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Background: Epigenetic changes may contribute importantly to cognitive decline in late life including Alzheimer’s disease (AD) and vascular dementia (VaD). Bromodomain and extra-terminal (BET) proteins are epigenetic “readers” that may distort normal gene expression and contribute to chronic disorders. Objective: To assess the effects of apabetalone, a small molecule BET protein inhibitor, on cognitive performance of patients 70 years or older participating in a randomized trial of patients at high risk for major cardiovascular events (MACE). Methods: The Montreal Cognitive Assessment (MoCA) was performed on all patients 70 years or older at the time of randomization. 464 participants were randomized to apabetalone or placebo in the cognition sub-study. In a prespecified analysis, participants were assigned to one of three groups: MoCA score≥26 (normal performance), MoCA score 25–22 (mild cognitive impairment), and MoCA score≤21 (dementia). Exposure to apabetalone was equivalent in the treatment groups in each MoCA-defined group. Results: Apabetalone was associated with an increased total MoCA score in participants with baseline MoCA score of≤21 (p = 0.02). There was no significant difference in change from baseline in the treatment groups with higher MoCA scores. In the cognition study, more patients randomized to apabetalone discontinued study drug for adverse effects (11.3% versus 7.9%). Conclusion: In this randomized controlled study, apabetalone was associated with improved cognition as measured by MoCA scores in those with baseline scores of 21 or less. BET protein inhibitors warrant further investigation for late life cognitive disorders.

Acute flaccid myelitis (AFM) is a disabling, polio-like illness mainly affecting children. Outbreaks of AFM have occurred across multiple global regions since 2012, and the disease appears to be caused by non-polio enterovirus infection, posing a major public health challenge. The clinical presentation of flaccid and often profound muscle weakness (which can invoke respiratory failure and other critical complications) can mimic several other acute neurological illnesses. There is no single sensitive and specific test for AFM, and the diagnosis relies on identification of several important clinical, neuroimaging, and cerebrospinal fluid characteristics. Following the acute phase of AFM, patients typically have substantial residual disability and unique long-term rehabilitation needs. In this Review we describe the epidemiology, clinical features, course, and outcomes of AFM to help to guide diagnosis, management, and rehabilitation. Future research directions include further studies evaluating host and pathogen factors, including investigations into genetic, viral, and immunological features of affected patients, host-virus interactions, and investigations of targeted therapeutic approaches to improve the long-term outcomes in this population.

Background After an acute coronary syndrome (ACS), patients with diabetes remain at high risk for additional cardiovascular events despite use of current therapies. Bromodomain and extra-terminal (BET) proteins are epigenetic modulators of inflammation, thrombogenesis, and lipoprotein metabolism implicated in atherothrombosis. The BETonMACE trial tests the hypothesis that treatment with apabetalone, a selective BET protein inhibitor, will improve cardiovascular outcomes in patients with diabetes after an ACS. Design Patients (n = 2425) with ACS in the preceding 7 to 90 days, with type 2 diabetes and low HDL cholesterol (≤40 mg/dl for men, ≤45 mg/dl for women), receiving intensive or maximum-tolerated therapy with atorvastatin or rosuvastatin, were assigned in double-blind fashion to receive apabetalone 100 mg orally twice daily or matching placebo. Baseline characteristics include female sex (25%), myocardial infarction as index ACS event (74%), coronary revascularization for index ACS (80%), treatment with dual anti-platelet therapy (87%) and renin-angiotensin system inhibitors (91%), median LDL cholesterol 65 mg per deciliter, and median HbA1c 7.3%. The primary efficacy measure is time to first occurrence of cardiovascular death, non-fatal myocardial infarction, or stroke. Assumptions include a primary event rate of 7% per annum in the placebo group and median follow-up of 1.5 years. Patients will be followed until at least 250 primary endpoint events have occurred, providing 80% power to detect a 30% reduction in the primary endpoint with apabetalone. Summary BETonMACE will determine whether the addition of the selective BET protein inhibitor apabetalone to contemporary standard of care for ACS reduces cardiovascular morbidity and mortality in patients with type 2 diabetes. Results are expected in 2019.

Background/Introduction Both major adverse cardiovascular events (MACE) and non-alcoholic fatty-liver disease (NAFLD) are highly prevalent in patients with high BMI and long-standing type 2 diabetes (T2DM). NAFLD is characterized by an augmented hepatic inflammation and fat deposition and is strongly associated with metabolic syndrome. Patients with NAFLD are at an increased risk of cardiovascular (CV) events, and MACE is the leading cause of death for patients with NAFLD. Apabetalone (APB) is a novel selective inhibitor of bromodomain and extra-terminal (BET) proteins, epigenetic regulators of gene expression. In the Phase 3 BETonMACE trial treatment of 2,425 T2DM patients post ACS with APB, resulted in hazard ratios (HR) of 0.82 (p=0.11) for the primary endpoint of ischemic MACE (CV death, non-fatal MI or stroke) and 0.59 (p=0.03) for the secondary endpoint of heart failure hospitalization (HFH) vs placebo (PBO). Transient elevations of alanine aminotransferase greater than 5xULN occurred in 3.3% of APB treated patients. Purpose In this exploratory post hoc analysis of BETonMACE we evaluated risk modification for a composite of MACE+HFH by APB based on the Angulo NAFLD fibrosis score (FS) using 6 variables (age, BMI, hyperglycemia/diabetes, AST/ALT ratio, platelet count, and albumin). The NAFLD FS categorizes individuals into groups that correlate with differing levels of fibrosis in biopsy studies: (FS F0-F2, no significant fibrosis; FS ID, indeterminant; and FS F3-F4, significant fibrosis). Methods Baseline characteristics and blood measurements were used to determine NAFLD FS at baseline. The incidence of MACE+HHF was compared between treatment groups. Results Based on FS, there were 618 pts were classified as FS F0-F2 (n=328 APB, n=290 PBO), 1,440 pts were classified as FS ID (n=708 APB, n=732 PBO) and 289 pts were classified as FS F3-F4 (n=144 APB, n=145). MACE+HHF in the PBO group was higher in FS ID and FS F3-F4 compared to FS F0-F2 (17.2% vs 15.0% vs 9.7%) and therefore the former two groups were combined into an elevated risk FS+ group. FS+ pts were older (63 vs 56), had longer duration of T2DM (9.0 vs 7.3 yrs), and higher BMI (30.8 vs 28.6) compared to FS- pts. Overall, APB was associated with fewer MACE+HHF (HR 0.78, 95% CI 0.60–1.01, p=0.06) compared to PBO in the FS+ pts with adjustment for age, duration of T2DM and BMI. Conclusions Patients with T2DM and ACS may share common risk factors with patients with NAFLD. Apabetalone appears to exert a favorable effect on MACE in patients with risk factors for NAFLD. Whether apabetalone has a modulatory effect on the development and progression of NAFLD is an important question requiring further investigation. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Resverlogix Corp.

