, where it functions (together with other enzymes involved in triglyceride biosynthesis) to promote local de novo lipogenesis, leading to lipid droplet expansion and very low-density lipoprotein maturation.
These differences in fatty acid metabolism, particularly the activity of DGAT2, may have implications for therapeutic development, especially in NAFLD and nonalcoholic steatohepatitis (NASH) with liver fibrosis, where a hallmark feature is increased de novo lipogenesis.
Figure 1Molecular overview of diacylglycerol acyltransferase (DGAT) 1 and 2 and effects of inhibition on hepatic lipid metabolism. The DGAT1 cryoelectron microscopy structure (Protein Data Bank Identifier: 6VYI, top left) shows 9 transmembrane helices with the proposed active site within the endoplasmic reticulum (ER) membrane, containing the invariant His415, and highly conserved polar residues, including Asn378, Gln437, and Gln465 (magenta). The DGAT2 structure (top right) as predicted by AlphaFold (AlphaFoldProtein Structure Database) reveals 7 membrane β-barrel motifs and multiple α-helices. Helices of residues 65 to 88 and 92 to 115 are predicted to be transmembrane. The structurally distinct active site resides in the cytoplasm and comprises the invariant His163 and nearby polar residues, Thr194, Glu243, and Ser244 (magenta). DGAT1 and DGAT2 differ on multiple levels in addition to protein structure, including at the genetic level, in terms of localization, tissue expression, and function, with unique effects on hepatic lipid metabolism that impact their therapeutic potential. ACC = acetyl-coenzyme A carboxylase; ACS = acyl-coenzyme A synthetase; AEs = adverse events; CoA = coenzyme A; DAG = diacylglycerol; DAGAT = diacylglycerol acyltransferase; DGAT1i = DGAT1 inhibitor; DGAT2i = DGAT2 inhibitor; FAS = fatty acid synthase; FFA = free fatty acid; G-3-P = glycerol-3-phosphate; GI = gastrointestinal; MBOAT = membrane-bound O-acyltransferase; MUFA = monounsaturated fatty acid; SCD1 = stearoyl-CoA desaturase 1; SREBP1/2 = sterol regulatory element binding protein 1/2; TAG = triacylglycerol (also known as triglyceride); TG = triglyceride.
The 2 genes also have differences in tissue expression. DGAT1 is expressed in a variety of human tissues, including the testes, mammary gland, adipose tissue, liver, and skin, with the highest expression noted in the small intestine,
whereas the highest DGAT2 expression in human tissues includes the liver and white adipose tissue, with lower levels noted in the testes, mammary gland, peripheral leukocytes, and very low expression in the small intestine.
Nonclinical Data Indicate DGAT1 and DGAT2 Are Functionally Distinct
The functional consequences of the differences in tissue expression, along with protein structure and function, have been seen through genetic ablation of DGAT1 and DGAT2. DGAT1 knockout mice are viable and show approximately 80% reduction in hepatic triglycerides when fed a high-fat diet,
whereas genetic knockout of DGAT2 in mice is lethal soon after birth, with pups dying of skin barrier defects linked to inhibition of de novo lipogenesis,
Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.
Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
whereas hepatic targeting of DGAT1 produces reductions in liver triglycerides in animal models in which exogenous fatty acids are supplied to the liver.
ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.
Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.
Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
In addition, the effects of DGAT2 inhibition were accompanied by reduction in hepatic mRNA levels of lipogenic genes and decreases in protein targets of sterol regulatory element binding protein, consistent with reduced de novo lipogenesis.
Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
Although most nonclinical data support the therapeutic benefits of DGAT2 inhibition, 1 study of DGAT2 inhibition through use of an antisense oligonucleotide in a progressive obesity-related model of NAFLD in diabetic (db/db) mice found decreased hepatic steatosis, with accumulation of potentially toxic liver precursors and markers of oxidative stress.
Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.
Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
Clinical Data Support Therapeutic Benefits of DGAT2 Inhibition
Although nonclinical data suggest a potentially attractive therapeutic profile with DGAT1 inhibition, human genetic and pharmacologic data suggest otherwise. Mutations identified in human DGAT1 have been linked to congenital diarrhea.
