BD2 (IC50 = 510±41 nM), BD1 (IC50 = 87±10 μM)

Apabetalone (RVX-208) competes with binding of an acetylated histone peptide to tandem BD1 BD2 protein constructions of the four BET proteins, exhibiting IC50s between 0.5 and 1.8 µM. Apabetalone promotes the formation of ApoA-I in hepatocytes in vitro, which leads in increased high density lipoprotein cholesterol (HDL-C). Apabetalone predominantly binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a position bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological effect. increases Apolipoprotein AI (ApoA-I) synthesis through an epigenetic mechanism and shows that BET inhibition may be a promising new approach to the treatment of atherosclerosis. Apabetalone promotes ApoA-I expression in liver cells[2].

In the study design for the preventive treatment of atherosclerosis, mice are given a Western diet along with 150 mg/kg/dose bid of medication for a duration of 12 weeks. A year after therapy, mice are killed. Both the vehicle treated and the Apabetalone (RVX-208) treated groups show a progressive increase in body weight. After 12 weeks on a Western diet, the Apabetalone treated group's body weight increased by 4 g (from 24 g to 28 g), while the vehicle treated group experienced a 9 g gain (from 25 g to 34 g). The notable reduction in body weight increase observed in mice treated with Apabetalone is not attributable to a decrease in feed intake, indicating a favorable characteristic of the compound. After six and twelve weeks of therapy with either the vehicle or apibetalone, plasma lipid measurements are performed. At six weeks into therapy, mice treated with apibetalone had a considerable rise (~200%) in their HDL-C levels compared to the vehicle control animals. This increase persisted until the 12-week study's conclusion[3].

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Despite the benefit of statins in reducing cardiovascular risk, a sizable proportion of patients still remain at risk. Since HDL reduces CVD risk through a process that involves formation of pre-beta particles that facilitates the removal of cholesterol from the lipid-laden macrophages in the arteries, inducing pre-beta particles, may reduce the risk of CVD. A novel BET bromodomain antagonist, Apabetalone (RVX-208), was reported to raise apoA-I and increase preβ-HDL particles in non-human primates and humans. In the present study, we investigated the effect of Apabetalone (RVX-208) on aortic lesion formation in hyperlipidemic apoE(-/-) mice. Oral treatments of apoE(-/-) mice with 150 mg/kg b.i.d RVX-208 for 12 weeks significantly reduced aortic lesion formation, accompanied by 2-fold increases in the levels of circulating HDL-C, and ∼50% decreases in LDL-C, although no significant changes in plasma apoA-I were observed. Circulating adhesion molecules as well as cytokines also showed significant reduction. Haptoglobin, a proinflammatory protein, known to bind with HDL/apoA-I, decreased >2.5-fold in the Apabetalone (RVX-208) treated group. With a therapeutic dosing regimen in which mice were fed Western diet for 10 weeks to develop lesions followed by switching to a low fat diet and concurrent treatment with RVX-208 for 14 weeks, RVX-208 similarly reduced lesion formation by 39% in the whole aorta without significant changes in the plasma lipid parameters. Apabetalone (RVX-208) significantly reduced the proinflammatory cytokines IP-10, MIP1(®) and MDC. These results show that the antiatherogenic activity of BET inhibitor, RVX-208, occurs via a combination of lipid changes and anti-inflammatory activities.[3]

Protein Stability Shift Assay (Tm Assay).[1]
Thermal melting experiments were carried out using an Mx3005p Real-Time PCR machine as previously described. Temperature shifts (An external file that holds a picture, illustration, etc. Object name is pnas.1310658110i2.jpg) for three independent measurements per protein/compound are summarized in Table S1.[1]

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Competitive Histone Displacement Assay (AlphaScreen Assay).[1]
Experiments were run on a PHERAstar FS plate reader using an AlphaScreen 680 excitation/570 emission filter set. IC50 values were calculated in Prism 5 after normalization against corresponding DMSO controls. Assays were performed as previously described, with minor modifications from the manufacturer’s protocol.[1]

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Isothermal Titration Calorimetry. [1]
Experiments were carried out on an ITC200 microcalorimeter from MicroCal at 15 °C in 50 mM Hepes, pH 7.5 (at 25 °C), 150 mM NaCl by titrating protein into ligand solutions (reverse titrations), and data were corrected for protein heats of dilution and deconvoluted using the MicroCal Origin software as previously described. Dissociation constants and thermodynamic parameters are listed in Tables S2 and S3.[1]