Background In stable patients with type 2 diabetes (T2D), insulin treatment is associated with elevated risk for major adverse cardiovascular events (MACE). Patients with acute coronary syndrome (ACS) and T2D are at particularly high risk for recurrent MACE despite evidence-based therapies. It is uncertain to what extent this risk is further magnified in patients with recent ACS who are treated with insulin. We examined the relationship of insulin use to risk of MACE and modification of that risk by apabetalone, a bromodomain and extra-terminal (BET) protein inhibitor. Methods The analysis utilized data from the BETonMACE phase 3 trial that compared apabetalone to placebo in patients with T2D, low HDL cholesterol, andACS. The primary MACE outcome (cardiovascular death, myocardial infarction, or stroke) was examined according to insulin treatment and assigned study treatment. Multivariable Cox regression was used to determine whether insulin use was independently associated with the risk of MACE. Results Among 2418 patients followed for median 26.5 months, 829 (34.2%) were treated with insulin. Despite high utilization of evidence-based treatments including coronary revascularization, intensive statin treatment, and dual antiplatelet therapy, the 3-year incidence of MACE in the placebo group was elevated among insulin-treated patients (20.4%) compared to those not-treated with insulin (12.8%, P = 0.0001). Insulin treatment remained strongly associated with the risk of MACE (HR 2.10, 95% CI 1.42–3.10, P = 0.0002) after adjustment for demographic, clinical, and treatment variables. Apabetalone had a consistent, favorable effect on MACE in insulin-treated and not insulin-treated patients. Conclusion Insulin-treated patients with T2D, low HDL cholesterol, and ACS are at high risk for recurrent MACE despite the use of evidence-based, contemporary therapies. A strong association of insulin treatment with risk of MACE persists after adjustment for other characteristics associated with MACE. There is unmet need for additional treatments to mitigate this risk. Trial registration ClinicalTrials.gov NCT02586155, registered October 26, 2015

Background and Aims Major adverse cardiac events (MACE) remain a leading cause of mortality in chronic kidney disease (CKD). Apabetalone is an orally available inhibitor of bromodomain & extraterminal (BET) proteins – epigenetic readers that modulate gene expression involved in fibrosis, inflammation and calcification. In the phase 3 BETonMACE trial, apabetalone treatment was associated with reduction in MACE in the subpopulation with CKD (eGFR < 60 mL/min/1.73m2; HR 0.50 95% CI 0.26,0.96 p=0.04]) implying favorable effects of apabetalone on cellular responses along the kidney-heart axis. This study examines effects of apabetalone on primary human renal mesangial cells (HRMCs) in culture on fibrosis, inflammation, reactive oxygen species (ROS) and calcification pathways that contribute to renal pathology. Method HRMCs from donors without kidney dysfunction were stimulated with TGF-β1 or lipopolysaccharide (LPS) ± 1-25µM apabetalone, 0.15-0.5µM JQ1 or 0.1µM MZ1 (BET inhibitors [BETi] with chemical scaffolds different than apabetalone). Gene expression was measured by real-time PCR and RNA-seq. Smooth muscle actin (α-SMA) was examined by immunofluorescence microscopy, and alkaline phosphatase enzyme activity in a biochemical assay. RNA-seq from TGF-β1 treated HRMC ± BETi was evaluated by Gene Ontology (GO) Enrichment and Ingenuity Pathway Analysis (IPA). Results TGF-β1 is a pro-fibrotic cytokine that activates HRMC to a fibroblast-like state which over-produces extracellular matrix (ECM). Apabetalone dose dependently suppressed TGF-β1 induced gene expression of (a) α-SMA, a marker of fibrotic activation, up to 90% p In GO Enrichment analysis of RNA-seq from TGF-β1 stimulated HRMCs, multiple gene sets associated with ECM were in the top 20 affected by BETi, supporting anti-fibrotic properties. IPA predicted NfkB-RelA and NFkB (complex) were upstream regulators inhibited by apabetalone, indicating suppression of NF-kB mediated inflammation. IPA also predicted apabetalone activated canonical pathways of glucose utilization & tolerance of ROS production, including Oxidative Phosphorylation (z-score 5.7, p0.05 at 5µM) and NRF2-Mediated Oxidative Stress Response (z score 2.3, p Conclusion Apabetalone downregulated responses to TGF-β1 or LPS in HRMCs that promote fibrotic, inflammatory and calcific processes which exacerbate kidney dysfunction. Changes in energy metabolism pathways predicted apabetalone facilitates adaptation to high glucose in the kidney. Together, our results provide mechanistic insight into reductions in MACE in CKD patients receiving apabetalone in the phase 3 BETonMACE trial. The effect of apabetalone on MACE in patients with diabetes and CKD will be further evaluated in the upcoming BETonMACE2 trial.