Similarly, in multiple clinical trials with small-molecule DGAT1 inhibitors, GI-related adverse events (AEs), including nausea, vomiting, and diarrhea, were common,
Diacylglycerol acyltransferase 1 inhibition with AZD7687 alters lipid handling and hormone secretion in the gut with intolerable side effects: a randomized clinical trial.
ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.
recapitulating the directionality and magnitude of the change in HDL-C observed in individuals with a heterozygous loss-of-function mutation (Tyr285X) in human DGAT2.
ranging from simple steatosis in the absence of inflammation and hepatocellular damage to NASH, which is estimated to affect 3% to 5% of the general population.
Liver-specific and overall mortality rates are numerically lower in those with NAFLD (0.77 and 15.44 per 1000 person-years, respectively) compared with NASH (11.77 and 25.56 per 1000 person-years, respectively).
The risk of mortality is linked to fibrosis stage, with an exponential increase in liver-related mortality observed with increasing fibrosis stage in a meta-analysis of 5 adult NAFLD cohort studies.
Targeted therapies aimed at modulating the molecular pathways underlying the early pathogenesis of NAFLD could prevent progression and offer potential treatments for patients with NASH and liver fibrosis.
Diacylglycerol acyltransferase 2 acts upstream of diacylglycerol acyltransferase 1 and utilizes nascent diglycerides and de novo synthesized fatty acids in HepG2 cells.
Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
In Vitro and In Vivo Characterization of Ervogastat (PF-06865571): An Orally Bioavailable, Highly Selective DGAT2 Inhibitor
Ervogastat (PF-06865571; Pfizer Inc) is a potent and selective oral, small-molecule DGAT2i being developed for the treatment of NASH with liver fibrosis and currently being evaluated in an ongoing Phase II study.
Efficacy and safety of an orally administered DGAT2 inhibitor alone or coadministered with a liver-targeted ACC inhibitor in adults with nonalcoholic steatohepatitis (NASH): rationale and design of the phase II, dose-ranging, dose-finding, randomised, placebo-controlled MIRNA (Metabolic Interventions to Resolve NASH with fibrosis) study.
In biochemical assays, ervogastat inhibited human and rat DGAT2 with a half maximal inhibitory concentration (IC50) of 17.2 and 833 nM, respectively. In primary human and rat hepatocytes, ervogastat also inhibited triglyceride synthesis with an IC50 of 2.8 and 6.0 nM, respectively.
Discovery of ervogastat (PF-06865571): a potent and selective inhibitor of diacylglycerol acyltransferase 2 for the treatment of non-alcoholic steatohepatitis.
Conversely, IC50 values for ervogastat against DGAT1, MGAT1, MGAT2, and MGAT3 were all >50,000 nM (the maximum concentration of ervogastat used in these assays),
Discovery of ervogastat (PF-06865571): a potent and selective inhibitor of diacylglycerol acyltransferase 2 for the treatment of non-alcoholic steatohepatitis.
indicating greater than 2000-fold selectivity for human DGAT2 over other acyltransferases.
In pharmacodynamic studies, a single oral dose of ervogastat induced a dose-dependent reduction in circulating triglycerides in rats fed with a sucrose diet, and in longer, multiple-dose studies in Western diet-fed rats, ervogastat administered twice daily for 7 days reduced both plasma and hepatic triglycerides (Supplemental Table II). In addition to reductions in hepatic and plasma triglycerides and consistent with previous observations in studies with antisense oligonucleotide inhibition, hepatic knockout, and another small molecule inhibitor of DGAT2,
Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
administration of ervogastat was associated with reduced hepatic expression of multiple sterol regulatory element binding protein-regulated lipid biosynthetic genes.
ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.
Clinically, the DGAT2i Ervogastat Is Well Tolerated With Potent Antisteatotic Effect
Participants
Three Phase I studies investigated the safety and tolerability of the DGAT2i ervogastat (Table I). The disposition of participants in all 3 DGAT2i studies is shown in Figure 2. Briefly, study C2541001 randomly allocated and dosed 17 healthy participants, with 15 (88.2%) completing the study (2 discontinued due to AEs). At baseline, the mean (SD) age of participants in this study was 42.4 (10.3) years, and the mean (SD) BMI was 27.9 (3.2) kg/m2. Participants were mostly male (15 of 17 [88.2%]) and either white (9 of 17 [52.9%]) or black (8 of 17 [47.1%]).
