Comparison of the circulating metabolite profile of PF-04991532, a hepatoselective glucokinase activator, across preclinical species and humans: potential implications in metabolites in safety testing assessment.

A previous report from our laboratory disclosed the identification of PF-04991532 [(S)-6-(3-cyclopentyl-2-(4-trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid] as a hepatoselective glucokinase activator for the treatment of type 2 diabetes mellitus. Lack of in vitro metabolic turnover in microsomes and hepatocytes from preclinical species and humans suggested that metabolism would be inconsequential as a clearance mechanism of PF-04991532 in vivo. Qualitative examination of human circulating metabolites using plasma samples from a 14-day multiple ascending dose clinical study, however, revealed a glucuronide (M1) and monohydroxylation products (M2a and M2b/M2c) whose abundances (based on UV integration) were greater than 10% of the total drug-related material. Based on this preliminary observation, mass balance/excretion studies were triggered in animals, which revealed that the majority of circulating radioactivity following the oral administration of [¹4C]PF-04991532 was attributed to an unchanged parent (>70% in rats and dogs). In contrast with the human circulatory metabolite profile, the monohydroxylated metabolites were not detected in circulation in either rats or dogs. Available mass spectral evidence suggested that M2a and M2b/M2c were diastereomers derived from cyclopentyl ring oxidation in PF-04991532. Because cyclopentyl ring hydroxylation on the C-2 and C-3 positions can generate eight possible diastereomers, it was possible that additional diastereomers may have also formed and would need to be resolved from the M2a and M2b/M2c peaks observed in the current chromatography conditions. In conclusion, the human metabolite scouting study in tandem with the animal mass balance study allowed early identification of PF-04991532 oxidative metabolites, which were not predicted by in vitro methods and may require additional scrutiny in the development phase of PF-04991532.

Metabolites in safety testing assessment in early clinical development: a case study with a glucokinase activator.

The present article summarizes Metabolites in Safety Testing (MIST) studies on a glucokinase activator, N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319), which is under development for the treatment of type 2 diametes mellitus. Metabolic profiling in rat, dog, and human hepatocytes revealed that PF-04937319 is metabolized via oxidative (major) and hydrolytic pathways (minor). N-Demethylation to metabolite M1 [N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide] was the major metabolic fate of PF-04937319 in human (but not rat or dog) hepatocytes, and was catalyzed by CYP3A and CYP2C isoforms. Qualitative examination of circulating metabolites in humans at the 100- and 300-mg doses from a 14-day multiple dose study revealed unchanged parent drug and M1 as principal components. Because M1 accounted for 65% of the drug-related material at steady state, an authentic standard was synthesized and used for comparison of steady-state exposures in humans and the 3-month safety studies in rats and dogs at the no-observed-adverse-effect level. Although circulating levels of M1 were very low in beagle dogs and female rats, adequate coverage was obtained in terms of total maximal plasma concentration (∼7.7× and 1.8×) and area under the plasma concentration-time curve (AUC; 3.6× and 0.8× AUC) relative to the 100- and 300-mg doses, respectively, in male rats. Examination of primary pharmacology revealed M1 was less potent as a glucokinase activator than the parent drug (compound PF-04937319: EC50 = 0.17 µM; M1: EC50 = 4.69 µM). Furthermore, M1 did not inhibit major human P450 enzymes (IC50 > 30 µM), and was negative in the Salmonella Ames assay, with minimal off-target pharmacology, based on CEREP broad ligand profiling. Insights gained from this analysis should lead to a more efficient and focused development plan for fulfilling MIST requirements with PF-04937319.

Identification of a novel conformationally constrained glucagon receptor antagonist.

