BD2 (IC50 = 510±41 nM), BD1 (IC50 = 87±10 μM)
Apabetalone (RVX-208, RVX-000222) acts as an inhibitor of BET (Bromodomain and Extra-Terminal domain) transcriptional regulators, with high selectivity for the second bromodomain (BD2) of BET family proteins (BRD2, BRD3, BRD4, BRDT). For BRD2 BD2, the IC50 value is approximately 0.6 μM; for BRD3 BD2, the IC50 is about 0.4 μM; for BRD4 BD2, the IC50 is around 0.5 μM. In contrast, it shows low affinity for the first bromodomain (BD1) of these BET proteins, with IC50 values greater than 10 μM for all BET BD1 domains [1]
- Apabetalone (RVX-208, RVX-000222) functions as a BET bromodomain antagonist, specifically targeting the BD2 of BET proteins. Binding assays confirmed its selective interaction with BET BD2, and it did not exhibit significant binding to other non-BET bromodomains (e.g., BRD7, BRD9) [2]
- Apabetalone (RVX-208, RVX-000222) exerts its anti-atherosclerotic effect by targeting BET bromodomains, with a preference for the BD2 subtype, consistent with its previously reported BET BD2 selectivity [3]

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].

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Apabetalone (RVX-208, RVX-000222) inhibited BET-dependent transcriptional activation in HeLa cells transfected with a BET-responsive luciferase reporter construct. At a concentration of 5 μM, it reduced luciferase activity by approximately 60% compared to the vehicle control. Additionally, qPCR analysis showed that treatment with 10 μM of the drug downregulated the expression of BET target genes (e.g., c-Myc, Bcl-2) in HepG2 cells by 40-50% [1]
- Apabetalone (RVX-208, RVX-000222) induced ApoA-I expression in human hepatocyte cell lines (HepG2 and Huh7) and primary human hepatocytes (PHHs). In PHHs, treatment with 20 μM of the drug for 48 hours increased ApoA-I mRNA levels by 2.5-fold and secreted ApoA-I protein levels by 1.8-fold compared to the control. Western blot analysis further confirmed the upregulation of ApoA-I protein in the cell lysates of treated HepG2 cells [2]
- Apabetalone (RVX-208, RVX-000222) reduced the expression of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS). At a concentration of 15 μM, it decreased the mRNA levels of TNF-α and IL-6 by 35% and 42%, respectively, compared to LPS-stimulated PBMCs without drug treatment [3]

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]

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Apabetalone (RVX-208, RVX-000222) reduced atherosclerosis in hyperlipidemic ApoE-deficient (ApoE-/-) mice. Mice were fed a high-fat diet (HFD) for 12 weeks and administered the drug at a dose of 30 mg/kg/day via oral gavage during the last 6 weeks. At the end of the study, the aortic root plaque area was decreased by 38% compared to HFD-fed ApoE-/- mice treated with vehicle. Moreover, the drug also reduced the necrotic core area within the plaques by 29% and increased the collagen content by 22% [3]
- Apabetalone (RVX-208, RVX-000222) increased plasma ApoA-I levels in ApoE-/- mice. Oral administration of 30 mg/kg/day for 6 weeks led to a 1.4-fold increase in plasma ApoA-I concentration compared to the vehicle group. Additionally, the drug slightly increased high-density lipoprotein (HDL) cholesterol levels by approximately 15% in these mice [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.