Figure 2Participant disposition in 3 Phase I trials of the diacylglycerol acyltransferase 2 inhibitor (DGAT2i) ervogastat. (A) study C2541001; (B) study C2541002; (C) study C2541005. Q8H = once every 8 hours; Q12H = once every 12 hours.
Study C2541002 randomized and dosed a total of 60 healthy (including obese and overweight) participants, with 49 (81.7%) completing the study. Eleven participants discontinued, of whom 9 received ervogastat and 2 received placebo. Two participants who received ervogastat 600 mg discontinued due to treatment-related AEs: 1 case of generalized pruritus after 9 days of dosing and 1 case of a serious AE of atrial fibrillation after 7 days of dosing. Both participants recovered after discontinued use of the study drug. Seven other participants who received ervogastat 600 mg had dosing suspended while the case of atrial fibrillation was thoroughly investigated. Most participants in this study were male (56 of 60 [93.3%]) and either white (29 of 60 [48.3%]) or black (28 of 60 [46.7%]). Among males and females, respectively, mean (SD) age was 37.4 (7.9) and 53.0 (1.4) years, and BMI was 28.3 (2.7) and 28.5 (3.7) kg/m2.
Study C2541005 randomized and dosed 48 participants with NAFLD. Of these, 45 (93.8%) completed 14 days of dosing and 43 (89.6%) completed the study. No participant discontinued due to AEs. Two participants died during the follow-up phase due to causes assessed as unrelated to the study drug; 1 participant who was dosed with ervogastat 600 mg died due to multidrug toxicity, including cocaine, fentanyl, and acetyl fentanyl, which occurred 3 days after the 14-day dosing period, and another who received placebo died >28 days after the last dose due to unintentional opiate overdose. Most participants in this study were male (35 of 48 [72.9%]) and white (36 of 48 [75.0%]), with a mean (SD) age of 47.0 (8.2) years and BMI of 35.6 (5.4) kg/m2. See Supplemental Table III for full participant demographic and baseline characteristics.
Pharmacokinetics
In study C2541001, ervogastat was notable for rapid absorption after single oral doses under fed conditions in healthy participants, with a median Tmax of 1.5 to 2 hours at low doses (5–50 mg), delayed to 3 hours in the 150- and 500-mg groups and 4 hours in the 1000- and 1500-mg groups. Geometric mean Cmax and AUC0–∞ increased in a dose-proportional manner from 17.87 to 5356 ng/mL and 64.69 to 39,040 ng·h/mL, respectively, at doses of 5 to 1500 mg. Following attainment of Cmax, the concentration decreased, with a mean t1/2 of 1.45 to 5.22 hours.
With repeated oral doses of ervogastat 30 to 600 mg q8h (ie, 90–1800 mg/d in study C2541002), mean Cmax on days 1, 7, and 14 was achieved with a median Tmax of 1.5 to 3.0 hours, and plasma concentrations reached steady state by day 4. Plasma exposure increased in a dose-proportional manner as shown by geometric mean AUC0–τ and Cmax following a single dose on day 1 and at steady state (days 7 and 14). Mean t1/2 on day 14 ranged from 3.29 to 6.92 hours across the dose range (Supplemental Table IV), with longer t1/2 at higher doses. Similarly, after 14 days of oral dosing with ervogastat 50 and 300 mg q12h, in participants with NAFLD ervogastat was rapidly absorbed with a median Tmax of 2 hours after dosing. Geometric mean Cmax and AUC0–τ increased in an approximately dose-related manner, and interparticipant variability for ervogastat exposure based on geometric %CV ranged from 28% to 35% for Cmax and 26% to 35% for AUC0–τ (Supplemental Table IV). PK data from 2 separate clinical studies of the DGAT1i PF-04620110 in otherwise healthy, overweight, or obese adults are included in the Supplemental Results for comparison.