Graphical abstract.

Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.Graphical Abstract

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory ‘cytokine-storm’, a cocktail of interferon gamma, interleukin 1β and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage., A combination of phosphoproteomics, drug screening and single-cell sequencing approaches identifies how cytokines elevated in COVID-19 patients drives cardiac dysfunction, with BET inhibitors serving as potential lead candidates decrease ACE2 cardiac expression and infection.

Introduction: Hyperactivation of the monocyte inflammatory response is partly controlled by epigenetic mechanisms and contributes to atherosclerotic plaque formation in patients with cardiovascular disease (CVD) and type 2 diabetes mellitus (DM2). Hypothesis: Monocyte activation in DM2+CVD is regulated by bromodomain and extraterminal (BET) epigenetic readers and can be inhibited by apabetalone - a clinical stage BET inhibitor. Methods: CD14+ monocytes from 14 DM2+CVD patients and 12 matched control subjects were treated ex vivo with 25 μM apabetalone ± 25 U/mL interferon γ (IFNγ) for 4h or 24h. Expressed genes (180) were analyzed with the Nanostring™ Innate Immunity Panel. Secreted cytokines were immunoprofiled with a Milliplex® array (42). Bioinformatics were performed with Ingenuity® Pathway Analysis (IPA®) software. Results: Unstimulated DM2+CVD monocytes had higher IL1A , IL1B and IL8 cytokine gene expression and Toll-like receptor (TLR) 2 surface abundance than control monocytes, indicating proinflammatory activation. Ex vivo apabetalone treatment reduced IL1A and IL8 mRNA (pex vivo stimulation with IFNγ, upregulating genes within cytokine and NF-κB pathways ( TNF , CCL7 , CCL8 , MYD88 , RELA ) >30% more than controls (pEx vivo apabetalone treatment also countered IFNγ induced secretion of IL-1β and TNFα in DM2+CVD monocytes (p Conclusions: Monocytes isolated from DM2+CVD patients receiving standard of care therapies are in a hyperinflammatory state and hyperactivate upon IFNγ stimulation. Apabetalone treatment diminishes this proinflammatory phenotype, providing mechanistic insight into how BET protein inhibition may reduce CVD risk in DM2 patients.

Introduction: Use of insulin has been associated with worse CV outcomes in patients (pts) with type 2 diabetes T2D. Apabetalone (APB) is a novel selective inhibitor of bromodomain and extra-terminal (BET) proteins, epigenetic regulators of gene expression. In the Phase 3 BETonMACE trial treatment with APB, compared with placebo, resulted in non-significantly fewer major adverse CV events (MACE: CV death, non-fatal MI or stroke) in 2425 pts with T2D and recent acute coronary syndrome (ACS). Objective: In this analysis of BETonMACE we examined the relationship of insulin use to MACE risk and its modification by APB. Methods: Baseline characteristics were compared in insulin-treated (INS) or not insulin-treated (no-INS) pts. The incidence of MACE and treatment hazard ratio (HR) were compared between these two subgroups. Results: 829 (34.2%) pts received insulin at baseline, with or without other diabetes drugs. INS vs no-INS pts were more likely to be female (29 vs 24%), had longer duration of T2D (12.6 vs 6.4 yrs), higher HbA1c (8.4 vs 6.9%) and baseline glucose (156 vs 126 mg/dL), lower use of metformin (73 vs 87%) and sulfonylureas (21 vs 33 %), and higher use of SGLT2 inhibitors (16 vs 6%) and GLP1 receptor agonists (10 vs 2%). MACE in the placebo group was higher in INS than no-INS (17.4% vs 9.7%; HR 1.94; 95% CI 1.39-2.73; p=0.0001). Overall, APB was associated with fewer MACE (HR 0.82, 95% CI 0.65-1.04, p=0.11). The relative reduction in MACE with ABP was similar in INS (HR 0.78, 95% CI. 0.55-1.10, p=0.16) and no-INS (HR 0.87, 95% CI 0.63-1.21, p = 0.42, p interaction =0.64). The absolute reduction in MACE with APB was numerically greater among INS than non-INS (3.69 vs 1.30%). Conclusions: Pts with T2D and recent ACS treated with insulin are at high risk for MACE. High risk of MACE with insulin use is likely through association with other clinical characteristics prognostic for MACE. Insulin treatment may be a marker to identify pts with potential for large absolute reduction in MACE with APB.