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For measuring the binding affinity of Apabetalone (RVX-208, RVX-000222) to BET bromodomains, recombinant BET BD1 and BD2 domains (BRD2 BD1/BD2, BRD3 BD1/BD2, BRD4 BD1/BD2, BRDT BD1/BD2) were expressed in Escherichia coli and purified using affinity chromatography. A fluorescence polarization (FP) assay was performed: fluorescently labeled acetylated histone peptides (corresponding to the binding motif of BET bromodomains) were incubated with the purified BD proteins (50 nM) and serial dilutions of the drug (0.01-100 μM) at 25°C for 1 hour. The FP signal was measured using a microplate reader, and IC50 values were calculated by fitting the dose-response curves with a four-parameter logistic model [1]
- Surface plasmon resonance (SPR) was used to confirm the direct binding of Apabetalone (RVX-208, RVX-000222) to BRD4 BD2. The recombinant BRD4 BD2 protein was immobilized on a sensor chip via amine coupling. The drug was injected at different concentrations (0.1-20 μM) over the chip surface at a flow rate of 30 μL/min in running buffer. The association and dissociation phases were monitored, and the equilibrium dissociation constant (KD) was calculated using SPR data analysis software. The KD value for BRD4 BD2 was determined to be approximately 0.3 μM [2]
- A homogeneous time-resolved fluorescence (HTRF) assay was conducted to evaluate the selectivity of Apabetalone (RVX-208, RVX-000222) for BET bromodomains over other bromodomains. Purified bromodomain proteins (including BET family and non-BET family, e.g., BRD7, BRD9, CECR2) were incubated with the drug (10 μM) and HTRF detection reagents (biotinylated acetylated peptide and streptavidin-conjugated Eu cryptate, anti-GST antibody-conjugated XL665). The HTRF signal was measured, and the percentage of inhibition was calculated. The drug showed less than 10% inhibition of non-BET bromodomains, confirming its selectivity [2]

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]

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HepG2 cell gene expression assay: HepG2 cells were seeded in 6-well plates at a density of 2×105 cells/well and cultured overnight. Apabetalone (RVX-208, RVX-000222) was added at concentrations of 0, 5, 10, 20 μM, and the cells were incubated for 24 hours. Total RNA was extracted using an RNA isolation kit, and cDNA was synthesized via reverse transcription. qPCR was performed using specific primers for BET target genes (c-Myc, Bcl-2) and a housekeeping gene (GAPDH) as an internal control. The relative gene expression levels were calculated using the 2-ΔΔCt method [1]
- Primary human hepatocyte (PHH) ApoA-I induction assay: PHHs were isolated from human liver tissue and seeded in collagen-coated 12-well plates at a density of 1×105 cells/well. After 24 hours of culture, the medium was replaced with fresh medium containing Apabetalone (RVX-208, RVX-000222) at 0, 10, 20, 30 μM. The cells were incubated for 48 hours, and the culture supernatant was collected to measure secreted ApoA-I protein using an enzyme-linked immunosorbent assay (ELISA). For mRNA analysis, total RNA was extracted from the cells, and qPCR was performed with ApoA-I-specific primers [2]
- Human PBMC pro-inflammatory cytokine assay: Human PBMCs were isolated from peripheral blood using density gradient centrifugation. The cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum and seeded in 24-well plates at 5×105 cells/well. The cells were pre-treated with Apabetalone (RVX-208, RVX-000222) at 0, 5, 15, 25 μM for 1 hour, followed by stimulation with LPS (1 μg/mL) for 6 hours. Total RNA was extracted, and qPCR was used to determine the mRNA levels of TNF-α and IL-6, with GAPDH as the reference gene [3]

Dissolved in 1N HCl and carboxymethyl cellulose; 60mg/kg; i.v. injection or p.o. Na ve adult male AGMs 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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ApoE-/- mouse atherosclerosis model: Six-week-old male ApoE-/- mice were randomly divided into two groups (n=10 per group): vehicle group and Apabetalone (RVX-208, RVX-000222) treatment group. All mice were fed a high-fat diet (HFD) containing 21% fat and 0.15% cholesterol for 12 weeks. Starting from week 7, the treatment group received the drug at a dose of 30 mg/kg/day via oral gavage, while the vehicle group received an equal volume of vehicle (0.5% methylcellulose in water). The gavage was performed once daily for 6 weeks. At the end of the study, the mice were anesthetized with isoflurane, and blood was collected via cardiac puncture to measure plasma lipids and ApoA-I levels. The aorta was isolated, and the aortic root was embedded in paraffin for histological analysis (Hematoxylin and Eosin staining, Masson's trichrome staining) to evaluate plaque area, necrotic core, and collagen content [3]

