BET bromodomain-containing proteins (BRD2, BRD3, BRD4), with selectivity for BD2 (second bromodomain). RVX-297 has 47-58 fold greater affinity for BD2 than for BD1. [1]

In synovial fibroblasts, RVX-297 (1-30 μM; 24 hours) decreases the expression of pro-inflammatory genes [1]. Similar to pan-BET inhibitors that non-selectively bind BET BD, RVX-297 displaces BET proteins from sensitive gene promoters and inhibits the recruitment of active RNA polymerase II [1]. In vitro, RVX-297 decreases the gene expression of inflammatory mediators. In a dose-dependent way, RVX-297 suppresses the induction of the IL-6 gene in human U937 macrophages, mice primary B cells derived from the spleen, mouse BMDM, and THP-1 monocytes. With an IC50 of 0.4-3 μM, RVX-297 suppresses the expression of IL-1β in LPS-stimulated mouse BMDM. RVX-297 has an IC50 of 0.4 μM for inhibiting MCP-1 expression in unstimulated human PBMC. RVX-297 suppresses T cell antigen stimulation and IL-17 expression induction [1].

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Suppressed inflammatory gene expression in multiple immune cell types. In human U937 macrophages, mouse primary B-cells, mouse bone marrow-derived macrophages (BMDMs), and THP-1 monocytes, RVX-297 suppressed IL-6 gene induction in a dose-dependent manner. In LPS-stimulated mouse BMDMs, IL-1β expression was repressed. It inhibited MCP1 expression in unstimulated human PBMCs. [1]
In human PBMCs stimulated with OKT3, RVX-297 inhibited IL-17 gene expression. [1]
In human rheumatoid arthritis synovial fibroblasts stimulated with TNFα, RVX-297 (1, 3, 10 μM) downregulated IL-6 and VCAM-1 gene expression in a dose-dependent manner after 24 hours. [1]
In primary mouse BMDMs stimulated with LPS, RVX-297, as well as pan-BETis JQ1 and I-BET762, inhibited the upregulation of IL-6, IL-1β, and TNFα gene expression. The IC50 for IL-6 inhibition in this system was 3.0 μM. [1]
Mechanism of action: Chromatin immunoprecipitation (ChIP) studies in mouse BMDMs showed that RVX-297 (10 μM) treatment reduced the association of BRD3 and BRD4, but not BRD2, with the IL-6 promoter; reduced BRD4 abundance on the IL-1β promoter; and reduced the amount of active RNA polymerase II (RNA pol II P-Ser2) on the IL-6 and IL-1β promoters. It did not affect BET protein abundance on the NF-κBia promoter, nor did it change the level of acetylated histone H4 on the IL-6 promoter. [1]

In an arthritic model caused by collagen in rats, RVX-297 (25–75 mg/kg; oral; once daily for 6 days) prevents disease advancement [1]. In mice with collagen-induced arthritis, RVX-297 (75–150 mg/kg) prevents the arthritis model's pathogenic advancement [1]. LPS-treated mice produce less cytokines when RVX-297 is administered [1].