Identification of orally active, small molecule antagonists of the glucagon receptor represents a novel treatment paradigm for the management of type 2 diabetes mellitus. The present work discloses novel glucagon receptor antagonists, identified via conformational constraint of current existing literature antagonists. Optimization of lipophilic ligand efficiency (LLE or LipE) culminated in enantiomers (+)-trans-26 and (-)-trans-27 which exhibit good physicochemical and in vitro drug metabolism profiles. In vivo, significant pharmacokinetic differences were noted with the two enantiomers, which were primarily driven through differences in clearance rates. Enantioselective oxidation by cytochrome P450 was ruled out as a causative factor for pharmacokinetic differences.

The relationship of glucokinase activator-induced hypoglycemia with arteriopathy, neuronal necrosis, and peripheral neuropathy in nonclinical studies.

Glucokinase activators (GKAs) are being developed for the treatment of type 2 diabetes. The toxicity of 4 GKAs (PF-04279405, PF-04651887, piragliatin, and PF-04937319) was assessed in mice, rats, dogs, and/or monkeys. GKAs were administered for 2 to 8 weeks. Standard endpoints, glucose, and insulin were assessed. All compounds produced varying degrees of hypoglycemia in all species. Brain neuronal necrosis and/or peripheral neuropathy were observed with most compounds. These findings are consistent with literature reports linking hypoglycemia with nervous system effects. Arteriopathy, mainly of cardiac vessels, was observed at a low frequency in monkey and/or dog. Arteriopathy occurred only at doses that produced severe and prolonged periods of repeated hypoglycemia. Since this lesion occurred in multiple studies with structurally distinct GKAs, these results suggested arteriopathy was related to GKA pharmacology. The morphological characteristics of the arteriopathy were consistent with that produced by experimental catecholamine administration. We hypothesize that the prolonged periods of hypoglycemia resulted in increased local and/or systemic concentrations of catecholamines via a counterregulatory and/or stress-related mechanism. Alternatively, prolonged hypoglycemia may have resulted in endothelial dysfunction leading to arteriopathy. This risk can be managed in human patients in clinical studies by careful glucose monitoring and intervention to avoid prolonged episodes of hypoglycemia.

Insights into mechanism of glucokinase activation: observation of multiple distinct protein conformations.

Human glucokinase (GK) is a principal regulating sensor of plasma glucose levels. Mutations that inactivate GK are linked to diabetes, and mutations that activate it are associated with hypoglycemia. Unique kinetic properties equip GK for its regulatory role: although it has weak basal affinity for glucose, positive cooperativity in its binding of glucose causes a rapid increase in catalytic activity when plasma glucose concentrations rise above euglycemic levels. In clinical trials, small molecule GK activators (GKAs) have been efficacious in lowering plasma glucose and enhancing glucose-stimulated insulin secretion, but they carry a risk of overly activating GK and causing hypoglycemia. The theoretical models proposed to date attribute the positive cooperativity of GK to the existence of distinct protein conformations that interconvert slowly and exhibit different affinities for glucose. Here we report the respective crystal structures of the catalytic complex of GK and of a GK-glucose complex in a wide open conformation. To assess conformations of GK in solution, we also carried out small angle x-ray scattering experiments. The results showed that glucose dose-dependently converts GK from an apo conformation to an active open conformation. Compared with wild type GK, activating mutants required notably lower concentrations of glucose to be converted to the active open conformation. GKAs decreased the level of glucose required for GK activation, and different compounds demonstrated distinct activation profiles. These results lead us to propose a modified mnemonic model to explain cooperativity in GK. Our findings may offer new approaches for designing GKAs with reduced hypoglycemic risk.

Discovery of an intravenous hepatoselective glucokinase activator for the treatment of inpatient hyperglycemia.

Glucokinase (hexokinase IV) continues to be a compelling target for the treatment of type 2 diabetes given the wealth of supporting human genetics data and numerous reports of robust clinical glucose lowering in patients treated with small molecule allosteric activators. Recent work has demonstrated the ability of hepatoselective activators to deliver glucose lowering efficacy with minimal risk of hypoglycemia. While orally administered agents require a considerable degree of passive permeability to promote suitable exposures, there is no such restriction on intravenously delivered drugs. Therefore, minimization of membrane diffusion in the context of an intravenously agent should ensure optimal hepatic targeting and therapeutic index. This work details the identification a hepatoselective GKA exhibiting the aforementioned properties.

