Dean Gilham

RVX-208, a BET-inhibitor for treating atherosclerotic cardiovascular disease, raises ApoA-I/HDL and represses pathways that contribute to cardiovascular disease

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.

Downregulation of the Complement Cascade In Vitro, in Mice and in Patients with Cardiovascular Disease by the BET Protein Inhibitor Apabetalone (RVX-208)

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.

Data on gene and protein expression changes induced by apabetalone (RVX-208) in ex vivo treated human whole blood and primary hepatocytes

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.

RVX-297, a BET bromodomain inhibitor, has therapeutic effects in preclinical models of acute inflammation and autoimmune disease

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

Abstract LB-038: Preclinical development and clinical validation of a whole blood pharmacodynamic marker assay for the BET bromodomain inhibitor ZEN-3694 in metastatic castration-resistant prostate cancer (mCRPC) patients

Detection of drug activity in patients is essential to confirm its mechanism of action, as well as to ensure proper target engagement at the selected dose to elicit optimal clinical activity. Pharmacodynamic (PD) markers are often developed to detect pharmacological responses and optimize drug dosing. Whole blood is an easily attainable and minimally invasive source of biological material to measure clinical activity of drugs. We designed, developed, and validated a whole blood PD marker assay to detect the activity of ZEN-3694, an orally available inhibitor of the bromodomain and extra-terminal (BET) domain family of proteins currently in phase I clinical trials in mCRPC (NCT02705469 and NCT02711956). Potential BET-specific PD markers were first identified via comparative microarray analysis using a PI3K inhibitor, a BET inhibitor, and a dual PI3K/BET inhibitor in an MV4-11 acute myeloid leukemia (AML) cell line. Further microarray analysis of subsequent in house data and published data of BET inhibitors from different chemical scaffolds in hematologic cell lines allowed us to develop a short list of ~20 candidate genes. Further testing was done by measuring the modulation of these PD markers by various Zenith BET inhibitors from different chemical scaffolds in a number of human cell lines derived from hematological cancers and solid tumors, as well as cryopreserved human peripheral blood mononuclear cells (PBMCs). In vivo validation was also done in whole blood obtained from xenograft mice, and cynomolgus monkeys that were dosed orally with ZEN-3694, as well as ex-vivo treated human blood derived from normal donors or patients diagnosed with either AML or diffuse large B cell lymphoma. There was also robust target engagement in tumors of mouse AML xenografts, making them suitable tumor PD markers. A quantitative real-time PCR assay was developed for human whole blood matrix with parameters defined based on the multiplex efficiency (85-115%), coefficient of correlation of the standard curve (R2>0.98), and dynamic copy number range (10-106). Assay validation testing demonstrated an inter-assay variability (operator/day/machine) of Citation Format: Laura Tsujikawa, Karen Norek, Cyrus Calosing, Sarah Attwell, Dean Gilham, Nimisha Sharma, Jennifer Tobin, Michelle Haager, Ravi Jahagirdar, Sanjay Lakhotia, Henrik C. Hansen, Eric Campeau. Preclinical development and clinical validation of a whole blood pharmacodynamic marker assay for the BET bromodomain inhibitor ZEN-3694 in metastatic castration-resistant prostate cancer (mCRPC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-038. doi:10.1158/1538-7445.AM2017-LB-038

Benefit of Apabetalone on Plasma Proteins in Renal Disease

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 <0.05) were differentially enriched in renally impaired patients versus control subjects, including cystatin C and β2 microglobulin, which correlate with renal function. Bioinformatics analysis of the plasma proteome revealed a significant activation of 42 pathways that control immunity and inflammation, oxidative stress, endothelial dysfunction, vascular calcification, and coagulation. At 12 hours postdose, apabetalone countered the activation of pathways associated with renal disease and reduced the abundance of disease markers, including interleukin-6, plasminogen activator inhibitor-1, and osteopontin. Conclusion These data demonstrated plasma proteome dysregulation in renally impaired patients and the beneficial impact of apabetalone on pathways linked to chronic kidney disease and its cardiovascular complications.

APABETALONE (RVX-208) LOWERS CARDIOVASCULAR DISEASE (CVD) IN DIABETES MELLITUS BY A MECHANISM INVOLVING MICROBIOME MEDIATED ACTIVITY ON THE COMPLEMENT PATHWAY

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