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Inhibition of Diacylglycerol Acyltransferase 2 Versus Diacylglycerol Acyltransferase 1: Potential Therapeutic Implications of Pharmacology

Open AccessPublished:January 21, 2023DOI:https://doi.org/10.1016/j.clinthera.2022.12.008

      Abstract

      Purpose

      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.

      Key words

      Introduction

      Nonalcoholic fatty liver disease (NAFLD) is a common condition that affects an estimated 25% to 34% of the general US population.
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      Association between noninvasive fibrosis markers and mortality among adults with nonalcoholic fatty liver disease in the United States.
      NAFLD is characterized by chronic lipid accumulation in the liver (hepatic steatosis)
      • Lambert JE
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      Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease.
      ,
      • Chalasani N
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      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      due to altered lipid metabolism, leading to accumulation of hepatic triglycerides.
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      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.
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      Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.
      Two DGAT enzymes, DGAT1 and DGAT2, have been characterized in mammals.
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      Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.
      ,
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      • Zhou P
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      Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members.
      Despite their similar names and data suggesting that each are able to compensate for the loss of the other in triglyceride synthesis,
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      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.
      Study number (NCT number) [sample size]DesignErvogastat doseParticipants exposed to ervogastatDuration of dosing
      Phase I studies in healthy adult participants
      C2541001 (NCT03092232)

      [N = 17
      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.
      ]
      Randomized, double-blind, 4-period, interleaving, single-ascending oral doseIn fed state: 5, 15, 50, 150, 500, 1000, and 1500 mg; in fasted state: 1000 mg17Single dose
      C2541002 (NCT03230383)