Introduction: Apabetalone is an orally available small molecule that affects gene transcription by inhibiting BET protein interactions with acetylated histones and transcription factors. Apabetalone is in late stage clinical development for the treatment of cardiovascular disease (CVD). Clinical trials show apabetalone is well tolerated and has a favorable safety profile. Recent evidence indicates this drug could be repurposed to treat COVID-19 by reducing expression of the angiotensin converting enzyme 2 (ACE2) receptor that is essential for SARS-CoV-2 viral entry and/or disrupting viral protein E interaction with BET proteins. The spike protein of SARS-CoV-2 engages with human ACE2 expressed in multiple organs such as lung, liver, kidney, heart and intestine to initiate infection in host cells. Ultimately, the infection leads to life threatening complications in COVID-19 patients. Hypothesis: Apabetalone downregulates ACE2 expression to protect human cells from SARS-CoV-2. Methods: Primary human kidney tubular epithelial cells (RPTEC) were stimulated with TNFα and co-treated with apabetalone overnight. Primary human hepatocytes (PHH) and the hepatoma cell line, HepG2, were treated up to 96 h with apabetalone or other BET inhibitors (BETi). Gene expression was analyzed by microarray or RT-PCR. Human aortic endothelial cells (HAEC) were pretreated with apabetalone for 1 h followed by TNFα stimulation for another 1h. Chromatin occupancy of the BET protein BRD4 was examined by ChIP-seq. Results: In RPTEC, 5μM apabetalone downregulated ACE2 mRNA by 50%. Apabetalone dose dependently reduced ACE2 gene expression in HepG2 or PHH from 3 independent donors by up to 90%. JQ1 is a pan BET inhibitor, whereas MZ1 promotes BET protein degradation. Both JQ1 and MZ1 treatments downregulated ACE2 in liver cells, indicating an on target BETi effect. In HAEC, apabetalone abolished BRD4 occupancy at an enhancer in proximity to the ACE2 gene. Conclusions: Apabetalone reduces ACE2 gene expression in multiple human cell types. ACE2 expression may be regulated by adjacent BRD4 enhancer occupancy. The impact of apabetalone on SARS-CoV-2 life cycle is under investigation. The results will provide mechanistic support for potential COVID-19 clinical trials.

Background Patients with cardiovascular disease (CVD) and type 2 diabetes (DM2) have a high residual risk for experiencing a major adverse cardiac event. Dysregulation of epigenetic mechanisms of gene transcription in innate immune cells contributes to CVD development but is currently not targeted by therapies. Apabetalone (RVX-208) is a small molecule inhibitor of bromodomain and extra-terminal (BET) proteins—histone acetylation readers that drive pro-inflammatory and pro-atherosclerotic gene transcription. Here, we assess the impact of apabetalone on ex vivo inflammatory responses of monocytes from DM2 + CVD patients. Results Monocytes isolated from DM2 + CVD patients and matched controls were treated ex vivo with apabetalone, interferon γ (IFNγ), IFNγ + apabetalone or vehicle and phenotyped for gene expression and protein secretion. Unstimulated DM2 + CVD monocytes had higher baseline IL-1α, IL-1β and IL-8 cytokine gene expression and Toll-like receptor (TLR) 2 surface abundance than control monocytes, indicating pro-inflammatory activation. Further, DM2 + CVD monocytes were hyper-responsive to stimulation with IFNγ, upregulating genes within cytokine and NF-κB pathways > 30% more than control monocytes (p Conclusions Monocytes isolated from DM2 + CVD patients receiving standard of care therapies are in a hyper-inflammatory state and hyperactive upon IFNγ stimulation. Apabetalone treatment diminishes this pro-inflammatory phenotype, providing mechanistic insight into how BET protein inhibition may reduce CVD risk in DM2 patients.

Background Despite established treatments, patients with type 2 diabetes mellitus (T2DM) and acute coronary syndrome (ACS) are at higher risk of ischemic cardiovascular (CV) events and hospitalization for heart failure (HHF) compared to those without T2DM. LDL-C lowering or use of GLP-1 agonists predominantly affects ischemic CV events, with little effect on HHF. Conversely, treatment with SGLT-2 inhibitors reduces HHF, with less effect on ischemic CV events. Preclinical studies indicate that bromodomain and extra-terminal (BET) proteins coordinate gene transcription for pathways that promote atherothrombotic events as well as heart failure. We assessed the clinical effect of apabetalone (APB), a novel BET protein inhibitor, on a composite of non-fatal ischemic CV events, HHF, and CV death in a post hoc analysis of the BETonMACE trial Methods BETonMACE was a double-blind, placebo-controlled phase 3 study in patients with T2DM and recent acute coronary syndrome receiving standard of care risk factor management. In 13 countries, 2425 patients were enrolled. We conducted a time-to-event analysis for first adjudicated CV death or non-fatal MI, stroke, or HHF using a log-rank test and Cox proportional hazards model. Results At baseline median age was 62 years, 25.6% were female, 87.6% white, and use of high intensity statin, ACE inhibitors/ angiotensin II blockers, dual antiplatelet therapy and beta blocker were 90, 88, 92 and 91% respectively. A total of 312 subjects had an endpoint event, with 139 (11.5%) patients in the ABP group and 173 (14.3%) among PBO (HR 0.78, 95% CI 0.63–0.98, p=0.03, Figure). At 26 months, the absolute risk reduction was 3.2% and number needed to treat was 31. Numerically favorable HRs were observed for each component endpoint except for stroke (Table). Conclusion This present analysis suggests that BET inhibition with APB may be a novel pathway through which to reduce both HHF and ischemic CV events in high risk patients with T2DM thus impacting broader clinical outcomes with potentially large benefits for patients and healthcare systems. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Resverlogix Corp

SUMMARYCardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory ‘cytokine-storm’, a cocktail of interferon gamma, interleukin 1β and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.