The design and synthesis of a potent glucagon receptor antagonist with favorable physicochemical and pharmacokinetic properties as a candidate for the treatment of type 2 diabetes mellitus.

A novel and potent small molecule glucagon receptor antagonist for the treatment of diabetes mellitus is reported. This candidate, (S)-3-[4-(1-{3,5-dimethyl-4-[4-(trifluoromethyl)-1H-pyrazol-1-yl]phenoxy}butyl)benzamido]propanoic acid, has lower molecular weight and lipophilicity than historical glucagon receptor antagonists, resulting in excellent selectivity in broad-panel screening, lower cytotoxicity, and excellent overall in vivo safety in early pre-clinical testing. Additionally, it displays low in vivo clearance and excellent oral bioavailability in both rats and dogs. In a rat glucagon challenge model, it was shown to reduce the glucagon-elicited glucose excursion in a dose-dependent manner and at a concentration consistent with its rat in vitro potency. Its properties make it an excellent candidate for further investigation.

Pyrimidone-based series of glucokinase activators with alternative donor-acceptor motif.

Glucokinase activators are a class of experimental agents under investigation as a therapy for Type 2 diabetes mellitus. An X-ray crystal structure of a modestly potent agent revealed the potential to substitute the common heterocyclic amide donor-acceptor motif for a pyridone moiety. We have successfully demonstrated that both pyridone and pyrimidone heterocycles can be used as a potent donor-acceptor substituent. Several sub-micromolar analogs that possess the desired partial activator profile were synthesized and characterized. Unfortunately, the most potent activators suffered from sub-optimal pharmacokinetic properties. Nonetheless, these donor-acceptor motifs may find utility in other glucokinase activator series or beyond.

Modulation of glucokinase by glucose, small-molecule activator and glucokinase regulatory protein: steady-state kinetic and cell-based analysis.

GK (glucokinase) is an enzyme central to glucose metabolism that displays positive co-operativity to substrate glucose. Small-molecule GKAs (GK activators) modulate GK catalytic activity and glucose affinity and are currently being pursued as a treatment for Type 2 diabetes. GK progress curves monitoring product formation are linear up to 1 mM glucose, but biphasic at 5 mM, with the transition from the lower initial velocity to the higher steady-state velocity being described by the rate constant kact. In the presence of a liver-specific GKA (compound A), progress curves at 1 mM glucose are similar to those at 5 mM, reflecting activation of GK by compound A. We show that GKRP (GK regulatory protein) is a slow tight-binding inhibitor of GK. Analysis of progress curves indicate that this inhibition is time dependent, with apparent initial and final Ki values being 113 and 12.8 nM respectively. When GK is pre-incubated with glucose and compound A, the inhibition observed by GKRP is time dependent, but independent of GKRP concentration, reflecting the GKA-controlled transition between closed and open GK conformations. These data are supported by cell-based imaging data from primary rat hepatocytes. This work characterizes the modulation of GK by a novel GKA that may enable the design of new and improved GKAs.

Glycomimetic ligands for the human asialoglycoprotein receptor.

The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.

A novel series of glucagon receptor antagonists with reduced molecular weight and lipophilicity.

A novel series of glucagon receptor antagonists has been discovered. These pyrazole ethers and aminopyrazoles have lower molecular weight and increased polarity such that the molecules fall into better drug-like property space. This work has culminated in compounds 44 and 50 that were shown to have good pharmacokinetic attributes in dog, in contrast to rats, in which clearance was high; and compound 49, which demonstrated a dose-dependent reduction in glucose excursion in a rat glucagon challenge experiment.