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Fluorescent Recovery After Photobleaching. [1]
Fluorescent recovery after photobleaching (FRAP) studies were performed in U2OS cells transfected with mammalian overexpression constructs encoding GFP chimeras of BRD3, using a Zeiss LSM 710 scanhead coupled to an inverted Zeiss Axio Observer.Z1 microscope equipped with a high-numerical-aperture (N.A. 1.3) 40× oil immersion objective equipped with a heated chamber set to 37 °C, using a protocol modified from previous studies.[1]

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Protein Thermal Denaturation Assay[2]
5 µM of purified bromodomain protein was incubated with 5X SYPRO® Orange at a final concentration of 20 mM HEPES pH 7.4, 100 mM NaCl in the presence of 100 µM compound or DMSO (0.2%) in a fast 96 well optical plate. Samples were incubated at room temperature for 30 minutes and ramped from 25°C to 95°C in a ViiA7 real-time PCR machine. The resulting fluorescence data was analyzed and melting temperatures calculated using Protein Thermal Shift™ Software v1.0.[2]

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Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay[2]
200 nM N-terminally His-tagged bromodomains or BRD4(BD1BD2) and 25–50 nM biotinylated tetra-acetylated histone H4 peptide were incubated in the presence of Europium Cryptate-labeled streptavidin and XL665-labeled monoclonal anti-His antibody in a white 96 well microtiter plate. For inhibition assays, serially diluted compound was added to these reactions in a 0.2% final concentration of DMSO. Final buffer concentrations were 30 mM HEPES pH 7.4, 30 mM NaCl, 0.3 mM CHAPS, 20 mM PO4 pH 7.0, 320 mM KF, 0.08% BSA). After 2 h incubation at room temperature, the fluorescence by FRET was measured at 665 and 620 nm by a SynergyH4 plate reader. IC50 values were determined from a dose response curve.[2]

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Isothermal Calorimetry[2]
150 µM BRD2[BD1], BRD2[BD2], BRD4[BD1] or BRD4[BD2]in 50 mM HEPES pH 7.5, 150 mM sodium chloride and 0.05% DMSO was injected into a solution containing 10 µM RVX-208 in the same buffer at 25°C, and the associated change in heat measured in a Microcal Auto-ITC instrument.

Cell culture[2]
Huh7 cells were plated at 23,000/well in a 96 well plate in DMEM +10% FBS before allowing to grow overnight. Cells were treated with compounds for 48 h in 0.1%DMSO with or without 5 µM Actinomycin D. U937 cells were differentiated for 3 days in 60 ng/mL PMA, 32,000 cells/well in 96-well format. Cells were then treated with compound in 0.1%DMSO in RPMI media +10%FBS, and after 1 h, lipopolysaccharide (LPS) was added to the cells at 1 µg/mL for 3 hours.[2]

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RT-PCR[2]
Cells were harvested by mRNA Catcher PLUS Kit followed by real-time PCR using the RNA UltraSense One-Step qRT-PCR System. ApoA-I, IL-6 and TNFα mRNA levels were measured relative to the endogenous control serpin A1 or cyclophilin in the same sample. Data was acquired using the 7500 Real Time PCR System.[2]

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Cell Culture and RNA Extraction. HepG2 cells were treated so that a final concentration of 0.1% DMSO was achieved. Cells were harvested, washed, and lysed in situ using standard protocols. Total RNA was extracted and prepared using RNeasy columns, and RNA was quantified and quality controlled using a Nanodrop spectrophotometer.[1]

The test article Apabetalone (RVX-208), 2-(4-(2-Hydroxymethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one, was prepared in formulation EA006, and stored in refrigerator at 4 °C for up to 1 week. Formulation EA006 includes an in situ HCl salt formation of Apabetalone (RVX-208) followed by suspension in a Polyethylene glycol-300/polysorbate-80 based vehicle after pH adjustment to 2.5–3.0. Each preparation of dosing solution was subjected to verification of the test compound concentration by collecting 0.2 mL of aliquot in duplicate.[3]

