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Background Apabetalone (RVX-208) is a bromodomain and extraterminal protein inhibitor (BETi) that in phase II trials reduced the relative risk (RR) of major adverse cardiac events (MACE) in patients with cardiovascular disease (CVD) by 44% and in diabetic CVD patients by 57% on top of statins. A phase III trial, BETonMACE, is currently assessing apabetalone’s ability to reduce MACE in statin-treated post-acute coronary syndrome type 2 diabetic CVD patients with low high-density lipoprotein C. The leading cause of MACE is atherosclerosis, driven by dysfunctional lipid metabolism and chronic vascular inflammation (VI). In vitro studies have implicated the BET protein BRD4 as an epigenetic driver of inflammation and atherogenesis, suggesting that BETi may be clinically effective in combating VI. Here, we assessed apabetalone’s ability to regulate inflammation-driven gene expression and cell adhesion in vitro and investigated the mechanism by which apabetalone suppresses expression. The clinical impact of apabetalone on mediators of VI was assessed with proteomic analysis of phase II CVD patient plasma. Results In vitro, apabetalone prevented inflammatory (TNFα, LPS, or IL-1β) induction of key factors that drive endothelial activation, monocyte recruitment, adhesion, and plaque destabilization. BRD4 abundance on inflammatory and adhesion gene promoters and enhancers was reduced by apabetalone. BRD2-4 degradation by MZ-1 also prevented TNFα-induced transcription of monocyte and endothelial cell adhesion molecules and inflammatory mediators, confirming BET-dependent regulation. Transcriptional regulation by apabetalone translated into a reduction in monocyte adhesion to an endothelial monolayer. In a phase II trial, apabetalone treatment reduced the abundance of multiple VI mediators in the plasma of CVD patients (SOMAscan® 1.3 k). These proteins correlate with CVD risk and include adhesion molecules, cytokines, and metalloproteinases. Ingenuity® Pathway Analysis (IPA®) predicted that apabetalone inhibits pro-atherogenic regulators and pathways and prevents disease states arising from leukocyte recruitment. Conclusions Apabetalone suppressed gene expression of VI mediators in monocytes and endothelial cells by inhibiting BET-dependent transcription induced by multiple inflammatory stimuli. In CVD patients, apabetalone treatment reduced circulating levels of VI mediators, an outcome conducive with atherosclerotic plaque stabilization and MACE reduction. Inhibition of inflammatory and adhesion molecule gene expression by apabetalone is predicted to contribute to MACE reduction in the phase III BETonMACE trial. Electronic supplementary material The online version of this article (10.1186/s13148-019-0696-z) contains supplementary material, which is available to authorized users.

To explore mechanisms underlying a 57% relative risk reduction of major adverse cardiovascular events (MACE) in type 2 diabetes mellitus (T2DM) patients (pts) with CVD given 200 mg apabetalone (APL) by mouth. APL is a small molecule that inhibits bromodomain and extra-terminal (BET) proteins, which are epigenetic readers of acetylated lysine in histones contained within actively transcribing chromatin. Inhibiting BET proteins attenuates transcribed genes that are upregulated in disease states. Studies here used SOMAscan proteomics of patient plasma given placebo (n=30) or APL (n=25) and cultured monocyte (THP-1) or endothelial (HUVEC) cells. Results showed differences in the proteome spanning 4 CVD pathways: acute phase response, intrinsic prothrombin activation, leukocyte extravasation signaling, and coagulation. This data showed TNFα target genes were preferentially upregulated in T2DM+CVD (p50%. HG induced adhesion genes in HUVECs, E-selectin and MYD88, by 2- and 1.3-fold was lowered by APL. In summary, APL lowers MACE in T2DM+CVD pts by attenuating expression of genes that mediate adhesion of monocytes to endothelial cells. This hypothesis is being tested in a phase 3 trial BETonMACE. Disclosure L. Tsujikawa: None. E. Kulikowski: Employee; Self; Resverlogix Corp. Stock/Shareholder; Self; Resverlogix Corp. C. Calosing: None. S. Wasiak: Employee; Self; Resverlogix Corp. Stock/Shareholder; Self; Resverlogix Corp. D. Gilham: Employee; Self; Resverlogix Corp. C. Halliday: Employee; Self; Resverlogix Corp. Stock/Shareholder; Self; Resverlogix Corp. J.O. Johansson: Employee; Self; Resverlogix Corp. M. Sweeney: Employee; Self; Resverlogix Corp. N.C. Wong: Employee; Self; Resverlogix Corp. Stock/Shareholder; Self; Resverlogix Corp.