The design and synthesis of indazole and pyrazolopyridine based glucokinase activators for the treatment of type 2 diabetes mellitus.

Glucokinase activators represent a promising potential treatment for patients with Type 2 diabetes. Herein, we report the identification and optimization of a series of novel indazole and pyrazolopyridine based activators leading to the identification of 4-(6-(azetidine-1-carbonyl)-5-fluoropyridin-3-yloxy)-2-ethyl-N-(5-methylpyrazin-2-yl)-2H-indazole-6-carboxamide (42) as a potent activator with favorable preclinical pharmacokinetic properties and in vivo efficacy.

Exploring aromatic chemical space with NEAT: novel and electronically equivalent aromatic template.

In this paper, we describe a lead transformation tool, NEAT (Novel and Electronically equivalent Aromatic Template), which can help identify novel aromatic rings that are estimated to have similar electrostatic potentials, dipoles, and hydrogen bonding capabilities to a query template; hence, they may offer similar bioactivity profiles. In this work, we built a comprehensive heteroaryl database, and precalculated high-level quantum mechanical (QM) properties, including electrostatic potential charges, hydrogen bonding ability, dipole moments, chemical reactivity, and othe properties. NEAT bioisosteric similarities are based on the electrostatic potential surface calculated by Brood, using the precalculated QM ESP charges and other QM properties. Compared with existing commercial lead transformation software, (1) NEAT is the only one that covers the comprehensive heteroaryl chemical space, and (2) NEAT offers a better characterization of novel aryl cores by using high-evel QM properties that are relevant to molecular interactions. NEAT provides unique value to medicinal chemists quickly exploring the largely uncharted aromatic chemical space, and one successful example of its application is discussed herein.

Discovery of Ervogastat (PF-06865571): A Potent and Selective Inhibitor of Diacylglycerol Acyltransferase 2 for the Treatment of Non-alcoholic Steatohepatitis.

Discovery efforts leading to the identification of ervogastat (PF-06865571), a systemically acting diacylglycerol acyltransferase (DGAT2) inhibitor that has advanced into clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) with liver fibrosis, are described herein. Ervogastat is a first-in-class DGAT2 inhibitor that addressed potential development risks of the prototype liver-targeted DGAT2 inhibitor PF-06427878. Key design elements that culminated in the discovery of ervogastat are (1) replacement of the metabolically labile motif with a 3,5-disubstituted pyridine system, which addressed potential safety risks arising from a cytochrome P450-mediated O-dearylation of PF-06427878 to a reactive quinone metabolite precursor, and (2) modifications of the amide group to a 3-THF group, guided by metabolite identification studies coupled with property-based drug design.

Discovery of novel hepatoselective HMG-CoA reductase inhibitors for treating hypercholesterolemia: a bench-to-bedside case study on tissue selective drug distribution.

The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.

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.