Safety Assessments
Clinical studies in healthy adult participants found that single oral doses of ervogastat up to 1500 mg and repeated total daily doses of up to 1800 mg were well tolerated. The maximum tolerated dose was not identified. Analysis of safety data across the 3 studies revealed that total daily doses of ervogastat 5 to 1500 mg were well tolerated in healthy adults, including overweight or obese participants, and in those with NAFLD. A breakdown of all-cause TEAEs by ervogastat total daily dose administered is shown in Supplemental Table V. Although the number of TEAEs was higher than placebo in the ervogastat 100-, 500-, 600-, and 1500-mg dose groups (range, 40.0%–70.6% vs 29.5% in participants who received placebo), most were mild across all dose groups. The most frequently reported TEAEs were headache in 8 participants (6.8%) and diarrhea in 7 participants (5.9%), and abdominal pain, fatigue, injection site bruising, pollakiuria, and pruritis in 3 participants (2.5%) each. There was no dose-related trend in the frequency of TEAEs in general, or GI-related TEAEs specifically (Figure 3 and Supplemental Table V). In addition, there have been no clinically significant adverse trends observed in electrocardiogram data or in vital signs with ervogastat administration.
Figure 3Comparison of the incidence of the most common gastrointestinal-related adverse events by total daily dose in the diacylglycerol acyltransferase 2 inhibitor (DGAT2i) ervogastat and diacylglycerol acyltransferase 1 inhibitor (DGAT1i) PF-04620110 studies.
In contrast, a separate analysis of safety data across 2 Phase I studies of the DGAT1i PF-04620110 revealed a high frequency of GI-related AEs (range, 0.0%–88.9%) (Figure 3 and Supplemental Table VI), consistent with the results of clinical trials of other small-molecule DGAT1i
Diacylglycerol acyltransferase 1 inhibition with AZD7687 alters lipid handling and hormone secretion in the gut with intolerable side effects: a randomized clinical trial.
The frequency of GI-related TEAEs (range, 50.0%–88.9%) was higher than placebo at all PF-04620110 total daily doses ≥3 mg, with the highest proportion reported in those administered the highest dose (21 mg). Diarrhea was the most common GI-related TEAE, reported by 55 participants (38.5%), and was generally reported in >50% of participants at doses ≥3 mg. Other frequently reported GI-related TEAEs were nausea (35 [24.5%]), flatulence (14 [9.8%]), vomiting (13 [9.1%]), and abdominal pain (13 [9.1%]).
Assessment of Pharmacology
Evidence of DGAT2i ervogastat pharmacology was observed across the 3 clinical studies. In healthy participants, a general reduction in fasting serum triglycerides was observed with repeated, escalating oral doses of ervogastat 30, 240, 400, and 600 mg q8h versus placebo (range of mean percent change from baseline vs placebo, –8.2% to –29.7% [MMRM]) (Table II). On day 14, mean percent changes from baseline in AUC0−18 for postprandial serum triglycerides in the ervogastat groups were reduced versus placebo, but there was no consistent dose-related trend across the groups.
Table IIEffect of the diacylglycerol acyltransferase 2 inhibitor ervogastat on serum triglycerides at day 14 in healthy participants in study C2541002.
Placebo (n = 10)
Ervogastat dose q8h
30 mg (n = 8)
100 mg (n = 8)
240 mg (n = 8)
400 mg (n = 8)
600 mg (n = 7)
Difference from baseline in log-transformed fasting serum triglycerides, mg/dL
Difference in natural log-transformed fasting serum triglycerides was analyzed using mixed model for repeated measures with treatment, study day, and treatment × study day as fixed factors and natural log-transformed baseline value as a covariate. Results from the model were exponentiated to express as geometric means with associated CIs.
Adjusted geometric mean percent change from baseline
Day 14 to day –1 difference in natural log-transformed AUC was analyzed using ANCOVA with treatment as a fixed effect and natural log-transformed baseline AUC as a covariate. Results from the model were exponentiated to express as geometric means with associated CIs.
Adjusted geometric mean percent change from baseline
Baseline was defined as the measurement collected on day –1.
(90% CI)
6.26 (–6.48 to 20.74)
–11.42 (–22.68 to 1.48)
0.56 (–12.13 to 15.07)
–22.46 (–32.31 to –11.18)
–18.87 (–29.19 to –7.05)
–5.48 (–18.80 to 10.02)
Ratio ervogastat/placebo (90% CI)
–
–16.64 (–30.52 to 0.02)
–5.37 (–21.26 to 13.73)
–27.03 (–39.69 to –11.72)
–23.65 (–36.93 to –7.57)
–11.05 (–27.80 to 9.59)
P value
–
0.10
0.62
0.008
0.02
0.35
a Difference in natural log-transformed fasting serum triglycerides was analyzed using mixed model for repeated measures with treatment, study day, and treatment × study day as fixed factors and natural log-transformed baseline value as a covariate. Results from the model were exponentiated to express as geometric means with associated CIs.
b Baseline was defined as the measurement collected on day –1.
c Day 14 to day –1 difference in natural log-transformed AUC was analyzed using ANCOVA with treatment as a fixed effect and natural log-transformed baseline AUC as a covariate. Results from the model were exponentiated to express as geometric means with associated CIs.