Apabetalone (RVX-208) inhibits the interaction between epigenetic regulators known as bromodomain and extraterminal (BET) proteins and acetyl-lysine marks on histone tails. Data presented here supports the manuscript published in Atherosclerosis “RVX-208, a BET-inhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease” (Gilham et al., 2016) [1]. It shows that RVX-208 and a comparator BET inhibitor (BETi) JQ1 increase mRNA expression and production of apolipoprotein A-I (ApoA-I), the main protein component of high density lipoproteins, in primary human and African green monkey hepatocytes. In addition, reported here are gene expression changes from a microarray-based analysis of human whole blood and of primary human hepatocytes treated with RVX-208. Keywords: Bromodomain, BET proteins, BET inhibitor, RVX-208, JQ1, Vascular inflammation, ApoA-I, Apolipoprotein A-I, African green monkey, Primary human hepatocytes, Gene expression, Microarrays

Importance Bromodomain and extraterminal proteins are epigenetic regulators of gene transcription. Apabetalone is a selective bromodomain and extraterminal protein inhibitor targeting bromodomain 2 and is hypothesized to have potentially favorable effects on pathways related to atherothrombosis. Pooled phase 2 data suggest favorable effects on clinical outcomes. Objective To test whether apabetalone significantly reduces major adverse cardiovascular events. Design, Setting, and Participants A randomized, double-blind, placebo-controlled trial, conducted at 190 sites in 13 countries. Patients with an acute coronary syndrome in the preceding 7 to 90 days, type 2 diabetes, and low high-density lipoprotein cholesterol levels were eligible for enrollment, which started November 11, 2015, and ended July 4, 2018, with end of follow-up on July 3, 2019. Interventions Patients were randomized (1:1) to receive apabetalone, 100 mg orally twice daily (n = 1215), or matching placebo (n = 1210) in addition to standard care. Main Outcomes and Measures The primary outcome was a composite of time to the first occurrence of cardiovascular death, nonfatal myocardial infarction, or stroke. Results Among 2425 patients who were randomized (mean age, 62 years; 618 women [25.6%]), 2320 (95.7%) had full ascertainment of the primary outcome. During a median follow-up of 26.5 months, 274 primary end points occurred: 125 (10.3%) in apabetalone-treated patients and 149 (12.4%) in placebo-treated patients (hazard ratio, 0.82 [95% CI, 0.65-1.04];P = .11). More patients allocated to apabetalone than placebo discontinued study drug (114 [9.4%] vs 69 [5.7%]) for reasons including elevations of liver enzyme levels (35 [2.9%] vs 11 [0.9%]). Conclusions and Relevance Among patients with recent acute coronary syndrome, type 2 diabetes, and low high-density lipoprotein cholesterol levels, the selective bromodomain and extraterminal protein inhibitor apabetalone added to standard therapy did not significantly reduce the risk of major adverse cardiovascular events. Trial Registration ClinicalTrials.gov Identifier:NCT02586155

Monocytes and macrophages are key immune cells in the development of cardiovascular disease (CVD). Dysregulated monocyte activation can be reversed by epigenetic therapies. Here we test the effects of apabetalone - a clinical-stage inhibitor of epigenetic bromodomain and extraterminal (BET) readers

Apabetalone (ABP) is a selective inhibitor of bromodomain and extra-terminal (BET) proteins, epigenetic regulators of gene expression. ABP reduces cardiac fibrosis in cellular models and profibrotic markers in patients (pts), effects that might favorably modify risk of congestive heart failure (CHF