Background and Aims Elevated serum alkaline phosphatase (ALP) independently predicts major adverse cardiac events (MACE) by contributing to vascular calcification and endothelial dysfunction arising in chronic kidney disease (CKD) and cardiovascular disease (CVD). Apabetalone is an orally active inhibitor of bromodomain and extraterminal (BET) proteins – epigenetic readers that modulate gene expression involved in vascular inflammation and calcification. Here we examined apabetalone’s effects on ALP post-hoc in recent clinical trials, then performed mechanistic studies into apabetalone’s impact on tissue non-specific ALP (TNALP) expression in mice and cell culture. Method Serum ALP was determined in CVD patients in phase 2 trials (3 month ASSERT and 6 month SUSTAIN & ASSURE) and in the phase 3 BETonMACE CVD outcomes trial, including subpopulations with CKD (eGFR Results In phase 2 trials, baseline serum ALP independently predicted MACE (hazard ratio [HR] 1.6, 95% CI 1.2-2.2, p=0.001). In the 3 month ASSERT trial, apabetalone dose dependently reduced serum ALP (p Liver-derived TNALP accounts for ≈50% of circulating ALP. In the liver of mice on high fat diet, apabetalone or JQ1 (BET inhibitors with different chemical scaffolds) reduced Alpl mRNA (p55%, p 40%; p Conclusion Apabetalone lowers serum ALP in clinical trials, which is consistent with reduced hepatic production of TNALP - the most abundant ALP isoform. Further, apabetalone downregulates ALPL gene expression in vascular cell types while reducing calcification. Together, BET-dependent epigenetic modulation of ALP by apabetalone can affect several pathogenetic processes, and thereby improve cardiovascular outcomes. This study provides insights to the CVD event reductions observed in the CKD subpopulation in the BETonMACE Phase 3 trial.

Background and Aims Patients with type 2 diabetes (T2D) and acute coronary syndrome (ACS) are at high risk for recurrent cardiovascular (CV) events, particularly in the presence of chronic kidney disease (CKD). Apabetalone (APB) is a novel inhibitor of bromodomain and extraterminal (BET) proteins. Its cardiovascular efficacy and safety were evaluated in a phase 3 trial, BETonMACE. Method BETonMACE was a randomized, double-blind, comparison of effects of ABP or placebo (PBO) on major adverse CV events (MACE) defined as CV-death, non-fatal myocardial infarct or stroke, in 2425 pts with T2D and recent ACS. Here we report MACE plus CHF hospitalization in subjects with or without CKD Stage 3. Results Baseline characteristics: median age 62 years, 25.6% female, 87.6% white, 90% high intensity statin use, mean LDL-C 70.3 and HDL-C 33.3 mg/dl, median HbA1c 7.3%, and 11% with CKD Stage 3. Overall in the trial, MACE plus CHF hospitalization occurred in 139 (11.5%) patients with ABP and 173 (14.3%) with PBO (HR 0.78, 95% CI 0.63-0.98). In the subgroup with CKD, MACE plus CHF hospitalization occurred in 16 (12.9%) on APB and 41 (25%) on PBO (HR 0.48, 95% CI 0.26-0.89). In the subgroup without CKD, MACE plus CHF hospitalization occurred in 123 (11.3%) and 132 (12.7%) with APB or PBO, respectively (HR 0.89, 95% CI 0.70-1. Conclusion Patients with T2D, ACS, and Stage 3 CKD have a very high risk of subsequent MACE plus CHF hospitalization. The BET protein inhibitor ABP may reduce this risk.

Background and Aims Fabry disease (FD) is a rare X-linked recessive genetic disorder caused by mutations in the α-galactosidase A gene. Absence or defects of this lysosomal enzyme lead to globotriaosylceramide (Gb3) accumulation. Gb3 disrupts basic cellular metabolic processes, inducing oxidative stress and promoting inflammation. Systemically, Gb3 accumulation leads to organ damage throughout the body with an increase in renal, cardiac, cerebrovascular and skin complications. Enzyme replacement therapy (ERT) is current standard of care. This therapy may reduce Gb3 deposits but the impact on cardiac and kidney function is unclear based on the limited studies. Further, ERT is less effective in the late phases of FD, partially due to the development of uncontrolled inflammation and fibrosis. Apabetalone is a clinical stage orally administered bromo and extra terminal domain inhibitor (BETi) that has beneficial effects on vascular inflammation and vascular calcification through an epigenetic pathway. Based on its mechanism of action and impact on multiple disease drivers in both preclinical and clinical studies, apabetalone has therapeutic potential to modulate inflammation in FD patients on ERT therapy. Method PBMCs and neutrophils were isolated from fresh blood of FD patients ± ERT therapy using density gradient centrifugation. The baseline immune status of PBMCs was compared in naïve FD patients (without ERT, n=3) and ERT treated patients (n=8) via flow cytometry. PBMCs or neutrophils from ERT patients were treated ex vivo with 5µM or 20µM apabetalone ± LPS stimulation. Expression of key pro-inflammatory mediators was analyzed with real time quantitative PCR. The production of reactive oxygen species (ROS) in neutrophils was assessed by flow cytometry. Results The PBMC inflammatory profile in baseline showed monocytes from ERT patients produced lower amounts of pro-inflammatory mediators (TNFα and IL6) than naïve patients (∼60%, trending p≤0.06 and p≤0.08 respectively). However, surface abundance of CCR2, a chemokine receptor for the MCP1 chemokine that promotes monocyte recruitment to local inflamed tissue during inflammatory process, was ∼2-fold greater on monocytes from ERT subjects than the naïve controls (trending p≤0.06). Gene expression of MCP1 was approximately 5-fold higher in PBMC from ERT patients versus naïve controls (p=0.01). In response to ex vivo LPS stimulation, PBMCs from FD patients on ERT showed a robust induction of pro-inflammatory responses with increases in gene expression of MCP1, IL12B, TNFA and IL6. Ex vivo treatment with apabetalone (5µM) blocked the LPS-induction of MCP1 gene expression (∼90%, p Conclusion Monocytes from FD patients on ERT therapy display an enhanced expression of the CCR2-MCP-1 axis as compared to naïve controls, indicating the potential for immune cell tissue infiltration and local inflammation. Apabetalone counters this increase by preventing MCP1 gene transcription. Apabetalone also reduces PBMC mediated pro-inflammatory gene transcription (TNFA, IL12B, IL6) and neutrophil mediated ROS production in response to LPS stimulation. Therefore, apabetalone treatment may reduce pathological inflammation in FD patients and thus complement ERT to optimize patient outcomes, which will be tested further with warranted clinical studies.