      [N = 60
      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.
      ]
      Randomized, double-blind, placebo-controlled, sequential, multiple-ascending oral dose30, 100, 240, 400, and 600 mg q8h (ie, TDD of 90, 300, 720, 1200, and 1800 mg)48Up to 14 days
      Phase I study in adults with NAFLD
      C2541005 (NCT03513588) (N = 48)Randomized, double-blind, placebo-controlled, parallel group, repeated dose50 and 300 mg q12h (ie, TDD of 100 and 600 mg)32Up to 14 days
      NAFLD = nonalcoholic fatty liver disease; TDD = total daily dose.
      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.
      • Cases S
      • Smith SJ
      • Zheng YW
      • et al.
      Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.
      ,
      • Cases S
      • Stone SJ
      • Zhou P
      • et al.
      Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members.
      Characterization revealed 2 distinct genes mapping to different chromosomes
      • Yen C-LE
      • Stone SJ
      • Koliwad S
      • Harris C
      • Farese Jr, RV
      Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.
      ,
      • Cases S
      • Smith SJ
      • Zheng YW
      • et al.
      Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.
      ,
      • Bhatt-Wessel B
      • Jordan TW
      • Miller JH
      • Peng L
      Role of DGAT enzymes in triacylglycerol metabolism.
      and belonging to unrelated gene families.
      • Yen C-LE
      • Stone SJ
      • Koliwad S
      • Harris C
      • Farese Jr, RV
      Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.
      Structurally, although both DGAT1 and DGAT2 are integral membrane proteins localized to the endoplasmic reticulum,
      • Yen C-LE
      • Stone SJ
      • Koliwad S
      • Harris C
      • Farese Jr, RV
      Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.
      ,
      • Bhatt-Wessel B
      • Jordan TW
      • Miller JH
      • Peng L
      Role of DGAT enzymes in triacylglycerol metabolism.
      ,
      • Cao H
      Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms.
      in general, DGAT1 is larger
      • Cao H
      Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms.
      and contains more transmembrane domains than DGAT2.
      • Cases S
      • Smith SJ
      • Zheng YW
      • et al.
      Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.
      ,
      • Bhatt-Wessel B
      • Jordan TW
      • Miller JH
      • Peng L
      Role of DGAT enzymes in triacylglycerol metabolism.
      • Cao H
      Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms.
      • Stone SJ
      • Levin MC
      • Farese Jr, RV
      Membrane topology and identification of key functional amino acid residues of murine acyl-CoA:diacylglycerol acyltransferase-2.
      • Sui X
      • Wang K
      • Gluchowski NL
      • et al.
      Structure and catalytic mechanism of a human triacylglycerol-synthesis enzyme.
      • McFie PJ
      • Stone SL
      • Banman SL
      • Stone SJ
      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.
      • Sui X
      • Wang K
      • Gluchowski NL
      • et al.
      Structure and catalytic mechanism of a human triacylglycerol-synthesis enzyme.
      The sequence of human DGAT2 predicts 2 transmembrane domains, resulting in both N- and C-termini exposed to the cytosol (Figure 1).
      • Bhatt-Wessel B
      • Jordan TW
      • Miller JH
      • Peng L
      Role of DGAT enzymes in triacylglycerol metabolism.
      These differences in membrane topology
      • Bhatt-Wessel B
      • Jordan TW
      • Miller JH
      • Peng L
      Role of DGAT enzymes in triacylglycerol metabolism.
      ,
      • Stone SJ
      • Levin MC
      • Farese Jr, RV
      Membrane topology and identification of key functional amino acid residues of murine acyl-CoA:diacylglycerol acyltransferase-2.
      • Sui X
      • Wang K
      • Gluchowski NL
      • et al.
      Structure and catalytic mechanism of a human triacylglycerol-synthesis enzyme.
      • McFie PJ
      • Stone SL
      • Banman SL
      • Stone SJ
      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.
      • Wurie HR
      • Buckett L
      • Zammit VA
      Evidence that diacylglycerol acyltransferase 1 (DGAT1) has dual membrane topology in the endoplasmic reticulum of HepG2 cells.
      are hypothesized to result in differences in terms of interactions with organelles, proteins,
      • Bhatt-Wessel B
      • Jordan TW
      • Miller JH
      • Peng L
      Role of DGAT enzymes in triacylglycerol metabolism.
      ,
      • Kuerschner L
      • Moessinger C
      • Thiele C
      Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets.
      • McFie PJ
      • Banman SL
      • Kary S
      • Stone SJ
      Murine diacylglycerol acyltransferase-2 (DGAT2) can catalyze triacylglycerol synthesis and promote lipid droplet formation independent of its localization to the endoplasmic reticulum.
      • Stone SJ
      • Levin MC
      • Zhou P
      • Han J
      • Walther TC
      • Farese Jr, RV
      The endoplasmic reticulum enzyme DGAT2 is found in mitochondria-associated membranes and has a mitochondrial targeting signal that promotes its association with mitochondria.
      • McFie PJ
      • Banman SL
      • Stone SJ
      Diacylglycerol acyltransferase-2 contains a c-terminal sequence that interacts with lipid droplets.
      and access to different fatty acid pools, resulting in distinct contributions to fatty acid metabolism.
      • Bhatt-Wessel B
      • Jordan TW
      • Miller JH
      • Peng L
      Role of DGAT enzymes in triacylglycerol metabolism.
      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.
      • Bhatt-Wessel B
      • Jordan TW
      • Miller JH
      • Peng L
      Role of DGAT enzymes in triacylglycerol metabolism.
      ,
      • Qi J
      • Lang W
      • Geisler JG
      • et al.
      The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2.
      ,
      • Wurie HR
      • Buckett L
      • Zammit VA
      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.
      • Li C
      • Li L
      • Lian J
      • et al.
      Roles of acyl-CoA:diacylglycerol acyltransferases 1 and 2 in triacylglycerol synthesis and secretion in primary hepatocytes.
      ,
      • Xu N
      • Zhang SO
      • Cole RA
      • et al.
      The FATP1-DGAT2 complex facilitates lipid droplet expansion at the ER-lipid droplet interface.
      ,
      • Wilfling F
      • Wang H
      • Haas JT
      • et al.
      Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.
      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.
      • Lambert JE
      • Ramos-Roman MA
      • Browning JD
      • Parks EJ
      Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease.
      ,
      • Chalasani N
      • Younossi Z
      • Lavine JE
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      Figure 1
      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.
      aAdaptive effects.
      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,
      • Yen C-LE
      • Stone SJ
      • Koliwad S
      • Harris C
      • Farese Jr, RV
      Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.
      ,
      • Cases S
      • Smith SJ
      • Zheng YW
      • et al.
      Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.
      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.
      • Yen C-LE
      • Stone SJ
      • Koliwad S
      • Harris C
      • Farese Jr, RV
      Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.
      ,
      • Cases S
      • Stone SJ
      • Zhou P
      • et al.
      Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members.