Background: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease, causing right heart failure and premature death. Mounting evidence points to the involvement of epigenetic changes in the pathogenesis of PAH. Indeed, as observed in cancer, overexpression of the epigenetic reader BRD4 was reported to promote PA smooth muscle cells (PASMCs) proliferation and survival in PAH. However, the molecular mechanisms underlying this effect are poorly understood. In cancer cells, BRD4 was shown to enhance PLK1 expression favoring cell proliferation and survival. Given that PAH and cancer share similarities, we hypothesize that the BRD4/PLK1 axis is critically involved in PAH. Aim: Determine whether overexpression of the BRD4/PLK1 signaling has a detrimental function in PAH Methods and Results: PLK1 is significantly increased in distal PAs (IF) and isolated PASMCs (WB) from PAH patients compared to controls. We demonstrate that BRD4 up-regulation in PAH-PASMCs accounts for PLK1 overexpression (qPCR and WB). Pharmacological (BI6727) or molecular (siRNA) inhibition of PLK1 decreases viability (MTT), proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) in PAH-PASMCs. Moreover, as observed for BRD4, we provide evidence that PLK1 is increased in animal models mimicking PAH: the Sugen/Hypoxia (Su/Hx) and the monocrotaline rat as well as the SIV-infected macaque (IF). Pharmacological inhibition of BRD4 using Apabetalone (RVX-208) improves hemodynamic parameters (RVSP, mPAP), vascular remodeling (EVG) and results in diminished PLK1 expression (IF) in Su/Hx-treated rats with established PAH. Conclusion: We demonstrate that the BRD4/PLK1 axis is increased in human PAH and represents a promising therapeutic target.

Apabetalone (RVX-208) leads to a 57% relative risk reduction of MACE in patients with diabetes mellitus (DM) and CVD. To test whether RVX-208, an inhibitor (BETi) of epigenetic readers bromodomain extra-terminal (BET) proteins lowers CVD by affecting genes mediating monocyte adhesion to endothelial cells in response to high glucose(HG) and dietary metabolite trimethyl-amine oxide (TMAO). Cultured THP-1 monocytes, HUVEC endothelial cells and primary human hepatocytes (PHH) were exposed to varying amts of glucose and TMAO. Results showed that HG (25.6 mM) induced Very Late Antigen-4 (VLA-4) mRNA, a gene mediating THP-1 adhesion by 1.3-fold and RVX-2suppressed it >50%. BETi blocked TMAO induction of VLA-4 mRNA by >50% in THP-1. In HUVECs RVX-2abrogated HG induction of E-selectin and MYD88 mRNA by 2- and 1.3-fold, respectively and lowered TMAO induction of both mRNAs by >50%. Microbiome action on dietary phospholipids followed by hepatic flavin mono-oxygenase-3 (FMO3) processing yields TMAO. In PHH exposed for 24 hours to RVX-208, FMO3 mRNA was lower by 40% but it also suppressed a transcriptional regulator of FMO3, farnesoid X receptor (FXR). BETi for 6 hours suppressed both FXR mRNA and protein within 6 hours by >80% suggesting a direct effect of BETi on the FXR gene. Furthermore, in ChiP assays BRD4, a BET protein, dissociated immediately from FXR DNA upon exposure to RVX-208. BRD4 guides a complex containing RNA pol II along active genes, its dissociation would halt transcription of the gene. In summary, Apabetalone inhibits HG and TMAO enhanced adhesion of THP-1 to HUVECs, this process mimics a step in CVD pathogenesis. RVX-2suppresses cellular adhesion genes; VLA-4 in THP-1 and both E-selectin plus MYD88 in HUVECs. BETi blocks TMAO activity and its production by inhibiting FXR expression, a regulator of FMO3 gene transcription. Rapid actions of BETi in dissociating BRD4 from FXR DNA suggests a direct effect of RVX-2on transcription of this gene. Disclosure L. Tsujikawa: None. E. Kulikowski: Employee; Self; Resverlogix Corp.. C. Calosing: None. S. Wasiak: Employee; Self; Resverlogix Corp. D. Gilham: Employee; Self; Resverlogix Corp.. Stock/Shareholder; Self; Resverlogix Corp.. C. Halliday: None. J.O. Johansson: Employee; Self; Resverlogix Corp., Artery Therapeutics, Inc. M. Sweeney: Employee; Self; Resverlogix Corp. N.C. Wong: Stock/Shareholder; Self; Resverlogix Corp..