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LPS-induced endotoxemia model: In C57Bl/6 mice stimulated with LPS, RVX-297 (75 mg/kg, oral, 4 hours pre- and at stimulation) reduced circulating serum levels of IL-6, IL-17, and IFNγ. It also decreased IL-6 and IL-17 gene expression in the spleen. Multi-analyte profiling showed lower levels of GM-CSF, MCP-1, MCP-5, IL-2, TNFα, and RANTES in the serum. [1]
Rat Collagen-Induced Arthritis (rCIA) model: In female Lewis rats with established arthritis, RVX-297 (25, 50, 75 mg/kg, oral, b.i.d. for 6 days) attenuated the increase in ankle diameter. It reduced synovitis, cartilage damage, pannus formation, and bone resorption in ankle and knee joints. It significantly decreased IL-1β, RANKL, MMP3, and MMP13 gene expression, and IL-6 and VCAM-1 protein levels in ankle joints. [1]
Mouse Collagen-Induced Arthritis (mCIA) model: RVX-297 (75, 150 mg/kg, oral, b.i.d.) prevented increases in clinical arthritis score and reduced histopathology scores in paws and knee. It also significantly lowered anti-collagen II IgG serum levels. [1]
Mouse Collagen Antibody-Induced Arthritis (mCAIA) model: In mice with established disease, RVX-297 (150 mg/kg, oral, b.i.d., for 9 days) prevented progression of clinical manifestations and significantly reduced histopathology scores. [1]
Mouse Experimental Autoimmune Encephalomyelitis (EAE) model:
Preventative regimen: RVX-297 (75, 125 mg/kg, oral, b.i.d., for 27 days) suppressed the rise in EAE clinical score and prevented EAE-associated weight loss. Histological examination of spinal cords showed no inflammatory foci, demyelination, or apoptotic cells, making them indistinguishable from naïve mice. It decreased inflammatory gene expression (IL-6, IL-17, MCP-1, GM-CSF, TNFα) in the spinal cord and reduced the percentage of CD4+ cells producing IL-6, GM-CSF, and IFNγ in the CNS. [1]
Therapeutic regimen: RVX-297 (75, 125/100 mg/kg, oral, b.i.d., for 18 days) countered the increase in EAE clinical scores in a dose-dependent manner and reduced EAE-associated weight loss. It significantly reduced inflammation, demyelination, and apoptotic cells in the spinal cord. [1]
Ex vivo assessment: Lymphocytes isolated from RVX-297-treated EAE mice and re-challenged with MOG35-55 showed substantially reduced secretion of IL-17a, IL-6, TNFα, and IFNγ into the culture media. [1]

Chromatin Immunoprecipitation (ChIP): Mouse bone marrow-derived macrophages (BMDMs) were plated and treated with compound (RVX-297 or DMSO) for 1 hour. LPS (1 μg/mL) was then added, and incubation continued for 3 hours. Cells were crosslinked with 1% formaldehyde, quenched with glycine, and chromatin was sheared by sonication. Immunoprecipitation was performed with antibodies against BRD2, BRD3, BRD4, RNA polymerase II, tetra-acetylated Histone H4, or IgG. After de-crosslinking, DNA was isolated and real-time PCR was performed to determine the relative abundance of these proteins at gene promoters (IL-6, IL-1β, NF-κBia). [1]

RT-PCR[1]
Cell Types: synovial fibroblasts
Tested Concentrations: 1-30 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: IL-6 and VCAM-1 gene expression was downregulated in synovial fibroblasts.

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U937 Cell Treatment: U937 cells were differentiated to macrophage-like cells with 60 ng/mL phorbol 12-myristate 13-acetate (PMA) for 3 days. Cells were pretreated for 1 hour with RVX-297 or DMSO, then LPS (1 μg/mL) was added. Cells were incubated for 3 hours before gene expression analysis by real-time PCR. [1]
Primary B-cell Isolation and Treatment: B-cells were isolated from C57/Bl6 mouse spleens using CD43 beads. Cells were plated and treated with RVX-297 or DMSO, LPS (1 μg/mL), and ionomycin (1 μM) for 3 hours, followed by gene expression analysis. [1]
IL-17 Expression in Human PBMCs: Human PBMCs were thawed and pre-treated with RVX-297 or DMSO for 1 hour. Media containing OKT3 (1 μg/mL) was then added, and incubation continued for 3 hours. IL-17 gene expression was analyzed by real-time PCR. [1]
MCP1 Assay in PBMCs: Cryopreserved human PBMCs were treated with RVX-297 or DMSO for 3 hours, and MCP1 gene expression was analyzed by real-time PCR. [1]
Mouse Bone Marrow-Derived Macrophage (BMDM) Isolation and Treatment: BMDMs were isolated from mouse femurs and differentiated for 6 days. Cells were re-plated and treated with RVX-297 or DMSO for 1 hour, followed by LPS (1 μg/mL) stimulation for 3 hours. Expression of IL-6 and IL-1β was analyzed by real-time PCR. [1]
Human Rheumatoid Arthritis Synovial Fibroblasts: Synovial fibroblasts were plated and treated identically to U937 cells, except stimulation was induced with 30 ng/mL TNFα. Real-time PCR was performed after 24 hours of treatment. [1]
Real-time PCR: mRNA was isolated from cell cultures. Gene expression was measured by TaqMan-based real-time PCR. mRNA levels were measured relative to an endogenous control. [1]

Animal/Disease Models: Female Lewis rat, 6-8 weeks old, approximately 150 g (rat collagen-induced arthritis) [1]
Doses: 25, 50 and 75 mg/kg
Route of Administration: Po; one time/day for 6 Day
Experimental Results: Prevents swelling and inflammation of ankle and knee joints.