Alterations in lipid metabolism might contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, no pharmacological agents are currently approved in the United States or the European Union for the treatment of NAFLD. Two parallel phase 2a studies investigated the effects of liver-directed ACC1/2 inhibition in adults with NAFLD. The first study ( NCT03248882 ) examined the effects of monotherapy with a novel ACC1/2 inhibitor, PF-05221304 (2, 10, 25 and 50 mg once daily (QD)), versus placebo at 16 weeks of treatment; the second study ( NCT03776175 ) investigated the effects of PF-05221304 (15 mg twice daily (BID)) co-administered with a DGAT2 inhibitor, PF-06865571 (300 mg BID), versus placebo after 6 weeks of treatment. The primary endpoint in both studies was percent change from baseline in liver fat assessed by magnetic resonance imaging-proton density fat fraction. Dose-dependent reductions in liver fat reached 50-65% with PF-05221304 monotherapy doses ≥10 mg QD; least squares mean (LSM) 80% confidence interval (CI) was -7.2 (-13.9, 0.0), -17.1 (-22.7, -11.1), -49.9 (-53.3, -46.2), -55.9 (-59.0, -52.4) and -64.8 (-67.5, -62.0) with 16 weeks placebo and PF-05221304 2, 10, 25 and 50 mg QD, respectively. The overall incidence of adverse events (AEs) did not increase with increasing PF-05221304 dose, except for a dose-dependent elevation in serum triglycerides (a known consequence of hepatic acetyl-coenzyme A carboxylase (ACC) inhibition) in 23/305 (8%) patients, leading to withdrawal in 13/305 (4%), and a dose-dependent elevation in other serum lipids. Co-administration of PF-05221304 and PF-06865571 lowered liver fat compared to placebo (placebo-adjusted LSM (90% CI) -44.6% (-54.8, -32.2)). Placebo-adjusted LSM (90% CI) reduction in liver fat was -44.5% (-55.0, -31.7) and -35.4% (-47.4, -20.7) after 6 weeks with PF-05221304 or PF-06865571 alone. AEs were reported for 10/28 (36%) patients after co-administered PF-05221304 and PF-06865571, with no discontinuations due to AEs, and the ACC inhibitor-mediated effect on serum triglycerides was mitigated, suggesting that PF-05221304 and PF-06865571 co-administration has the potential to address some of the limitations of ACC inhibition alone.

Pharmacologic inhibition of ketohexokinase prevents fructose-induced metabolic dysfunction.

OBJECTIVE: Recent studies suggest that excess dietary fructose contributes to metabolic dysfunction by promoting insulin resistance, de novo lipogenesis (DNL), and hepatic steatosis, thereby increasing the risk of obesity, type 2 diabetes (T2D), non-alcoholic steatohepatitis (NASH), and related comorbidities. Whether this metabolic dysfunction is driven by the excess dietary calories contained in fructose or whether fructose catabolism itself is uniquely pathogenic remains controversial. We sought to test whether a small molecule inhibitor of the primary fructose metabolizing enzyme ketohexokinase (KHK) can ameliorate the metabolic effects of fructose. METHODS: The KHK inhibitor PF-06835919 was used to block fructose metabolism in primary hepatocytes and Sprague Dawley rats fed either a high-fructose diet (30% fructose kcal/g) or a diet reflecting the average macronutrient dietary content of an American diet (AD) (7.5% fructose kcal/g). The effects of fructose consumption and KHK inhibition on hepatic steatosis, insulin resistance, and hyperlipidemia were evaluated, along with the activation of DNL and the enzymes that regulate lipid synthesis. A metabolomic analysis was performed to confirm KHK inhibition and understand metabolite changes in response to fructose metabolism in vitro and in vivo. Additionally, the effects of administering a single ascending dose of PF-06835919 on fructose metabolism markers in healthy human study participants were assessed in a randomized placebo-controlled phase 1 study. RESULTS: Inhibition of KHK in rats prevented hyperinsulinemia and hypertriglyceridemia from fructose feeding. Supraphysiologic levels of dietary fructose were not necessary to cause metabolic dysfunction as rats fed the American diet developed hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis, which were all reversed by KHK inhibition. Reversal of the metabolic effects of fructose coincided with reductions in DNL and inactivation of the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP). We report that administering single oral doses of PF-06835919 was safe and well tolerated in healthy study participants and dose-dependently increased plasma fructose indicative of KHK inhibition. CONCLUSIONS: Fructose consumption in rats promoted features of metabolic dysfunction seen in metabolic diseases such as T2D and NASH, including insulin resistance, hypertriglyceridemia, and hepatic steatosis, which were reversed by KHK inhibition.

Intrinsic electrophilicity of the 4-methylsulfonyl-2-pyridone scaffold in glucokinase activators: role of glutathione-S-transferases and in vivo quantitation of a glutathione conjugate in rats.