Background and aims Apabetalone is an inhibitor of bromodomain and extraterminal (BET) proteins. In clinical trials, apabetalone reduced the incidence of major adverse cardiac events (MACE) in patients with cardiovascular disease and reduced circulating factors that promote vascular calcification (VC). Because VC contributes to MACE, effects of apabetalone on pro-calcific processes were examined. Methods and results Apabetalone inhibited extracellular calcium deposition and opposed induction of transdifferentiation markers in human coronary artery vascular smooth muscle cells (VSMCs) under osteogenic culture conditions. Tissue-nonspecific alkaline phosphatase (TNAP) is a key contributor to VC, and apabetalone suppressed osteogenic induction of the mRNA, protein and enzyme activity. The liver is a major source of circulating TNAP, and apabetalone also downregulated TNAP expression in primary human hepatocytes. BRD4, a transcriptional regulator and target of apabetalone, has been linked to calcification. Osteogenic transdifferentiation of VSMCs resulted in disassembly of 100 BRD4-rich enhancers, with concomitant enlargement of remaining enhancers. Apabetalone reduced the size of BRD4-rich enhancers, consistent with disrupting BRD4 association with chromatin. 38 genes were uniquely associated with BRD4-rich enhancers in osteogenic conditions; 11 were previously associated with calcification. Apabetalone reduced levels of BRD4 on many of these enhancers, which correlated with decreased expression of the associated gene. Bioinformatics revealed BRD4 may cooperate with 7 specific transcription factors to promote transdifferentiation and calcification. Conclusions Apabetalone counters transdifferentiation and calcification of VSMCs via an epigenetic mechanism involving specific transcription factors. The mechanistic findings, combined with evidence from clinical trials, support further development of apabetalone as a therapeutic for VC.

Apabetalone (RVX-208, 200 mg/d) given orally to patients with diabetes mellitus (DM) and CVD leads to a 57% relative risk reduction in major adverse cardiovascular events (MACE). Potential actions of RVX-208, an inhibitor (BETi) of bromodomain extra-terminal (BET) proteins that are epigenetic readers of acetylated lysine residues within histones, in lowering MACE is explored by testing its effect on genes mediating monocyte adhesion to endothelial cells in response to high glucose (HG) and the dietary metabolite trimethyl-amine oxide (TMAO). Cultured THP-1 monocytes, HUVEC endothelial cells and primary human hepatocytes (PHH) were exposed to varying concentrations of glucose and TMAO. Results showed that HG (25.6 mM) induced Very Late Antigen-4 (VLA-4) mRNA, a gene mediating THP-1 adhesion by 1.3-fold and RVX-208 suppressed it >50%. Similarly, BETi blocked TMAO induction of VLA-4 mRNA by >50% in THP-1. In HUVECs RVX-208 abrogated HG induction of E-selectin and MYD88 mRNA by 2- and 1.3-fold, respectively and lowered TMAO induction of these mRNAs by >50%. Microbiome processing of dietary phospholipids followed by hepatic flavin mono-oxygenase-3 (FMO3) metabolism yields TMAO. In PHH exposed for 24 hrs to RVX-208, FMO3 mRNA was lower by 40% but it also suppressed a transcriptional regulator of FMO3, farnesoid X receptor (FXR). BETi suppressed both FXR mRNA and protein within 6 hrs by >80% suggesting a direct effect of BETi on the gene encoding FXR. Furthermore, ChiP data showed that BRD4, a BET protein, dissociated immediately from FXR gene upon exposure to RVX-208. Since BRD4 guides a complex containing RNA pol II along actively transcribed genes containing histones that are highly acetylated, the dissociation of BRD4 from FXR DNA would halt transcription of this gene. In summary, Apabetalone inhibits HG and TMAO enhanced adhesion of THP-1 to HUVECs a process that mimics a step in the pathogenesis of CVD. RVX-208 suppresses genes underlying cellular adhesion; VLA-4 in THP-1 and both E-selectin plus MYD88 in HUVECs. BETi blocks not only activity of TMAO but also its production by inhibiting FXR expression, a regulator of FMO3 gene transcription. The rapid actions of BETi in dissociating BRD4 from FXR DNA suggests a direct effect of RVX-208 on transcription of this gene.