      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,
      • Villanueva CJ
      • Monetti M
      • Shih M
      • et al.
      Specific role for acyl CoA:Diacylglycerol acyltransferase 1 (Dgat1) in hepatic steatosis due to exogenous fatty acids.
      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,
      • Stone SJ
      • Myers HM
      • Watkins SM
      • et al.
      Lipopenia and skin barrier abnormalities in DGAT2-deficient mice.
      ,
      • Chitraju C
      • Walther TC
      • Farese Jr, RV
      The triglyceride synthesis enzymes DGAT1 and DGAT2 have distinct and overlapping functions in adipocytes.
      observations that delayed investigations into DGAT2 as a pharmacologic target.
      • Stone SJ
      • Myers HM
      • Watkins SM
      • et al.
      Lipopenia and skin barrier abnormalities in DGAT2-deficient mice.
      Multiple nonclinical approaches to assess the pharmacologic inhibition profile of DGAT1
      • Villanueva CJ
      • Monetti M
      • Shih M
      • et al.
      Specific role for acyl CoA:Diacylglycerol acyltransferase 1 (Dgat1) in hepatic steatosis due to exogenous fatty acids.
      ,
      • Choi CS
      • Savage DB
      • Kulkarni A
      • et al.
      Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
      • Chitraju C
      • Mejhert N
      • Haas JT
      • et al.
      Triglyceride synthesis by DGAT1 protects adipocytes from lipid-induced ER stress during lipolysis.
      • Ables GP
      • Yang KJZ
      • Vogel S
      • et al.
      Intestinal DGAT1 deficiency reduces postprandial triglyceride and retinyl ester excursions by inhibiting chylomicron secretion and delaying gastric emptying.
      and DGAT2
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      • Yu XX
      • Murray SF
      • Pandey SK
      • et al.
      Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.
      • Futatsugi K
      • Kung DW
      • Orr STM
      • et al.
      Discovery and optimization of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2).
      • Calle RA
      • Amin NB
      • Carvajal-Gonzalez S
      • et al.
      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.
      • Liu Y
      • Millar JS
      • Cromley DA
      • et al.
      Knockdown of acyl-CoA:diacylglycerol acyltransferase 2 with antisense oligonucleotide reduces VLDL TG and ApoB secretion in mice.
      • Gluchowski NL
      • Gabriel KR
      • Chitraju C
      • et al.
      Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
      • Imbriglio JE
      • Shen D-M
      • Liang R
      • et al.
      Discovery and pharmacology of a novel class of diacylglycerol acyltransferase 2 inhibitors.
      have been published. Data indicate that reduced DGAT1 activity results in a reduction in plasma triglycerides,
      • Ables GP
      • Yang KJZ
      • Vogel S
      • et al.
      Intestinal DGAT1 deficiency reduces postprandial triglyceride and retinyl ester excursions by inhibiting chylomicron secretion and delaying gastric emptying.
      whereas hepatic targeting of DGAT1 produces reductions in liver triglycerides in animal models in which exogenous fatty acids are supplied to the liver.
      • Villanueva CJ
      • Monetti M
      • Shih M
      • et al.
      Specific role for acyl CoA:Diacylglycerol acyltransferase 1 (Dgat1) in hepatic steatosis due to exogenous fatty acids.
      DGAT2 inhibition also produces a consistent reduction in plasma and liver triglycerides,
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      • Yu XX
      • Murray SF
      • Pandey SK
      • et al.
      Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.
      • Futatsugi K
      • Kung DW
      • Orr STM
      • et al.
      Discovery and optimization of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2).
      • Calle RA
      • Amin NB
      • Carvajal-Gonzalez S
      • et al.
      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.
      • Liu Y
      • Millar JS
      • Cromley DA
      • et al.
      Knockdown of acyl-CoA:diacylglycerol acyltransferase 2 with antisense oligonucleotide reduces VLDL TG and ApoB secretion in mice.
      • Gluchowski NL
      • Gabriel KR
      • Chitraju C
      • et al.
      Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
      • Imbriglio JE
      • Shen D-M
      • Liang R
      • et al.
      Discovery and pharmacology of a novel class of diacylglycerol acyltransferase 2 inhibitors.
      with no adverse effects) reported in most studies.
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      ,
      • Yu XX
      • Murray SF
      • Pandey SK
      • et al.
      Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.
      ,
      • Calle RA
      • Amin NB
      • Carvajal-Gonzalez S
      • et al.
      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.
      ,
      • Gluchowski NL
      • Gabriel KR
      • Chitraju C
      • et al.
      Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
      ,
      • Futatsugi K
      • Huard K
      • Kung DW
      • et al.
      Small structural changes of the imidazopyridine diacylglycerol acyltransferase 2 (DGAT2) inhibitors produce an improved safety profile.
      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.
      • Choi CS
      • Savage DB
      • Kulkarni A
      • et al.
      Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
      ,
      • Yu XX
      • Murray SF
      • Pandey SK
      • et al.
      Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.
      ,
      • Gluchowski NL
      • Gabriel KR
      • Chitraju C
      • et al.
      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.
      • Yamaguchi K
      • Yang L
      • McCall S
      • et al.
      Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
      This finding has not been replicated by other studies.
      • Choi CS
      • Savage DB
      • Kulkarni A
      • et al.
      Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
      ,
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      ,
      • Yu XX
      • Murray SF
      • Pandey SK
      • et al.
      Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.
      ,
      • Calle RA
      • Amin NB
      • Carvajal-Gonzalez S
      • et al.
      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.
      ,
      • Gluchowski NL
      • Gabriel KR
      • Chitraju C
      • et al.
      Hepatocyte deletion of triglyceride-synthesis enzyme acyl CoA: diacylglycerol acyltransferase 2 reduces steatosis without increasing inflammation or fibrosis in mice.
      ,
      • Futatsugi K
      • Huard K
      • Kung DW
      • et al.
      Small structural changes of the imidazopyridine diacylglycerol acyltransferase 2 (DGAT2) inhibitors produce an improved safety profile.
      The reasons for this disparity are unclear but may be related to the use of a methionine and choline deficient diet in db/db mice,
      • Yamaguchi K
      • Yang L
      • McCall S
      • et al.
      Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
      as well as the potential for hepatotoxicity known to be associated with certain antisense oligonucleotide sequences,
      • Kamola PJ
      • Maratou K
      • Wilson PA
      • et al.
      Strategies for in vivo screening and mitigation of hepatotoxicity associated with antisense drugs.
      an interpretation that is confounded by the absence of a negative control antisense sequence.
      • Yamaguchi K
      • Yang L
      • McCall S
      • et al.
      Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
      Such toxicity was not observed in other studies using DGAT2 antisense oligonucleotides.
      • Choi CS
      • Savage DB
      • Kulkarni A
      • et al.
      Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
      ,
      • Yu XX
      • Murray SF
      • Pandey SK
      • et al.
      Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.