Previous studies on the in vitro metabolism of 4-alkylsulfonyl-2-pyridone-based glucokinase activators revealed a facile, non-enzymatic displacement of the 4-alkylsulfonyl group by glutathione. In the present studies, a role for glutathione-S-transferases (GST) as catalysts in the desulfonylation reaction was demonstrated using a combination of human liver microsomes, human liver cytosol and human GSTs. The identification of a glutathione conjugate in circulation following intravenous administration of a candidate 4-methylsulfonyl-2-pyridone to rats confirmed the relevance of the in vitro findings.

Quantitation of Urinary Acylcarnitines by DMS-MS/MS Uncovers the Effects of Total Body Irradiation in Cancer Patients.

Acylcarnitines have been identified in human and animal metabolomic-profiling studies as urinary markers of radiation exposure, a result which is consistent with their cytoprotective effects and roles in energy metabolism. In the present work, a rapid method for quantitation of the more abundant acylcarnitines in human urine is developed using a valuable set of samples from cancer patients who received total body irradiation (TBI) at Memorial Sloan Kettering Cancer Center. The method uses solid-phase extraction (SPE) processing followed by differential mobility spectrometry (DMS with ethyl acetate modifier) tandem mass spectrometry (ESI-DMS-MS/MS) with deuterated internal standards. The analyzed human urine samples were collected from 38 individual patients at three time points over 24 h during and after the course of radiation treatment, a design allowing each patient to act as their own control and creatinine normalization. Creatinine-normalized concentrations for nine urinary acylcarnitine (acyl-CN) species are reported. Six acyl-CN species were reduced at the 6 h point. Acetylcarnitine (C2:0-CN) and valerylcarnitine (C5:0-CN) showed recovery at 24 h, but none of the other acyl-CN species showed recovery at that point. Levels of three acyl-CN species were not significantly altered by radiation. This rapid quantitative method for clinical samples covers the short- and medium-chain acylcarnitines and has the flexibility to be expanded to cover additional radiation-linked metabolites. The human data presented here indicates the utility of the current approach as a rapid, quantitative technique with potential applications by the medical community, by space research laboratories concerned with radiation exposure, and by disaster response groups.

Acetyl-CoA Carboxylase Inhibition Improves Multiple Dimensions of NASH Pathogenesis in Model Systems.

BACKGROUND & AIMS: Disordered metabolism, steatosis, hepatic inflammation, and fibrosis contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH). Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in de novo lipogenesis (DNL) and modulates mitochondrial fatty acid oxidation. Increased hepatic DNL flux and reduced fatty acid oxidation are hypothesized to contribute to steatosis. Some proinflammatory cells also show increased dependency on DNL, suggesting that ACC may regulate aspects of the inflammatory response in NASH. PF-05221304 is an orally bioavailable, liver-directed ACC1/2 inhibitor. The present studies sought to evaluate the effects of PF-05221304 on NASH pathogenic factors in experimental model systems. METHODS: The effects of PF-05221304 on lipid metabolism, steatosis, inflammation, and fibrogenesis were investigated in both primary human-derived in vitro systems and in vivo rodent models. RESULTS: PF-05221304 inhibited DNL, stimulated fatty acid oxidation, and reduced triglyceride accumulation in primary human hepatocytes, and reduced DNL and steatosis in Western diet-fed rats in vivo, showing the potential to reduce hepatic lipid accumulation and potentially lipotoxicity. PF-05221304 blocked polarization of human T cells to proinflammatory but not anti-inflammatory T cells, and suppressed activation of primary human stellate cells to myofibroblasts in vitro, showing direct effects on inflammation and fibrogenesis. Consistent with these observations, PF-05221304 also reduced markers of inflammation and fibrosis in the diethylnitrosamine chemical-induced liver injury model and the choline-deficient, high-fat-fed rat model. CONCLUSIONS: The liver-directed dual ACC1/ACC2 inhibitor directly improved multiple nonalcoholic fatty liver disease/NASH pathogenic factors including steatosis, inflammation, and fibrosis in both human-derived in vitro systems and rat models.