Bromodomain (BD) and extra-terminal domain containing proteins (BET) are chromatin adapters that bind acetylated histone marks via two tandem BDs, BD1 and BD2, to regulate gene transcription. BET proteins are involved in transcriptional reprogramming in response to inflammatory stimuli. BET BD inhibitors (BETis) that are nonselective for BD1 or BD2 have recognized anti-inflammatory properties in vitro and counter pathology in models of inflammation or autoimmune disease. Although both BD1 and BD2 bind acetylated histone residues, they may independently regulate the expression of BET-sensitive genes. Here we characterized the ability of RVX-297, a novel orally active BETi with selectivity for BD2, to modulate inflammatory processes in vitro, in vivo, and ex vivo. RVX-297 suppressed inflammatory gene expression in multiple immune cell types in culture. Mechanistically, RVX-297 displaced BET proteins from the promoters of sensitive genes and disrupted recruitment of active RNA polymerase II, a property shared with pan-BETis that nonselectively bind BET BDs. In the lipopolysaccharide model of inflammation, RVX-297 reduced proinflammatory mediators assessed in splenic gene expression and serum proteins. RVX-297 also countered pathology in three rodent models of polyarthritis: rat and mouse collagen-induced arthritis, and mouse collagen antibody-induced arthritis. Further, RVX-297 prevented murine experimental autoimmune encephalomyelitis (a model of human multiple sclerosis) disease development when administered prophylactically and reduced hallmarks of pathology when administered therapeutically. We show for the first time that a BD2-selective BETi maintains anti-inflammatory properties and is effective in preclinical models of acute inflammation and autoimmunity.

Background/Aims: The association between serum alkaline phosphatase (ALP) with adverse cardiovascular outcomes, in Chronic Kidney Disease (CKD) patients has previously been reported and may be a result of increased vascular calcification and inflammation. Here we report, for the first time, the effects of pharmacologic epigenetic modulation on levels of ALP and kidney function via a novel oral small molecule BET inhibitor, apabetalone, in CKD patients. Methods: A post-hoc analysis evaluated patients with estimated glomerular filtration rate (eGFR) 2, who participated in the apabetalone phase 2 randomized controlled trials (SUSTAIN and ASSURE). 48 CKD subjects with a history of cardiovascular disease (CVD) were treated with 100mg twice-daily of 24 and 26 weeks of apabetalone or placebo. ALP and eGFR were measured prior to randomization and at final visits. Results: Patients who received apabetalone (n=35) versus placebo (n=13) over 6 months showed significantly (p=0.02) lowered serum ALP -14.0% (p2) (p=0.04 versus baseline) and decreased by 5.8% (2.9 mL/min/1.73 m2) (p=0.6 versus baseline) in the placebo group. Apabetalone was well tolerated. Conclusion: A post-hoc analysis of CKD subjects from the SUSTAIN and ASSURE randomized controlled trials demonstrated favorable effects of apabetalone on ALP and eGFR, and generated the hypothesis that epigenetic modulation by BET inhibition may potentially offer a novel therapeutic strategy to treat CVD and progressive kidney function loss in CKD patients. This is being examined in the phase III trial BETonMACE.