Chronic systemic inflammation contributes to cardiovascular disease (CVD) and correlates with the abundance of acute phase response (APR) proteins in the liver and plasma. Bromodomain and extraterminal (BET) proteins are epigenetic readers that regulate inflammatory gene transcription. We show that BET inhibition by the small molecule apabetalone reduces APR gene and protein expression in human hepatocytes, mouse models, and plasma from CVD patients. Steady-state expression of serum amyloid P, plasminogen activator inhibitor 1, and ceruloplasmin, APR proteins linked to CVD risk, is reduced by apabetalone in cultured hepatocytes and in humanized mouse liver. In cytokine-stimulated hepatocytes, apabetalone reduces the expression of C-reactive protein (CRP), alpha-2-macroglobulin, and serum amyloid P. The latter two are also reduced by apabetalone in the liver of endotoxemic mice. BET knockdown in vitro also counters cytokine-mediated induction of the CRP gene. Mechanistically, apabetalone reduces the cytokine-driven increase in BRD4 BET occupancy at the CRP promoter, confirming that transcription of CRP is BET-dependent. In patients with stable coronary disease, plasma APR proteins CRP, IL-1 receptor antagonist, and fibrinogen γ decrease after apabetalone treatment versus placebo, resulting in a predicted downregulation of the APR pathway and cytokine targets. We conclude that CRP and components of the APR pathway are regulated by BET proteins and that apabetalone counters chronic cytokine signaling in patients.

Apabetalone is a selective bromodomain and extra-terminal (BET) inhibitor which modulates lipid and inflammatory pathways implicated in atherosclerosis. The impact of apabetalone on attenuated coronary atherosclerotic plaque (AP), a measure of vulnerability, is unknown. The ApoA-1 Synthesis Stimulation and intravascular Ultrasound for coronary atheroma Regression Evaluation (ASSURE; NCT01067820) study employed serial intravascular ultrasound (IVUS) measures of coronary atheroma in 281 patients treated with apabetalone or placebo for 26 weeks. AP was measured at baseline and follow-up. Factors associated with changes in AP were investigated. AP was observed in 31 patients (11%) [27 (13.0%) in the apabetalone group and four (5.5%) in the placebo group]. The apabetalone group demonstrated reductions in AP length by − 1 mm [interquartile range (IQR) − 4, 1] (p = 0.03), AP arc by − 37.0° (IQR − 59.2, 8.2) (p = 0.003) and the AP index by − 34.6 mm° (IQR − 52.6, 10.1) (p = 0.003) from baseline. The change in AP index correlated with on-treatment concentration of high-density lipoprotein (HDL) particles (r = − 0.52, p = 0.006), but not HDL cholesterol (r = − 0.11, p = 0.60) or apolipoprotein A-1 (r = − 0.16, p = 0.43). Multivariable analysis revealed that on-treatment concentrations of HDL particles (p = 0.03) and very low-density lipoprotein particles (p = 0.01) were independently associated with changes in AP index. Apabetalone favorably modulated ultrasonic measures of plaque vulnerability in the population studied, which may relate to an increase in HDL particle concentrations. The clinical implications are currently being investigated in the phase 3 major adverse cardiac event outcomes trial BETonMACE.

Key Teaching Points • In its early phase, cardiac sarcoidosis can present with isolated premature ventricular contractions, which makes the differentiation from benign premature ventricular contraction challenging. • The clinical course of cardiac sarcoidosis can resemble that of arrhythmogenic right ventricular cardiomyopathy (ARVC), evolving over years. • Myocardial scar in cardiac sarcoidosis can evolve with epicardial predominance, which also resembles ARVC.

Introduction: Apabetalone, a small molecule inhibitor, targets epigenetic readers termed BET proteins that contribute to gene dysregulation in human disorders. Apabetalone has in vitro and in vivo anti-inflammatory and antiatherosclerotic properties. In phase 2 clinical trials, this drug reduced the incidence of major adverse cardiac events in patients with cardiovascular disease. Chronic kidney disease is associated with a progressive loss of renal function and a high risk of cardiovascular disease. We studied the impact of apabetalone on the plasma proteome in patients with impaired kidney function. Methods: Subjects with stage 4 or 5 chronic kidney disease and matched controls received a single dose of apabetalone. Plasma was collected for pharmacokinetic analysis and for proteomics profiling using the SOMAscan 1.3k platform. Proteomics data were analyzed with Ingenuity Pathway Analysis to identify dysregulated pathways in diseased patients, which were targeted by apabetalone. Results: At baseline, 169 plasma proteins (adjusted P value

Introduction: Obesity increases the risk of type 2 diabetes (DM2) and cardiovascular disease (CVD), due to associated insulin resistance, dyslipidemia, high blood pressure and chronic inflammation....

