ATP-citrate lyase (ACL); AMPK

Bempedoic acid (ETC-1002) activates AMP-activated protein kinase in a Ca2+/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge.It has been demonstrated that bempedoic acid quickly converts to a CoA thioester in the liver, which directly inhibits ATP-citrate lyase[1]. Increased AMP-activated protein kinase (AMPK) phosphorylation is associated with decreased MAP kinase activity and decreased production of proinflammatory cytokines and chemokines in cells treated with bempedoic acid (ETC-1002)[2].

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ETC-1002 is an investigational drug currently in Phase 2 development for treatment of dyslipidemia and other cardiometabolic risk factors. In dyslipidemic subjects, ETC-1002 not only reduces plasma LDL cholesterol but also significantly attenuates levels of hsCRP, a clinical biomarker of inflammation. Anti-inflammatory properties of ETC-1002 were further investigated in primary human monocyte-derived macrophages and in in vivo models of inflammation. In cells treated with ETC-1002, increased levels of AMP-activated protein kinase (AMPK) phosphorylation coincided with reduced activity of MAP kinases and decreased production of proinflammatory cytokines and chemokines. AMPK phosphorylation and inhibitory effects of ETC-1002 on soluble mediators of inflammation were significantly abrogated by siRNA-mediated silencing of macrophage liver kinase B1 (LKB1), indicating that ETC-1002 activates AMPK and exerts its anti-inflammatory effects via an LKB1-dependent mechanism. In vivo, ETC-1002 suppressed thioglycollate-induced homing of leukocytes into mouse peritoneal cavity. Similarly, in a mouse model of diet-induced obesity, ETC-1002 restored adipose AMPK activity, reduced JNK phosphorylation, and diminished expression of macrophage-specific marker 4F/80. These data were consistent with decreased epididymal fat-pad mass and interleukin (IL)-6 release by inflamed adipose tissue. Thus, ETC-1002 may provide further clinical benefits for patients with cardiometabolic risk factors by reducing systemic inflammation linked to insulin resistance and vascular complications of metabolic syndrome.[2]

Bempedoic acid (ETC-1002) treatment for two weeks causes a noticeable and long-lasting increase in AMPK and ACC phosphorylation in the livers of rats. In rat liver, Bempedoic acid is more prevalent >100-fold than CoA thioester and is connected to AMPK activation[1]. Leukocytes' ability to home into the mouse peritoneal cavity is suppressed by bempedoic acid (ETC-1002). In a mouse model of diet-induced obesity, Bempedoic acid improves adipose AMPK activity, lowers JNK phosphorylation, and decreases expression of the macrophage-specific marker 4F/80[2].

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ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.[1]

Glucose production assay[1]
Glucose production was measured in primary rat hepatocyte cultures. Cells were cultured in glucose- and phenol red-free DMEM, containing 10 mM lactate, 1 mM pyruvate, and nonessential amino acids (glucose production buffer, GPB). To assess the effects of ETC-1002 on glucagon-stimulated glucose production, cells were incubated with and without 0.3 μM glucagon with various concentrations of ETC-1002 (0.1 to 100 μM). Media was sampled over time. Following specified treatments, cells were washed twice in GPB. Cells were then incubated for an additional hour to assess glucose production by adding GPB containing equivalent glucagon concentrations without ETC-1002. Cells were incubated for 1 h, and the concentration of glucose in the media was determined using a glucose oxidase assay kit.

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ETC-1002 formulation / ETC-1002-CoA synthesis[2]
For in vitro assays, ETC-1002 was formulated using aseptic technique at 30 and 100 mM in sterile dimethylsulfoxide (DMSO) and stored in sterile microcentrifuge tubes at 4°C for up to four weeks (stability was assessed). Working solutions of ETC-1002 were prepared in serum-free RPMI 1640 containing 12 mM HEPES, 10,000 U/ml penicillin, and 100 μg/ml streptomycin. ETC-1002-CoA was synthesized using rat liver microsomes as described.

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7.5× compounds are added to a 96-well PolyPlate containing 60 μL of Buffer per well with substrates CoA (200 μM), ATP (400 μM), and [14C]citrate. Reaction is started with 4 μL (300 ng/well) ACL, and the plate is incubated at 37°C for 3 h.

Glucose production is measured in primary rat hepatocyte cultures. It contains nonessential amino acids, 10 mM lactate, 1 mM pyruvate, and is free of glucose and phenol red. Cells are cultured in this mixture. Bempedoic acid (0.1 to 100 μM) is incubated with the cells in a variety of concentrations[1].