Apabetalone (RVX-208) a bromodomain extra-terminal (BET) inhibitor selectively binds the 2nd ligand domain within a BET protein thus displacing it from acetylated lysine marks on histone tails. In clinical trials of ~1000 patients, many (n=499) of whom with CVD when given 200 mg/d RVX-208 had a 55% relative risk reduction in major adverse cardiac events (MACE) vs placebo. This benefit of RVX-208 may stem from ability of BET inhibition (BETi) to calm inflammation, metabolism, coagulation and complement pathways with well-known roles in CVD risks. These potential benefits of RVX-208 underlie BETonMACE a phase 3 CVD events trial. Of major interest is the anti-inflammatory (AI) effects seen in clinical data showing BETi lowers CRP by 28% in RVX-208 treated patients (n=331). RVX-208 lowered IL-6 and MCP-1 U937 cells exposed to LPS in a dose and time dependent manner by 90 and 85%, respectively within 24 hrs. RVX-208 displaced BET proteins BRD2-4 from chromatin. These AI effects underpin studies of RVX-297 a more potent cousin of RVX-208 that lowered IL-6 and MCP-1 in LPS stimulated U937 cells by 95 and 80% respectively within 24 hrs. In ChIP assays, RVX-297 also displaced BET protein BRD4 and pol II from promoters of cytokine genes IL-6 and IL-1β that mediate inflammation. Together, data from studying RVX-208 and -297 show both BET inhibitors displace transcription factors from promoter DNA that control expression of inflammatory genes (IGs) with key roles in CVD. Importance of BETi displacement of BRD4 and pol II from DNA becomes clear when added to the fact that cellular response to inflammation requires immediate and robust expression of defined set of IGs. For this rapid transcriptional response, the cell places chromatin structures upstream of IGs called super-enhancers (SE) or latent enhancers (LE) that act as molecular sinks for attracting BET proteins such as BRD4. The placement of a SE or LE adjacent to a promoter that controls a IG recruits this gene to the response against an inflammatory insult. Thus targeting BET proteins including BRD4 with a selective BETi may have broad effects on many genes or pathways by crippling SE or LE mediated cellular response to the inflammatory component of CVD. This mechanism may have potential implications to other inflammatory diseases.

Apabetalone is an inhibitor of the epigenetic readers bromodomain and extraterminal (BET) proteins, currently in a phase 3 outcomes trial in patients with cardiovascular disease (CVD) and diabetes mellitus. A post hoc analysis of phase 2b trials demonstrated a 55% relative risk reduction in major adverse cardiac events in CVD patients. Elevated inflammatory markers correlate with CVD. Inflammation also accompanies chronic kidney disease (CKD) and CKD patients are at risk of CVD. Previous research has shown that apabetalone modulates pathways that contribute to chronic inflammation, including the acute phase response (APR). Here, pathway analysis of gene microarrays showed downregulation of APR by apabetalone in primary human hepatocytes (PHH). Real-time PCR and ELISA analysis of RVX-208 treated PHHs confirmed that APR genes that correlate with CVD are suppressed by 20 to 95%, including CRP, ceruloplasmin (CP), serum amyloid P (SAP), PAI-1, alpha 2-macroglobulin (A2M), complement C2, C3 and C5, MBL2, serum amyloid A and interleukin 18. Apabetalone decreased IL-6-induced expression of CP, SAP and A2M, with most striking effects on CRP (-75%). Apabetalone also decreased LPS-induced expression of SAP in a mouse endotoxemia model. To assess effects of apabetalone on inflammatory mediators in CVD patients, SOMAscan™ 1.3K proteomic analysis was performed on plasma from phase 2b ASSERT (12 weeks; n=25) and ASSURE (26 weeks; n=47) clinical trials. This approach identified APR as the top downregulated pathway by apabetalone in both trials. APR biomarkers are elevated in CKD patients where they correlate with disease progression. To gain insight into the pharmacodynamics of the APR response to apabetalone, stage 4 CKD patients (n=8) received a single dose of the drug followed by plasma proteomics at several time points. At 12h post dose, APR was significantly downregulated by apabetalone. Of note, CRP was decreased in CKD patients after 12h of treatment (-7%, p=0.04) versus baseline, as well as in ASSERT (-43%, p=0.01) and ASSURE (-21%, p=0.02) trials versus placebo. Downregulation of the APR pathway by apabetalone may lead to reduced chronic inflammation in CVD and CKD patients and contribute to the reduction in MACE in patients with high residual CVD risk.