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Compound Formulation:** RVX-297 was formulated for oral administration in formulation EA006. The concentration of the test compound in each preparation was verified by LC/MS/MS. [1]
* **LPS Induced Endotoxemic Mouse Model:** Eight-week-old male C57Bl/6 mice received LPS by intraperitoneal injection. RVX-297 was administered orally at 75 mg/kg 4 hours before and again at the time of LPS stimulation. For serum cytokine determinations, animals received 5 μg of LPS, and serum was collected 4 hours post-LPS. For gene expression analysis, animals received 10 μg of LPS, and spleens were collected 3 hours post-LPS. [1]
* **Rat Collagen-Induced Arthritis (rCIA):** Female Lewis rats (6-8 weeks old) were immunized with bovine type II collagen emulsified with incomplete Freund's adjuvant (IFA) via intradermal injection on day 0, with a booster on day 7. Beginning on day 11 (arthritis phase), RVX-297 was administered orally twice per day (b.i.d.) for 6 days at 25, 50, and 75 mg/kg/dose. Efficacy was evaluated by body weight, ankle diameter measurement, histopathology of ankle and knee, and gene expression/cytokine levels in the ankle. [1]
* **Mouse Collagen Antibody Induced Arthritis (mCAIA):** Female BALB/cAnNHsd mice were injected intravenously with a cocktail of four arthritogenic monoclonal antibodies to collagen-II. After 24 hours, mice received 25 μg of LPS by intraperitoneal injection. Twice per day oral administration of RVX-297 (150 mg/kg) was initiated 30 hours following LPS and continued for 9 days. Efficacy was evaluated by body weight, clinical arthritis scores, combined hind paw weights, and histologic assessment. [1]
* **Murine Experimental Autoimmune Encephalomyelitis (EAE):** EAE was induced in female C57Bl/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) in complete Freund's adjuvant (CFA). Pertussis toxin was administered by intraperitoneal injection 2 and 24 hours after immunization. RVX-297 was delivered by oral gavage at 75 and 125 mg/kg twice per day (b.i.d.) for 18 days in a therapeutic regimen (starting at the first sign of EAE) or for 27 days in a preventative regimen (starting immediately after immunization). In the therapeutic regimen, the 125 mg/kg dose was adjusted to 100 mg/kg b.i.d. on day 10 due to tolerability. EAE development and clinical score were evaluated based on clinical manifestations, and spinal cords were collected for gene expression analysis, flow cytometry, and histological assessment. [1]