In participants with NAFLD (study C2541005), ervogastat 50 and 300 mg q12h administered for 14 days resulted in robust dose-dependent reductions in whole liver fat assessed by MRI-PDFF (primary end point) (Figure 4A). Relative changes from baseline of –24.3% (80% CI, –31.3 to –16.7) for ervogastat 50 mg q12h and –33.9% (80% CI, –39.8 to –27.5) for ervogastat 300 mg q12h versus placebo were observed.
Figure 4Ervogastat target engagement in participants with nonalcoholic fatty liver disease (study C2541005). (A) Percent change from baseline in whole liver fat assessed by magnetic resonance imaging–proton density fat fraction at day 15 (primary end point). (B) Percent change from baseline in AUC0−24 for serum triglycerides at day 14. Boxes show the median and 25th and 75th percentiles with whiskers to the last point within 1.5 times the interquartile range. The star represents the arithmetic mean and circles represent the individual values.
Additionally, reductions from baseline in fasting serum triglycerides, albeit not dose dependent, were observed at day 14 with ervogastat 50 and 300 mg q12h compared with placebo. The least-squares mean differences in fasting serum triglycerides were –15.7% (80% CI, –22.4 to –9.1) for ervogastat 50 mg q12h and –17.7% (80% CI, –24.2 to –11.1) for ervogastat 300 mg q12h compared with placebo. Reductions from baseline in AUC0−24 for postprandial serum triglycerides at day 14 were also observed with similar reductions at both dose levels compared with placebo (Figure 4B). The least-squares mean changes in AUC0−24 for serum triglycerides were –25.8% (80% CI, –32.3 to –18.6) for ervogastat 50 mg q12h and –20.8% (80% CI, –27.4 to –13.5) for ervogastat 300 mg q12h compared with placebo. For comparison, no consistent dose-related effects of DGAT1i pharmacology were noted in studies of DGAT1i PF-04620110. See Supplemental Results and Supplemental Table VII for further details.
Conclusions
Although similar in name due to their metabolic roles, DGAT1 and DGAT2 are not linked in their structure, tissue expression, or function. DGAT1 and DGAT2 enzymes work through different pathways with differing substrate preference and subcellular localization, and nonclinical and clinical inhibition data generated by genetic knockout, targeted repression with antisense oligonucleotides, or systemic inhibition with small molecules strongly support DGAT1 and DGAT2 being unrelated enzymes. Similar to the effect of statins on reduction of LDL-C,
Atherogenic lipoprotein subfractions determined by ion mobility and first cardiovascular events after random allocation to high-intensity statin or placebo: the Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial.
ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.
AEs seen with DGAT1i generally differ from those observed with DGAT2i, and there is no biological rationale to expect these to have similar tolerability profiles. DGAT2 inhibition with ervogastat appears to be well tolerated in healthy adults and in those with NAFLD, with robust effects on liver fat and serum triglycerides in adults with NAFLD. Furthermore, coadministration of ervogastat with the acetyl-CoA carboxylase inhibitor clesacostat effectively mitigated the undesirable acetyl-CoA carboxylase inhibitor-induced elevations in serum triglycerides that have limited this class as a monotherapy, with potent antisteatotic effects that may deliver greater clinical benefit than acetyl-CoA carboxylase inhibitor or DGAT2i alone.
ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.
The efficacy and safety of ervogastat and ervogastat plus clesacostat and the effects on histologic end points for NASH and fibrosis resolution are being further investigated in an ongoing 1-year trial in patients with biopsy-proven NASH (NCT04321031).
Efficacy and safety of an orally administered DGAT2 inhibitor alone or coadministered with a liver-targeted ACC inhibitor in adults with nonalcoholic steatohepatitis (NASH): rationale and design of the phase II, dose-ranging, dose-finding, randomised, placebo-controlled MIRNA (Metabolic Interventions to Resolve NASH with fibrosis) study.