      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.
      • Haas JT
      • Winter HS
      • Lim E
      • et al.
      DGAT1 mutation is linked to a congenital diarrheal disorder.
      ,
      • Gluchowski NL
      • Chitraju C
      • Picoraro JA
      • et al.
      Identification and characterization of a novel DGAT1 missense mutation associated with congenital diarrhea.
      Similarly, in multiple clinical trials with small-molecule DGAT1 inhibitors, GI-related adverse events (AEs), including nausea, vomiting, and diarrhea, were common,
      • Meyers CD
      • Tremblay K
      • Amer A
      • Chen J
      • Jiang L
      • Gaudet D
      Effect of the DGAT1 inhibitor pradigastat on triglyceride and apoB48 levels in patients with familial chylomicronemia syndrome.
      • Sanyal AJ
      • Cusi K
      • Patel S
      • Wright M
      • Liu C
      • Keefe DL
      Effect of pradigastat, a diacylglycerol acyltransferase 1 inhibitor, on liver fat content in nonalcoholic fatty liver disease.
      • Stroes ESG
      • Patel S
      • Bernelot-Moens S
      • et al.
      The diacylglycerol acyltransferase 1 inhibitor, pradigastat, was well tolerated in a 52-week clinical trial in FCS patients.
      • Denison H
      • Nilsson C
      • Löfgren L
      • et al.
      Diacylglycerol acyltransferase 1 inhibition with AZD7687 alters lipid handling and hormone secretion in the gut with intolerable side effects: a randomized clinical trial.
      limiting dose escalation in a previous study.
      • Denison H
      • Nilsson C
      • Kujacic M
      • et al.
      Proof of mechanism for the DGAT1 inhibitor AZD7687: results from a first-time-in-human single-dose study.
      In contrast, DGAT2 inhibition clinically reduces hepatic steatosis in healthy participants after 2 weeks
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      and in participants with NAFLD after 6 weeks,
      • Calle RA
      • Amin NB
      • Carvajal-Gonzalez S
      • et al.
      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.
      as well as after 13 weeks of dosing.
      • Loomba R
      • Morgan E
      • Watts L
      • et al.
      Novel antisense inhibition of diacylglycerol O-acyltransferase 2 for treatment of non-alcoholic fatty liver disease: a multicentre, double-blind, randomised, placebo-controlled phase 2 trial.
      Encouragingly, DGAT2 inhibition in these studies was well tolerated, with no dose-limiting AEs reported.
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      ,
      • Loomba R
      • Morgan E
      • Watts L
      • et al.
      Novel antisense inhibition of diacylglycerol O-acyltransferase 2 for treatment of non-alcoholic fatty liver disease: a multicentre, double-blind, randomised, placebo-controlled phase 2 trial.
      A reduction in HDL-C was also observed,
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      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.
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      ,
      • Liu DJ
      • Peloso GM
      • Yu H
      • et al.
      Exome-wide association study of plasma lipids in >300,000 individuals.
      ,
      • McLaren DG
      • Han S
      • Murphy BA
      • et al.
      DGAT2 inhibition alters aspects of triglyceride metabolism in rodents but not in non-human primates.