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Protein arrays[2]
At the end of differentiation period, macrophages were washed with PBS and switched to RPMI 1640 containing 5% autologous serum and supplemented with 14 mM HEPES, 100 U/ml penicillin, 50 U/ml streptomycin, and 2 mM L-glutamine. ETC-1002 at various concentrations (50 μM and 100 μM) was added to the media 1 h prior to stimulation with 100 ng/ml of lipopolysaccharide (LPS) from Escherichia coli 0111:B4. Media conditioned by MDMs was collected 12 h following LPS stimulation and assayed with Proteome Profiler Human Cytokine Array Kit, Panel A, and Human Matrix Metalloproteinase Array, according to the manufacturer's instructions. Data for cytokine and matrix metalloproteinase (MMP) arrays were captured and analyzed with a Kodak 4000MM Image Station. Net signal intensity for each analyte was expressed as percentage of the internal reference standard for each individual array membrane. Data are presented as mean ± SEM. Comparisons between groups were performed by one-way ANOVA. A Bonferroni's post hoc multiple comparison test was used to assess significant differences revealed by the ANOVA. Significance was accepted at P ≤ 0.05.[2]

Rats: Male Wistar Han rats are fasted for 48 hours and then given a single dose of bempedoic acid before receiving a second 48-hour feeding of a high-carbohydrate diet. Rats are kept on a standard chow diet and given oral gavage doses of bempedoic acid for a two-week assessment. The dose is 30 mg/kg/day given in the morning. Food is discontinued two hours before the final oral dose of engine control or bempedoic acid after nutritional staging and/or dosing[1].

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For in vivo experiments, ETC-1002 dosing solutions were formulated by preparing a disodium salt aqueous solution using 2:1 molar ratio of NaOH to ETC-1002 in water. Carboxymethyl cellulose (CMC) and Tween-20 were added to make a final solution containing 0.5% CMC and 0.025% Tween with a final pH 7–8. Compound concentrations in dosing solutions were administered at a volume of 10 ml/kg body.[1],2]

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Wistar rats.[1]
Male Wistar Han [Crl:WI] rats weighing 225–250 g were acclimated to the laboratory environment for seven days, housed 2–3 per cage in a temperature controlled room, and maintained on a 12 h light and dark cycle with ad libitum access to food and water. Prior to single-dose ETC-1002 administration, rats were fasted for 48 h and refed a high-carbohydrate diet for an additional 48 h. For two-week assessment, rats were maintained on standard chow diet (Purina 5001) and dosed by oral gavage with ETC-1002 at 30 mg/kg/day for two weeks in the morning. Following nutritional staging and/or dosing, food was withdrawn 2 h prior to last the oral dose of vehicle control or ETC-1002. Blood and liver were collected from isofluorane-anesthetized animals 2 or 8 h after the last dose, blood was collected from the subclavian vein, and liver tissue was harvested by freeze clamp. The freeze-clamped liver samples were held frozen in liquid nitrogen immediately following excision and stored at −70°C. Plasma triglycerides, β-hydroxybutyrate (β-HBA), and total cholesterol levels were measured with commercially available kits (Wako Diagnostics, Richmond, VA) adapted to a 96-well format.

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Golden Syrian hamsters.[1]
Male golden Syrian hamsters were obtained from Charles River (Montreal, QC) at 8–10 weeks of age and weighed 100–120 g. Animals were maintained on Prolab RMH 1000 standard rodent chow diet during a seven-day quarantine period. Following randomization into treatment groups (n = 6), hyperlipidemia was induced by feeding high-fat, high-cholesterol (HFHC) Prolab RMH 1000 diet containing: 11.5% coconut oil, 11.5% corn oil, 5% fructose, and 0.5% cholesterol. During the study, animals were individually housed in an environmentally controlled room with a 12 h light and dark cycle. Following two weeks on HFHC diet, hamsters were dosed by oral gavage once daily with vehicle (0.5% carboxymethyl cellulose and 0.025% Tween-20, pH 7–8) or vehicle plus ETC-1002 (30 mg/kg) for three weeks. Body weights were recorded every two days at the beginning of dosing, and food consumption was measured every four days. Blood samples were collected by administering isoflurane anesthesia and bleeding from the orbital venous plexus in lithium heparinized tubes during the study and by cardiac puncture under anesthesia at the end of the study. Plasma samples were analyzed for triglycerides, total cholesterol, nonesterified fatty acids, and β-hydroxybutyrate on an automated chemistry analyzer. Liver and epididymal fat were collected, weighed, frozen in liquid nitrogen, and stored at −80°C until processing. All hamster procedures were conducted in accordance with the current guidelines for animal welfare at the Hospital for Sick Children and were in compliance with National Institutes of Health Publication 86-23, 1985; Animal Welfare act, 1966, as amended in 1970, 1976, and 1985, 9 CFR Parts 1, 2, and 3.