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Atherosclerosis prophylactic study design[3]
Eight week old apoE−/− mice were first acclimated for a week on rodent chow diet and following a 4 h fast blood was drawn under ether anesthesia and lipid measurements were done. Based on the body weight and lipid values, mice were divided into 2 groups (n = 12): group 1, vehicle; and group 2, test agent, Apabetalone (RVX-208). Mice were then switched to Western diet (0.15% cholesterol and 42% calories from fat) and concurrently treated orally by gavage with either vehicle or the test agent, Apabetalone (RVX-208) (150 mg/kg/dose b.i.d) for 12 weeks. After 6 week of treatment, an interim blood draw was done to monitor serum lipid levels. After 12 weeks of treatment mice were sacrificed to measure blood lipid parameters, aortic lesion, and liver and aortic RNA. Eight mice were used for enface (aortic plaque) analysis, 4 mice for tissue collection for mRNA and all 12 mice used for aortic sinus lesion area measurement.

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Atherosclerosis therapeutic study design[3]
Eight week old apoE−/− mice were first acclimated for a week on rodent chow diet. Mice were then switched to Western diet (0.15% cholesterol and 42% calories from fat) ad libitum for 10 weeks in order to allow the development of lesion formation. Baseline lipid measurements after feeding Western diet for 10 weeks were carried out following 4 h fast, and all mice were switched to rodent chow diet and concurrently treated with either vehicle or Apabetalone (RVX-208) (150 mg/kg/dose, b.i.d) by oral gavage for 14 weeks. After 6 week of treatment, an interim blood draw was done to monitor serum lipid levels. After 14 weeks of treatment mice were sacrificed to measure blood lipid parameters, aortic plaque and sinus lesion, liver and aortic mRNA as described above in section.

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Dissolved in 1N HCl and carboxymethyl cellulose; 60mg/kg; i.v. injection or p.o.

Na ve adult male AGMs

[1]. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain. Proc Natl Acad Sci U S A. 2013 Dec 3;110(49):19754-9.

[2]. RVX-208, an inducer of ApoA-I in humans, is a BET bromodomain antagonist. PLoS One. 2013 Dec 31;8(12):e83190.

[3]. A novel BET bromodomain inhibitor, RVX-208, shows reduction of atherosclerosis in hyperlipidemic ApoE deficient mice. Atherosclerosis. 2014 Sep;236(1):91-100.

Apabetalone has been investigated for the treatment of Diabetes, Atherosclerosis, and Coronary Artery Disease.

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Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.[1]

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Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.[2]

C20H22N2O5

370.4

370.152

C, 64.85; H, 5.99; N, 7.56; O, 21.60

1044870-39-4

135564749

Typically exists as White to yellow solids at room temperature

1.3±0.1 g/cm3

1.596

2.04

2

6

6

27

543

0

O(C([H])([H])C([H])([H])O[H])C1C(C([H])([H])[H])=C([H])C(=C([H])C=1C([H])([H])[H])C1=NC2C([H])=C(C([H])=C(C=2C(N1[H])=O)OC([H])([H])[H])OC([H])([H])[H]

NETXMUIMUZJUTB-UHFFFAOYSA-N

InChI=1S/C20H22N2O5/c1-11-7-13(8-12(2)18(11)27-6-5-23)19-21-15-9-14(25-3)10-16(26-4)17(15)20(24)22-19/h7-10,23H,5-6H2,1-4H3,(H,21,22,24)

2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one

RVX-000222; RVX208; RVX 000222; RVX 208; RVX000222; RVX-208; 2-(4-(2-Hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one; RVX-000222; RVX 208; Apabetalone [INN]; RVX000222; Apabetalone.

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.75 mM) (saturation unknown) 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 2: ≥ 2.5 mg/mL (6.75 mM) (saturation unknown) 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 3: ≥ 2.5 mg/mL (6.75 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 4: ≥ 2.5 mg/mL (6.75 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 5: ≥ 2.5 mg/mL (6.75 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: ≥ 0.5 mg/mL (1.35 mM) (saturation unknown) in 1% DMSO 99% 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 7: 0.5% CMC Na (1N HCl, PH 2.5-3.0):8 mg/mL

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Solubility in Formulation 8: 15.15 mg/mL (40.90 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.)