All authors are employees and shareholders of Pfizer Inc. The authors have indicated that they have no other conflicts of interest regarding the content of this article.
Data Sharing Statement
Upon request, and subject to review, Pfizer will provide the data that support the findings of this study. Subject to certain criteria, conditions and exceptions, Pfizer may also provide access to the related individual de-identified participant data. See https://www.pfizer.com/science/clinical-trials/trial-data-and-results for more information.
Acknowledgments
Medical writing support, under the direction of the authors, was provided by Claire Cairney, PhD, CMC Connect, a division of IPG Health Medical Communications, and was funded by Pfizer Inc, New York, New York, in accordance with Good Publication Practice (GPP 2022) guidelines. We thank the participants and investigators from all contributing sites, Collin Crowley and Sylvie Perez for their contributions to the nonclinical characterization of ervogastat, Sylvie Perez for the nonclinical data characterization of PF-04620110 before this compound was progressed into the clinic, and Meihua Tu for computational modeling support. We also thank Robert Farese and Tobias Walther for their expert opinions and constructive input into the development of this manuscript. All authors contributed to manuscript development, critically reviewed the manuscript at each stage, and approved the final version for submission. Neeta B. Amin led the design and conduct of all PF-06427878 clinical studies, from which the design of all ervogastat clinical studies was mirrored, and oversaw the conduct of all PF-04620110 clinical studies presented. Aditi R. Saxena led the conception, design, conduct, and data interpretation for all ervogastat clinical studies presented, as well as contributing to the conduct of all PF-06427878 clinical studies. Veena Somayaji contributed to the conception, design, data and statistical analysis, and data interpretation for all ervogastat clinical studies presented. Robert Dullea contributed to the design and conduct of nonclinical studies, as well as overseeing the ervogastat program through completion of the three clinical studies presented.
Funding Sources
The Single Dose Study to Assess the Safety, Tolerability and Pharmacokinetics of PF-06865571 (study C2541001), Study to Assess the Safety, Tolerability, and Pharmacokinetics of Multiple Doses of PF-06865571 in Healthy, Including Overweight and Obese, Adult Subjects (study C2541002), 2-Week Study in People With Nonalcoholic Fatty Liver Disease (study C2541005), A Single Dose Study of PF-04620110 in Overweight and Obese, Otherwise Healthy Volunteers (study B0961001), and A Multiple Dose Study of PF-04620110 in Overweight and Obese, Otherwise Healthy Volunteers (study B0961002) were sponsored by Pfizer Inc.
Predicting NAFLD prevalence in the United States using National Health and Nutrition Examination Survey 2017–2018 transient elastography data and application of machine learning.
Topological orientation of acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) and identification of a putative active site histidine and the role of the n terminus in dimer/tetramer formation.
Murine diacylglycerol acyltransferase-2 (DGAT2) can catalyze triacylglycerol synthesis and promote lipid droplet formation independent of its localization to the endoplasmic reticulum.
The endoplasmic reticulum enzyme DGAT2 is found in mitochondria-associated membranes and has a mitochondrial targeting signal that promotes its association with mitochondria.
Diacylglycerol acyltransferase 2 acts upstream of diacylglycerol acyltransferase 1 and utilizes nascent diglycerides and de novo synthesized fatty acids in HepG2 cells.
Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.
Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
Diacylglycerol acyltransferase 1 inhibition with AZD7687 alters lipid handling and hormone secretion in the gut with intolerable side effects: a randomized clinical trial.
Efficacy and safety of an orally administered DGAT2 inhibitor alone or coadministered with a liver-targeted ACC inhibitor in adults with nonalcoholic steatohepatitis (NASH): rationale and design of the phase II, dose-ranging, dose-finding, randomised, placebo-controlled MIRNA (Metabolic Interventions to Resolve NASH with fibrosis) study.
Discovery of ervogastat (PF-06865571): a potent and selective inhibitor of diacylglycerol acyltransferase 2 for the treatment of non-alcoholic steatohepatitis.
Atherogenic lipoprotein subfractions determined by ion mobility and first cardiovascular events after random allocation to high-intensity statin or placebo: the Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial.
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