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Diet-induced obesity in mice.[1]
Male C57BL/6N mice were obtained from Taconic at 8 weeks of age and singly housed on α-dri paper bedding on a normal 12 h light and dark cycle (6 AM to 6 PM). Upon arrival mice, were fed a high-fat diet (HFD) containing 60% kcal fat for 12 weeks. Mice were randomized into two treatment arms at 20 weeks of age based on 4 h fasted blood glucose and body weight and received oral dosing of either CMC/Tween vehicle or 30 mg/kg/day ETC-1002 q.d in the morning for an additional two weeks. Body weight and food consumption were monitored throughout the study. Following the two-week dosing period, food was removed at 8 AM, and bedding was changed 2 h prior to oral administration of ETC-1002. Two hours post dose, fasting samples were collected. Fasting blood glucose levels were measured immediately prior to anesthesia using a hand-held Alphatrak glucometer (Abbott, Chicago, IL), with blood collected by unrestrained tail snip. For insulin determinations, blood was collected under isoflurane anesthesia via retro-orbital sinus into EDTA-coated tubes, and plasma was isolated by centrifugation. Plasma insulin levels were measured with a commercially available ELISA

Absorption, Distribution and Excretion
Bempedoic acid is rapidly absorbed in the small intestine. The Tmax of the 180mg tablet is estimated at 3.5 hours.
Bempedoic acid's conjugates are primarily eliminated via the urine (70%) and the feces (30%). A total of 5% of the unchanged drug is excreted in the urine and feces, combined.
The apparent volume of distribution of bempedoic acid is about 18L.
The clearance (CL/F) of bempedoic acid at steady state was estimated at 11.2 mL/min during clinical trials.

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Metabolism / Metabolites
The two main metabolites of bempedoic metabolism are ETC-1002-CoA and ESP15228. Bempedoic acid is primarily eliminated via the metabolism of its acyl glucuronide. This drug is reversibly converted to an active metabolite (ESP15228) based on observations during in vitro studies. Both compounds resulting from the metabolism of bempedoic acid are metabolized to become inactive glucuronide conjugates by the enzyme UGT2B7.

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Biological Half-Life
The half-life of bempedoic acid ranges between 15 and 24 hours. Prescribing information indicates a clearance of 21 hours +/- 11 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No relevant published information exists on the use of bempedoic acid during breastfeeding. Bempedoic acid and its metabolites are 99% plasma protein bound, so amounts in milk are likely very low. However, because of a concern with disruption of infant lipid metabolism, bempedoic acid is best avoided during breastfeeding. An alternate drug is preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
The plasma protein binding of bempedoic acid and its metabolites is about 99%.

[1]. AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism. J Lipid Res. 2013 Jan;54(1):134-51.

[2]. ETC-1002 regulates immune response, leukocyte homing, and adipose tissue inflammation via LKB1-dependent activation of macrophage AMPK. J Lipid Res. 2013 Aug;54(8):2095-108.

Pharmacodynamics
Bempedoic acid inhibits the synthesis of cholesterol in the liver, reducing LDL-C levels. This reduces the development of atherosclerotic plaques that may increase the risk of cardiovascular events. Earlier clinical trials studying the effects of bempedoic acid showed a dose‐dependent reduction of LDL‐C levels in addition to decreased LDL particle number, and reduced levels of apolipoprotein B, non–HDL cholesterol, and high‐sensitivity C‐reactive protein. Due to its unique mechanism of action, bempedoic acid is not associated with myositis, an adverse effect that frequently accompanies statin therapy. More recent trials have supported that this drug significantly decreases LDL-C levels after 12 weeks of therapy and provides additional lowering of LDL-C when combined with ezetimibe and statin therapy. The effects of bempedoic acid on mortality are currently unknown.

C19H36O5

344.4861

344.256

C, 66.25; H, 10.53; O, 23.22

738606-46-7

Bempedoic acid-d4;2408131-70-2;Bempedoic acid-d5;2408131-71-3

10472693

White to light yellow solid powder

87-92

4.469

3

5

14

24

351

0

OC(CCCCCC(C(=O)O)(C)C)CCCCCC(C(=O)O)(C)C

HYHMLYSLQUKXKP-UHFFFAOYSA-N

InChI=1S/C19H36O5/c1-18(2,16(21)22)13-9-5-7-11-15(20)12-8-6-10-14-19(3,4)17(23)24/h15,20H,5-14H2,1-4H3,(H,21,22)(H,23,24)

8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid

ETC-1002; ETC 1002; ETC1002; ESP-55016; Bempedoate; 8-Hydroxy-2,2,14,14-tetramethylpentadecanedioic acid; Nexletol; Nilemdo; ESP-55016; Bempedoic acid; ETC-1002ESP55016; ETC1002ESP

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~290.3 mM)

Solubility in Formulation 1: ≥ 2.87 mg/mL (8.33 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.87 mg/mL (8.33 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.26 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (7.26 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.5 mg/mL (7.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 0.57 mg/mL (1.65 mM) in 1% DMSO + 99% Saline (add these co-solvents sequentially from left to right, and one by one),suspension solution;clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)