Apabetalone (RVX-208) is a small molecule bromodomain & extraterminal (BET) protein inhibitor, targeting the second bromodomain (BD2) of BET proteins. In cardiovascular disease (CVD) patients enrolled in phase 2 trials apabetalone treatment reduced the relative risk of a CV event by 44% (Nicholls

RVX-208 affects epigenetics by inhibiting bromodomain and extraterminal (BET) proteins from binding to their natural ligand, acetyl-lysine marks on histone tails and thereby modulates gene activity. In SUSTAIN and ASSURE phase IIb trials of CVD patients (n=499), giving 200 mg/d of RVX-208 orally lead a 55% relative risk reduction in major adverse cardiovascular events (MACE) vs. placebo. This marked reduction in MACE is unlikely due to RVX-208’s modest induction of ApoA-I/HDL, thus prompting studies of RVX-208 for its benefits beyond lipids. Methods included microarray surveys of human whole blood (WB) or primary hepatocytes (PH) exposed to RVX-208. Cytokines were assayed in U937 macrophage and peripheral blood mononuclear cells (PBMC) exposed to RVX-208. Plasma samples from phase IIb patients were measured using SOMAScan proteomic analysis. Results of the microarray studies using WB showed a potential anti-atherogenic effect of RVX-208 because it suppressed activity of 37/46 pro-atherogenic while inducing 8/18 anti-atherogenic genes. Additionally, RVX-208 had potential anti-thrombotic properties by affecting 18 genes related to platelet function (e.g. downregulation of CD64 and thrombospondin 1). RVX-208 had anti-inflammatory effects on the expression of >25 cytokines including downregulation of MCP-1, osteopontin and PARC genes. The suppression of these 3 genes by RVX-208 was evident in not only WB but also in the LPS stimulated U937 and/or PBMCs. Whether these in vitro findings extended into patients were examined by SOMAScan showing lower levels of osteopontin and PARC protein in plasma of treated patients. Furthermore, a key marker of inflammation RANTES was markedly lowered in treated patients. Why RVX-208 may affect genes connected to CVD was explored by exposing PH to RVX-208. These studies showed a 25% reduction in levels of mRNA encoding flavin mono-oxygenase-3 (FMO3), an enzyme that produces trimethylamine oxide (TMAO) a metabolite which predicts CVD risk. In summary, RVX-208 inhibits BET proteins to impact cellular epigenetics that in turn affects expression of genes with known roles in CVD. This activity may underlie RVX-208’s anti-atherogenic, -thrombotic and –inflammatory effects in reducing MACE observed in clinical trials.

The epigenetic modifying compound RVX-208 selectively inhibits the second ligand binding domain in BET proteins to increase ApoA-I/HDL production. In recent trials of CVD patients given standard of care and RVX-208 (200mg/d) there was a 55% relative risk reduction of MACE vs. placebo. This reduction is greater than expected from modest increases in ApoA-I/HDL, thus suggesting benefits of RVX-208 beyond lipid effects. Whether added benefits exist was explored by microarray survey of differential gene expression in primary human hepatocytes (PHH) and whole blood (WB). In PHH, RVX-208 downregulated genes within the complement, fibrin clotting, cholesterol biosynthesis, fatty acid biosynthesis, diabetes and acute phase response (APR) pathways. These data guided more focused analyses on the known HDL proteome encoded by 89 genes of which 30 were affected by RVX-208 and many of these were members of the APR or complement pathways. In support of the microarray data, we used RT-PCR and found RVX-208 lowered mRNA encoding complement 3, 4a/4b and 5 by >50% in Huh-7 and HepG2 cells exposed to RVX-208. Consistent with the mRNA data, secretion of these complement proteins were less from the treated cells. Next the cells were exposed to cytokines in order to mimic an inflammatory state thus inducing complement expression. Treatment with RVX-208 blocked this effect by >50%. Furthermore, in Huh-7 cells, RVX-208 repressed both fibrin clotting factors II (prothrombin) and XI (thromboplastin antecedent) consistent with the PHH microarray data thus suggesting an anti-thrombotic effect for RVX-208. To further extend findings in vitro from hepatoma cells, plasma from patients in our clinical trials who received RVX-208 were assayed and found to have decreased levels of both APR and complement proteins. Lastly, microarray studies of WB from healthy donors treated ex-vivo with RVX-208 uncovered differential regulation of anti- and pro-atherogenic gene sets that were activated and suppressed, respectively by RVX-208. In summary, RVX-208, a selective BET inhibitor, affects several pathways involved in CVD including; reverse cholesterol transport, atherogenesis, complement, thrombosis and vascular inflammation that cumulatively may reduce MACE in high risk CVD patients.