      Fatty Liver Disease: A Target for DGAT2 Inhibition

      NAFLD is a progressive spectrum of liver disease associated with hepatic triglyceride accumulation,
      • Chalasani N
      • Younossi Z
      • Lavine JE
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      ,
      • Brunt EM
      • Tiniakos DG
      Histopathology of nonalcoholic fatty liver disease.
      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.
      • Younossi ZM
      • Tampi R
      • Priyadarshini M
      • Nader F
      • Younossi IM
      • Racila A
      Burden of illness and economic model for patients with nonalcoholic steatohepatitis in the United States.
      ,
      • Vernon G
      • Baranova A
      • Younossi ZM
      Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults.
      NASH is characterized histologically by the presence of hepatocyte ballooning, inflammation, and often progressive fibrosis,
      • Chalasani N
      • Younossi Z
      • Lavine JE
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      ,
      • Brunt EM
      • Tiniakos DG
      Histopathology of nonalcoholic fatty liver disease.
      ultimately leading in some cases to cirrhosis
      • Chalasani N
      • Younossi Z
      • Lavine JE
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      or hepatocellular carcinoma.
      • Khan FZ
      • Perumpail RB
      • Wong RJ
      • Ahmed A
      Advances in hepatocellular carcinoma: nonalcoholic steatohepatitis-related hepatocellular carcinoma.
      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).
      • Younossi ZM
      • Koenig AB
      • Abdelatif D
      • Fazel Y
      • Henry L
      • Wymer M
      Global epidemiology of nonalcoholic fatty liver disease – meta-analytic assessment of prevalence, incidence, and outcomes.
      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.
      • Dulai PS
      • Singh S
      • Patel J
      • et al.
      Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: systematic review and meta-analysis.
      Globally, there are currently no approved agents for the treatment of NASH with liver fibrosis, with improvement based on histologic changes,
      • Sharma M
      • Premkumar M
      • Kulkarni AV
      • Kumar P
      • Reddy DN
      • Rao NP
      Drugs for non-alcoholic steatohepatitis (NASH): quest for the holy grail.
      ,
      • Sumida Y
      • Yoneda M
      • Ogawa Y
      • Yoneda M
      • Okanoue T
      • Nakajima A
      Current and new pharmacotherapy options for non-alcoholic steatohepatitis.
      and there is a regulatory-defined medical need for treatment of patients at risk for progression with significant fibrosis (fibrosis stage ≥2),
      • Chalasani N
      • Younossi Z
      • Lavine JE
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      who may benefit most from treatment.
      • Dulai PS
      • Singh S
      • Patel J
      • et al.
      Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: systematic review and meta-analysis.
      There is a single approved agent for the treatment of NASH in India, based on clinical findings of a reduction in alanine aminotransferase.
      • Gawrieh S
      • Noureddin M
      • Loo N
      • et al.
      Saroglitazar, a PPAR-α/γ agonist, for treatment of nonalcoholic fatty liver disease: a randomized controlled double-blind phase 2 trial.
      ,
      • Padole P
      • Arora A
      • Sharma P
      • Chand P
      • Verma N
      • Kumar A
      Saroglitazar for nonalcoholic fatty liver disease: a single center experience in 91 patients.
      De novo lipogenesis is a hallmark feature of NAFLD.
      • Lambert JE
      • Ramos-Roman MA
      • Browning JD
      • Parks EJ
      Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease.
      As in other conditions, such as atherosclerosis, the initial lipid insult may lead to inflammation and disease progression.
      • Geovanini GR
      • Libby P
      Atherosclerosis and inflammation: overview and updates.
      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.
      • Esler WP
      • Bence KK
      Metabolic targets in nonalcoholic fatty liver disease.
      ,
      • Noureddin M
      • Anstee QM
      • Loomba R
      Review article: emerging anti-fibrotic therapies in the treatment of non-alcoholic steatohepatitis.
      The key role of DGAT2 in hepatic steatosis, and in de novo lipogenesis specifically, makes DGAT2 an attractive therapeutic target.
      • Yen C-LE
      • Stone SJ
      • Koliwad S
      • Harris C
      • Farese Jr, RV
      Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.
      ,
      • Wurie HR
      • Buckett L