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Compound Formulation: RVX-297 was formulated for oral administration in formulation EA006. The concentration of the test compound in each preparation was verified by LC/MS/MS. [1]
LPS Induced Endotoxemic Mouse Model: Eight-week-old male C57Bl/6 mice received LPS by intraperitoneal injection. RVX-297 was administered orally at 75 mg/kg 4 hours before and again at the time of LPS stimulation. For serum cytokine determinations, animals received 5 μg of LPS, and serum was collected 4 hours post-LPS. For gene expression analysis, animals received 10 μg of LPS, and spleens were collected 3 hours post-LPS. [1]
Rat Collagen-Induced Arthritis (rCIA): Female Lewis rats (6-8 weeks old) were immunized with bovine type II collagen emulsified with incomplete Freund's adjuvant (IFA) via intradermal injection on day 0, with a booster on day 7. Beginning on day 11 (arthritis phase), RVX-297 was administered orally twice per day (b.i.d.) for 6 days at 25, 50, and 75 mg/kg/dose. Efficacy was evaluated by body weight, ankle diameter measurement, histopathology of ankle and knee, and gene expression/cytokine levels in the ankle. [1]
Mouse Collagen Antibody Induced Arthritis (mCAIA): Female BALB/cAnNHsd mice were injected intravenously with a cocktail of four arthritogenic monoclonal antibodies to collagen-II. After 24 hours, mice received 25 μg of LPS by intraperitoneal injection. Twice per day oral administration of RVX-297 (150 mg/kg) was initiated 30 hours following LPS and continued for 9 days. Efficacy was evaluated by body weight, clinical arthritis scores, combined hind paw weights, and histologic assessment. [1]
Murine Experimental Autoimmune Encephalomyelitis (EAE): EAE was induced in female C57Bl/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) in complete Freund's adjuvant (CFA). Pertussis toxin was administered by intraperitoneal injection 2 and 24 hours after immunization. RVX-297 was delivered by oral gavage at 75 and 125 mg/kg twice per day (b.i.d.) for 18 days in a therapeutic regimen (starting at the first sign of EAE) or for 27 days in a preventative regimen (starting immediately after immunization). In the therapeutic regimen, the 125 mg/kg dose was adjusted to 100 mg/kg b.i.d. on day 10 due to tolerability. EAE development and clinical score were evaluated based on clinical manifestations, and spinal cords were collected for gene expression analysis, flow cytometry, and histological assessment. [1]

Systemic Exposure in Rats: In the rat collagen-induced arthritis (rCIA) model, plasma exposure of RVX-297 (AUC0-12) was determined to be 23,165 hrng/mL at a dose of 25 mg/kg b.i.d., 52,145 hrng/mL at 50 mg/kg b.i.d., and 86,452 hrng/mL at 75 mg/kg b.i.d. [1]

In the therapeutic regimen of the mouse EAE model, RVX-297 was not well tolerated at 125 mg/kg b.i.d., leading to accelerated weight loss. The dose was adjusted to 100 mg/kg b.i.d. on day 10 of treatment. [1]

[1]. RVX-297, a BET Bromodomain Inhibitor, Has Therapeutic Effects in Preclinical Models of Acute Inflammation and Autoimmune Disease. Mol Pharmacol. 2017;92(6):694-706.

[2]. RVX-297- a novel BD2 selective inhibitor of BET bromodomains. Biochem Biophys Res Commun. 2016;477(1):62-67.

Background: Bromodomain and extra-terminal (BET) domain proteins are chromatin adapters that regulate gene transcription. RVX-297 is a novel, orally active BET inhibitor with selectivity for the BD2 bromodomain. [1]
Mechanism: RVX-297 reduces inflammatory gene expression by displacing BET proteins from acetylated histones on the promoters of sensitive genes, disrupting the recruitment of active RNA polymerase II. [1]
Efficacy: The study demonstrates for the first time that a BD2-selective BET inhibitor maintains anti-inflammatory properties and is effective in preclinical models of acute inflammation and autoimmunity (polyarthritis, multiple sclerosis). [1]
Comparison: In the EAE model, the efficacy of RVX-297 was comparable or superior to the S1P1 agonist FTY720 (fingolimod), an approved therapeutic for multiple sclerosis. [1]
Funding: This research was funded by Resverlogix Corp. [1]

C24H29N3O4

423.504766225815

423.215

1044871-04-6

135567084

White to light brown solid powder

3.5

1

6

7

31

639

0

O(C1C(C)=CC(C2=NC3C=C(C=C(C=3C(N2)=O)OC)OC)=CC=1C)CCN1CCCC1

PQZDYFRDRHRZGF-UHFFFAOYSA-N

InChI=1S/C24H29N3O4/c1-15-11-17(12-16(2)22(15)31-10-9-27-7-5-6-8-27)23-25-19-13-18(29-3)14-20(30-4)21(19)24(28)26-23/h11-14H,5-10H2,1-4H3,(H,25,26,28)

2-[3,5-dimethyl-4-(2-pyrrolidin-1-ylethoxy)phenyl]-5,7-dimethoxy-3H-quinazolin-4-one

RVX-297 RVX297 RVX 297

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 : ~50 mg/mL (~118.06 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.91 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 20.8 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: ≥ 2.08 mg/mL (4.91 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 20.8 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: ≥ 2.08 mg/mL (4.91 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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