Inhibition of bromodomain and extra-terminal (BET) proteins can modulate lipoprotein and inflammatory factors that mediate atherosclerosis. The impact of the BET inhibitor, apabetalone, on cardiovascular events is unknown. Our objective was to investigate the impact of apabetalone on cardiovascular event rates in a pooled analysis of clinical studies in patients with established coronary artery disease. We conducted a pooled analysis of patients (n = 798) with coronary artery disease who participated in clinical trials (ASSERT, ASSURE, SUSTAIN) that evaluated the impact of 3–6 months of treatment with apabetalone on lipid parameters and coronary atherosclerosis. The incidence of major adverse cardiovascular events (death, myocardial infarction, coronary revascularization, hospitalization for cardiovascular causes) in the treatment groups was evaluated. At baseline, patients treated with apabetalone were more likely to be Caucasian, have a history of dyslipidemia, and be undertreated with s-blocker and anti-platelet agents. Treatment with apabetalone produced the following dose-dependent changes compared with placebo: increases in apolipoprotein A-I (apoA-I) of up to 6.7% (P

Apabetalone (RVX-208) is an epigenetic regulator developed to treat cardiovascular disease (CVD) that targets BET proteins. Through transcriptional regulation RVX-208 modulates pathways that underlie CVD including reverse cholesterol transport, vascular inflammation, coagulation, and complement. Using transcriptomics and proteomics we show that complement is one of the top pathways downregulated by RVX-208 in primary human hepatocytes (PHH) and in plasma from CVD patients. RVX-208 reduces basal and cytokine-driven expression of complement factors in PHH and in chimeric mice with humanized livers. Plasma proteomics of CVD patients shows that RVX-208 decreases complement proteins and regulators, including complement activators SAP and CRP. Circulating activated fragments C5a, C3b, and C5b-C6 are reduced by 51, 32, and 10%, respectively, indicating decreased activity of complement in patients. As complement components are linked to CVD and metabolic syndrome, including major acute cardiac events, modulating their levels and activity by RVX-208 may alleviate risks associated with these diseases. Electronic supplementary material The online version of this article (doi:10.1007/s12265-017-9755-z) contains supplementary material, which is available to authorized users.

Background Diabetes (DM) is associated with increased risk of macro/microvascular disease and cognitive decline. Inflammation and vascular calcification may be contributing factors. Bromodomain and extraterminal (BET) proteins coordinate gene transcription and modify the transcriptional response to hyperglycemia, and inflammation. Apabetalone competitively and selectively inhibits binding between BET proteins and acetyl-lysine marks on histone tails: normalizing transcriptional profiles to physiological levels; reducing in vitro alkaline phosphatase (ALP) transcription and in vivo plasma ALP in a dose-dependent manner. Phase 2 trials with apabetalone show improved renal function in the chronic kidney disease (CKD) subgroups. Furthermore, treatment showed a 55% reduction in CVD events with more pronounced benefit among patients with DM, low HDL-cholesterol (HDL-C) and high sensitivity C-reactive protein (hsCRP). Methods The double-blind, placebo controlled phase 3 BETonMACE trial is testing the hypothesis that apabetalone 100 mg b.i.d., added to standard care, reduces major adverse cardiovascular events (MACE: CV death, non-fatal myocardial infarction or stroke) in patients with DM, acute coronary syndrome (ACS) within the preceding 7–90 days, low HDL-C (30 mL/min/1.7m2. The trial will continue until at least 250 MACE, providing 80% power to detect a 30% reduction. Secondary endpoints include changes in eGFR in patients with baseline eGFR 30 to Results Enrollment of 2425 patients across 13 countries and 195 centers is now complete. Baseline characteristics [median (IQR)] include LDL-C 65.0 (36) mg/dL, HDL-C 33.0 (7) mg/dL, HbA1c 7.3 (2.3) %, hsCRP 2.8 (4.9) mg/L, mean blood pressure 129/76 mmHg, and CKD in 266 patients (10.8%). Background care was based on guideline recommendations. Diabetes medications include metformin (79%), insulin (36%), sulfonylureas (28%), DPP4 inhibitors (11%), SGLT2 inhibitors (9.7%) and GLP1 receptor agonists (0.3%). The CKD subpopulation vs. total population differed significantly from the whole population with regard to age (71 vs. 62 y. o.), male sex (58% vs. 75%), history of hypertension (46% vs. 88%), history of stroke (1.5% vs. 7.5%), and current smokers (6.1% vs. 13%). In the 70 year and older (n=466, 19%) population 54% (n=243) showed a baseline MoCA score 25 and lower suggesting cognitive impairment. Summary The BETonMACE trial is testing the hypothesis that selective BET-inhibition with apabetalone, added to established, evidence-based treatment, reduces MACE in high-risk patients with DM, recent ACS, and low HDL-C. The study will also assess apabetalone's effect on renal function and cognition.

