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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.

A new approach for delineating burned areas in savannas is presented using a series of Sentinel-1. SAR images and object-based image analysis (OBIA). Using analysis-ready Sentinel-1 data increases the frequency of observation to several scenes per month, enabling more timely and precise detection of fire events during a fire season. Using an object-based segmentation, classified using backscatter indices calculated between consecutive image pairs, there was 87.6% spatial agreement with expert interpretation of cloud-free sentinel-2 imagery. Additionally, we map the timing and extent of individual burnt areas and their coalescence. We apply the method to produce more detailed and precise maps of the progression of fires during 2019 in Belize than previously available from passive multispectral sensors. We discuss the scope for extending the method and draw preliminary conclusions on the effectiveness of the temporal indices and OBIA for detecting burns in this landscape.

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.

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.

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.

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.

Carbon dioxide (CO2) using capillary blood gas (CBG) analysis is commonly used children with acute bronchiolitis. Evidence to support its use is limited. A retrospective observational study was conducted over two bronchiolitis seasons (2014 -2016) of infants admitted to a tertiary teaching hospital using patient electronic medical records. Using logistical regression models (STATA/IC 12.1) the association between CBG pCO2 and markers of disease severity (length of stay (LOS) and high dependency admission (HDU)) was examined. 332 children were assessed with 526 CBG performed in 158 infants (mean age 0.31 years, 54% male, 27% premature, 77% RSV positive). The initial CBG pCO2 was a mean 5.9kPa (SD1.1) and a maximum mean of 6.4kPa (SD1.5). Median LOS was 3 days (range 0–35). A CBG pCO2 >7.0kPa during the admission (in 23% infants (36/158)) was significantly associated with younger age (OR 0.005 (95%CI 0.0007, 0.03); p 7kPa (N=26/98) in ED was significantly associated with LOS (IRR 1.4 (95%CI 1.1,1.8); p=0.008) and HDU admission (OR 3.5 (95%CI 1.7,7.8); p=0.001). CBG pCO2 >7 kPa identifies children in ED with more severe disease with longer length of stay and risk of admission to HDU. Our results suggest that CBG pCO2 may be a possible marker of severity in future intervention trials for bronchiolitis.

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

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.

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

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.

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

Utilisation of blood gas analysis(BGA) in acute bronchiolitis is common but evidence for current guidelines is sparse. This study investigated indicators demarcating clinically important rise in carbon dioxide(CO 2 ). A prospective observational study assessed children admitted from an emergency department(ED) to a tertiary care hospital with bronchiolitis (10/14–01/15). Hospital charts and electronic patient data were analysed using logistic regression models to examine relationships between rise in CO 2 and possible clinical indicators. 220 patients with bronchiolitis were admitted (mean age 0.57years (95%CI:0.05,0.64)). 113(51%) had at least one BGA. 14% required high dependency care(HDU). In ED CO 2 >7kPA was associated with HDU admission (OR 3.75(95%CI:1.13,12.47),p=0.031); prematurity and young age also independently predicted HDU (IRR 1.53(95%CI:1.21,1.93),p 2 >7kPa in ED were 2 >7kPa (OR 2.46(95%CI:1.42,4.26),p=0.001 and OR 0.08(95%CI:0.03,0.17),p 2 . Overall, LOS was significantly higher in those who had BGA during admission (4.0days (d) (95%CI 3.5,4.5) versus 2.3d (95%CI:2.0,2.6)). Age under 3m, history of prematurity and CO 2 >7kPa in ED identify those with prolonged LOS and/or HDU admission. Significantly raised CO 2 is not seen without oxygen requirement. Further work aims to elucidate when BGA may be helpful in bronchiolitis.

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)

This paper examines the emerging cultural patterns and interpretative repertoires in reports of an impending pandemic of avian flu in the UK mass media and scientific journals at the beginning of 2005, paying particular attention to metaphors, pragmatic markers ('risk signals'), symbolic dates and scare statistics used by scientists and the media to create expectations and elicit actions. This study complements other work on the metaphorical framing of infectious disease, such as foot and mouth disease and SARS, tries to link it to developments in the sociology of expectations and applies insights from pragmatics both to the sociology of metaphor and the sociology of expectations.

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.