      • Zammit VA
      Diacylglycerol acyltransferase 2 acts upstream of diacylglycerol acyltransferase 1 and utilizes nascent diglycerides and de novo synthesized fatty acids in HepG2 cells.
      ,
      • Gluchowski NL
      • Gabriel KR
      • Chitraju C
      • et al.
      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.
      • Amin NB
      • Darekar A
      • Anstee QM
      • et al.
      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.
      • Futatsugi K
      • Cabral S
      • Kung DW
      • et al.
      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),
      • Futatsugi K
      • Cabral S
      • Kung DW
      • et al.
      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,
      • Choi CS
      • Savage DB
      • Kulkarni A
      • et al.
      Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.
      ,
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      ,
      • Yu XX
      • Murray SF
      • Pandey SK
      • et al.
      Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.
      ,
      • Gluchowski NL
      • Gabriel KR
      • Chitraju C
      • et al.
      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.
      • Calle RA
      • Amin NB
      • Carvajal-Gonzalez S
      • et al.
      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 2
      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.
      aOccurred during the follow-up phase.
      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 3
      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
      • Meyers CD
      • Tremblay K
      • Amer A
      • Chen J
      • Jiang L
      • Gaudet D
      Effect of the DGAT1 inhibitor pradigastat on triglyceride and apoB48 levels in patients with familial chylomicronemia syndrome.
      • Sanyal AJ
      • Cusi K
      • Patel S
      • Wright M
      • Liu C
      • Keefe DL
      Effect of pradigastat, a diacylglycerol acyltransferase 1 inhibitor, on liver fat content in nonalcoholic fatty liver disease.
      • Stroes ESG
      • Patel S
      • Bernelot-Moens S
      • et al.
      The diacylglycerol acyltransferase 1 inhibitor, pradigastat, was well tolerated in a 52-week clinical trial in FCS patients.
      • Denison H
      • Nilsson C
      • Löfgren L
      • et al.
      Diacylglycerol acyltransferase 1 inhibition with AZD7687 alters lipid handling and hormone secretion in the gut with intolerable side effects: a randomized clinical trial.
      and with the finding of congenital diarrhea linked to mutation of the human DGAT1 gene.
      • Haas JT
      • Winter HS
      • Lim E
      • et al.
      DGAT1 mutation is linked to a congenital diarrheal disorder.
      ,
      • Gluchowski NL
      • Chitraju C
      • Picoraro JA
      • et al.
      Identification and characterization of a novel DGAT1 missense mutation associated with congenital diarrhea.
      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
      Baseline was defined as the measurement collected on day –1.
      (90% CI)
      8.42 (–6.02 to 25.07)–0.44 (–15.62 to 17.51)9.41 (–7.44 to 29.33)–6.72 (–21.33 to 10.59)–11.86 (–25.32 to 4.03)–23.79 (–34.01 to –11.99)
      Ratio ervogastat/placebo (90% CI)–8.17 (–26.17 to 14.21)0.92 (–18.78 to 25.39)–13.96 (–31.44 to 7.97)–18.70 (–34.70 to 1.21)–29.71 (–42.75 to –13.70)
      P value0.520.940.270.120.006
      Difference from baseline in log-transformed serum triglycerides AUC0–18
      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 value0.100.620.0080.020.35
      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 4
      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,
      • Mora S
      • Caulfield MP
      • Wohlgemuth J
      • et al.
      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.
      DGAT2i will not completely repress but rather dampen triglyceride production, an effect that is well tolerated according to clinical studies to date.
      • Amin NB
      • Carvajal-Gonzalez S
      • Purkal J
      • et al.
      Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis.
      ,
      • Calle RA
      • Amin NB
      • Carvajal-Gonzalez S
      • et al.
      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.
      • Calle RA
      • Amin NB
      • Carvajal-Gonzalez S
      • et al.
      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).
      • Amin NB
      • Darekar A
      • Anstee QM
      • et al.
      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.

      Declaration of Interest

      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.

      Appendix. Supplementary materials

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