Apabetalone (RVX-208) is an orally available small molecule bromodomain & extraterminal (BET) protein inhibitor that targets the second bromodomain (BD2) of BET proteins. Apabetalone returns dysregulated BET-dependent transcription toward normal physiological levels. In phase 2 trials, apabetalone treatment reduced the incidence of major adverse cardiac events by 44% in CVD patients and by 57% in diabetic CVD patients. Previous studies have highlighted apabetalone's positive impact on vascular calcification (VC) and inflammation (VI) marker expression in vitro, as well as its ability to lower serum alkaline phosphatase (ALP) levels, and improve atherosclerotic plaque stability parameters in treated patients. In CVD, elevated inflammatory mediators and cell surface adhesion molecules drive VI, resulting in leukocyte adhesion, infiltration, uptake of oxLDL, and ultimately plaque formation. Here we show in vitro that THP-1 monocyte adhesion to human aortic endothelial cells (HAECs) increases with TNFα stimulation and is attenuated by apabetalone treatment, with fewer monocytes attaching to HAECs under flow conditions. This functional outcome is attributed to apabetalone's reduction of key endothelial adhesion genes, VCAM-1 (50%, p=0.0001) and SELE (37%, p=9x10–5). Apabetalone also prevents TNFα induction of endothelial recruitment genes (MCP-1; 75%, p=0.0002) and genes involved in plaque rupture (IL8; 24%, p=2x10–5). Basal HAEC ALP expression, a potential contributor to endothelial dysfunction and VC, also decreases with apabetalone treatment (70%, p=0.005). Induction of VI genes by TNFα is BET-dependent as degradation of BET proteins by MZ-1 prevents an increase in transcripts in response to TNFα treatment. Ingenuity® Pathway Analysis (IPA®), GSEA, and GO analysis of HAEC gene expression data predicts apabetalone inhibition of pro-atherogenic pathways, gene sets, and upstream regulators induced by TNFα. These include cytokine and chemokine, Toll-Like Receptor (TLR), NFkβ, Interferon and TNFα signaling. In addition, IPA® disease and biological function analysis predicts inhibition of immune cell activation and recruitment by apabetalone. Plasma proteomics (SOMAscan®) and IPA® analysis from apabetalone-treated CVD patients in ASSERT and ASSURE phase 2 trials indicate that apabetalone inhibits pro-atherogenic upstream regulators (IL-6 and IFNy), canonical pathways, and diseases and functions. Serum ALP also decreases dose dependently with apabetalone treatment (ASSERT). Epigenetic inhibition of VI and VC driven atherogenesis likely contributes to the reduction in MACE observed in phase 2 apabetalone treated patients. The ongoing phase 3 post-acute coronary syndrome (ACS) clinical trial in T2DM patients, BETonMACE, is currently testing this hypothesis.

BACKGROUND AND AIMS: In patients with cardiovascular disease, considerable residual risk remains despite evidence-based secondary prevention measures. Alkaline phosphatase (ALP) has been suggested as a modifiable cardiovascular risk factor. We sought to determine whether cardiovascular risk reduction by the bromodomain and extra-terminal (BET) protein inhibitor apabetalone is associated with the concomitant lowering of serum ALP. METHODS: In a post-hoc analysis of 795 patients with established coronary heart disease and statin treatment, who participated in phase 2 placebo-controlled trials of apabetalone, we determined the effect of assigned treatment for up to 24 weeks on the incidence of major adverse cardiovascular events (MACE) and serum ALP. RESULTS: Baseline ALP (median 72 U/L) predicted MACE (death, non-fatal myocardial infarction, coronary revascularization, or hospitalization for cardiovascular causes), independent of high-sensitivity C-reactive protein (hsCRP), sex, age, race, study, cardiovascular risk factors, chronic kidney disease (CKD), liver function markers and treatment allocation (hazard ratio [HR] per standard deviation [SD] 1.6, 95% CI 1.19-2.16, p = 0.002). Mean placebo-corrected decreases in ALP from baseline were 9.2% (p

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 (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.

Objectives This study was undertaken to compare efficacy, tolerability, and pharmacokinetics of DM-1992, an extended-release formulation of carbidopa/levodopa (CD/l-dopa) with immediate-release (IR) CD/l-dopa in patients with advanced Parkinson's disease. Methods This randomized, open-label, crossover study included a 3-d baseline and two 10-d treatment periods. Patients with daily OFF time of 2.5 h or more taking 400 mg or more l-dopa/d in four or more divided doses were titrated to stable regimens of DM-1992 2 times per day or CD/l-dopa IR 3 times to 8 times per day. Patients were allowed to take rescue CD/l-dopa as needed. Using home diaries, patients recorded OFF time and ON time with or without troublesome dyskinesia during baseline and treatment days 7 through 9. During 12-h clinic visits on day 10, plasma samples were collected for pharmacokinetics, and motor performance was assessed hourly. Results Thirty-four patients were enrolled; mean baseline l-dopa dosage was 968 mg/d. After titration, CD/l-dopa IR was dosed 4.8 times per day and DM-1992, 2 times per day. Rescue CD/l-dopa IR was given 1.3 times during the DM-1992 arm and 0.2 times during the CD/l-dopa IR arm. The reduction from baseline in % OFF time was greater for DM-1992 compared with CD/l-dopa IR (−5.52% vs. +1.33%; P = 0.0471). At steady-state, compared with CD/l-dopa IR, DM-1992 exhibited a smoother plasma l-dopa concentration profile mostly because of a significantly higher (day 10) predose l-dopa concentration, associated with enhanced motor performance. Although more patients taking DM-1992 had one or more adverse events (AEs) than CD/l-dopa IR patients (35% vs. 15%), no pattern to the AEs was seen, nor any resulting discontinuations. Conclusions DM-1992 was associated with a reduction in %OFF time compared with CD/l-dopa IR despite a reduced dosing frequency. Although the open-label study design and the greater number of rescue doses during the DM-1992 arm call for caution in interpreting the results, the elevated predose plasma l-dopa concentration (12 h after DM-1992 administration) lends objective support to our findings, suggesting that phase 3 studies are warranted. © 2015 International Parkinson and Movement Disorder Society

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.