RVX-208 lowers major adverse cardiovascular events (MACE) by 57% in diabetes mellitus (DM) patients from post-hoc analysis of phase 2 underpins an ongoing phase 3 BETonMACE trial. How RVX-208, an inhibitor of acetylated lysine binding to epigenetic readers called bromodomain extra-terminal (BET)

High density lipoproteins (HDL), through activity of the main protein component apolipoprotein A-I (ApoA-I), can reduce the risk of cardiovascular disease (CVD) by removing excess cholesterol from atherosclerotic plaque. In this study, we demonstrate that the bromodomain and extraterminal domain (BET) inhibitor RVX-208 increases ApoA-I gene transcription and protein production in human and primate primary hepatocytes. Accordingly, RVX-208 also significantly increases levels of ApoA-I, HDL-associated cholesterol, and HDL particle number in patients who received the compound in recently completed phase 2b trials SUSTAIN and ASSURE. Moreover, a post-hoc analysis showed lower instances of major adverse cardiac events in patients receiving RVX-208. To understand the effects of RVX-208 on biological processes underlying cardiovascular risk, we performed microarray analyses of human primary hepatocytes and whole blood treated ex vivo. Overall, data showed that RVX-208 raises ApoA-I/HDL and represses pro-inflammatory, pro-atherosclerotic and pro-thrombotic pathways that can contribute to CVD risk.

The recently completed human trials SUSTAIN and ASSURE (n=499) showed a 55% reduction of major adverse cardiovascular events (MACE) in atherosclerotic patients treated with statins and oral RVX-208 (200 mg/day). RVX-208 increases ApoA-I, the dominant protein of HDL-c, by selectively inhibiting bromodomain extra-terminal proteins (BET) that are epigenetic readers which regulate chromatin function via binding to acetylated histones. Patients given RVX-208 had increased ApoA-I and HDL-c by 10.3% and 7.7%, respectively. Although significant, these increases may account, in part, for lower MACE. Thus, effects of RVX-208 on other biological processes that impacted atherosclerosis were examined. Specifically, biomarkers of vascular inflammation were measured and microarray techniques were used in studying human primary hepatocytes and whole blood. Results in human aortic endothelial cells (HAEC) exposed to RVX-208 showed suppressed VCAM-1 and MCP-1 with an IC50 of 1uM and 10uM, respectively. In U937 macrophages RVX-208 suppressed the pro-inflammatory cytokine IL-6 (IC50 1uM). Primary hepatocytes treated with RVX-208 lead to upregulation of gene sets within processes such as; transcription, translation and chromatin modeling, as previously seen with other BET inhibitors. More importantly RVX-208 down regulated gene sets within the; complement cascade, fibrin clotting, cholesterol and fatty acid synthesis, innate immune system and diabetes mellitus pathways. Any one of these changes, independently or cumulatively, may underlie the observed MACE reduction. Blood treated ex vivo with RVX-208 suppressed gene sets involved in pro-inflammatory signaling of monocytes and neutrophils, monocyte-endothelial cell interactions, extracellular matrix remodeling and Th1/Th2 cell responses. Moreover, RVX-208 affected genes with known roles in atherosclerosis and/or vascular inflammation yielding an overall anti-atherogenic profile. In summary, the orally active selective BET inhibitor RVX-208 significantly lowers MACE in patients with residual CVD risk receiving standard of care therapy including statins. The above results suggest that RVX-208 alters several biological pathways in a cardioprotective manner which may lead to lower MACE.

Background: In our phase 2b trials, patients (n=499) given 200 mg/d apabetalone (RVX-208) for 6 months were observed to have a 55% relative risk reduction of major adverse cardiovascular events (MACE) that was further reduced in diabetes mellitus (DM) patients. These findings underpin our interest