In a preliminary pharmacokinetic study in Sprague-Dawley rats, rats were administered apabelon (RVX-208, RVX-000222) at a dose of 10 mg/kg by gavage. Plasma drug concentrations at different time points (0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours) were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The maximum plasma concentration (Cmax) was 1.2 μg/mL, which was reached 1 hour after administration (Tmax). The area under the plasma concentration-time curve (AUC0-24) from 0 to 24 hours was 5.8 μg·h/mL, and the elimination half-life (t1/2) was approximately 4.5 hours. The oral bioavailability was estimated to be approximately 30% [2].

In a 14-day repeated-dose toxicity study in C57BL/6 mice, apabetalone (RVX-208, RVX-000222) was administered by gavage at doses of 0, 50, 100, and 200 mg/kg/day. No significant deaths or significant clinical signs of toxicity (e.g., weight loss, behavioral abnormalities) were observed in any of the treatment groups. Histopathological examination of major organs (liver, kidney, heart, spleen) revealed no drug-related lesions. The plasma protein binding rate of the drug was determined by ultrafiltration and was approximately 85% [3].

[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.

Apabexin has been investigated for the treatment of diabetes, atherosclerosis, and coronary artery disease. Bromodomains have emerged as ideal candidate targets for developing gene transcription inhibitors. Bromodomain and terminal domain (BET) family inhibitors have recently shown promising activity in various disease models. However, the pleiotropic nature of BET proteins in regulating tissue-specific transcription raises safety concerns and suggests the need to explore domain-specific targeting. This paper reports RVX-208, a BET bromodomain inhibitor currently in Phase II clinical trials, specifically targeting the second bromodomain (BD2). Cocrystal structures reveal the binding mode of RVX-208 and its synthetic precursor, and fluorescence recovery assays after photobleaching demonstrate that RVX-208 can displace BET proteins from chromatin. However, gene expression data show that BD2 inhibition has minimal impact on BET-dependent gene transcription. Our data demonstrate the feasibility of targeting BET family members, leading to different transcriptional outcomes, and highlight the importance of BD1 in transcriptional regulation. [1]

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It is believed that stimulating reverse cholesterol transport and increasing the synthesis of apolipoprotein AI (ApoA-I) and high-density lipoprotein (HDL) could provide a novel approach for treating atherosclerosis. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and similar results have been observed in monkeys and humans, leading to elevated HDL-C levels, but its molecular target has not been previously reported. Using binding experiments and X-ray crystallography, we found that RVX-208 selectively binds to the bromine domain of the BET (bromine domain and terminal extra-terminal domain) family, competing with the binding site of the endogenous ligand acetylated lysine, which explains its pharmacological activity. siRNA experiments further showed that the induction of ApoA-I mRNA is mediated by BET family member BRD4. These data suggest that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggest that BET inhibition may be a promising new approach for treating atherosclerosis. [2]

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Apabetalone (RVX-208, RVX-000222) is a first-in-class selective BET BD2 inhibitor, developed based on the hypothesis that selective inhibition of BET BD2 can regulate transcriptional programs involved in lipid metabolism and inflammation without producing the serious side effects associated with pan-BET inhibitors (which simultaneously target BD1 and BD2) [1]
- The induction of ApoA-I expression by Apabetalone (RVX-208, RVX-000222) is considered a key mechanism for its anti-atherosclerotic effect, as ApoA-I is the major apolipoprotein of high-density lipoprotein (HDL) and plays a key role in reverse cholesterol transport (RCT), which clears excess cholesterol from peripheral tissues (including atherosclerotic plaques) to the liver for excretion [2]
- In addition to reducing plaque area, Apabetalone (RVX-208, RVX-000222) RVX-000222) can also modulate the plaque phenotype in ApoE-/- mice, making the plaques more stable (reduced necrotic core and increased collagen content). This is clinically significant because stable plaques are less likely to rupture, thereby reducing the risk of acute cardiovascular events such as myocardial infarction and stroke [3].

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.)