Oral communications

S3- EPIGENETICS AND THE BET-SYSTEM IN VASCULAR DEMENTIA, ALZHEIMER’S DISEASE AND MIXED DEMENTIA – THE PROBLEM AND POTENTIAL REMEDIES. Bengt WINBLAD (1), Charles DECARLI (2), Henrik ZETTERBERG (3), Ewelina KULIKOWSKI (4), Jeffrey CUMMINGS (5) ((1) Karolinska Institute, Sweden, (2) UC Davis, United States, (3) Sahlgrenska Academy, Sweden, (4) Resverlogix Corp., Canada, (5) Cleveland Clinics, United States)

The current world-wide prevalence of dementia is estimated at 35 million, and this number is projected to rise to over 100 million by 2050 if means of preventing, delaying, slowing or improving cognitive symptoms are not found. Most dementia is attributable to mixed age-related pathologies with Alzheimer’s disease (AD) and vascular pathology being the two most common contributing elements. Vascular risk factors such as age, lack of exercise, cigarette smoking, hypertension, and obesity are associated with the risk of cognitive decline, dementia, vascular cognitive impairment (VCI), and AD. There is a need to detect and differentiate disease early and to treat its’ root cause. Serum biomarkers that relate to different aspects of AD and VCI pathology include markers of neurodegeneration: neurofilament light chain and visininlike protein (VILIP-1); markers of amyloidogenesis and brain amyloidosis: apolipoproteins; markers of inflammation: YKL-40 and monocyte chemoattractant protein 1; marker of synaptic dysfunction: neurogranin. Serum alkaline phosphatase (ALP) has emerged as a marker of global dementia potentially by effects on tau processing and/or vascular calcification. These markers can highlight on the state and stage-associated changes that occur in AD, VCI and mixed disease with disease progression. Recent data suggest that epigenetic regulation is important in vascular pathophysiology, cerebral small vessel disease and vascular health. Gene expression mediated by activated BET system results in medial vascular calcification, increased levels of cytokines and endothelial adhesion molecules which are associated with compromised blood flow, neuroinflammation and cognitive impairment in nonclinical animal models. Bromodomain and extraterminal domain (BET) proteins are transcription-readers. They decondence/open chromatin and activate cytokine-associated transcription. BET proteins have two bromodomains (BD1 and 2) that bind acetylated lysines on transcription factors and chromatin with high affinity and are recruited through these interactions to the promoters and enhancers of genes that control cell identity, differentiation, and proliferation. On the promoters and enhancers, the BET proteins act as a scaffold, binding positive transcription elongation factor b to stimulate RNA polymerase II dependent transcription of the proximal genes. Many diseases alter acetylation marks, directing BET proteins to inappropriate genes, and pathological protein production. Apabetalone is a BD2-selective BET-inhibitor that returns mRNA and protein production towards physiological levels leading to improvement in vascular integrity, reduction in medial vascular calcification and decreased expression of inflammatory cytokines. Intensive research is ongoing in discerning their effects on neuron and glial cell (patho-) physiology. Bromodomain and extraterminal domain (BET) proteins are a family of four epigenetic readers (BRD2, BRD3, BRD4 and BRDT) that regulate gene transcription. Apabetalone modulates the expression of immune, inflammatory and pro-atherosclerotic genes in ex vivo treated human whole blood cells, as well as in the apoE knockout mouse model of atherosclerosis. Prophylactic and therapeutic treatment with apabetalone significantly reduced aortic lesion formation and lowered levels of circulating adhesion molecules and cytokines in hyperlipidemic apoE-/- mice. Apabetalone also impacts gene transcription within the acute phase response, complement and coagulation pathways in primary human hepatocytes, and vascular calcification in vascular smooth muscle cells. As part of correcting acute phase reactants apabetalone induces hepatic synthesis of apolipoprotein (apo) A-I enhancing cholesterol efflux capacity of high density lipoprotein (HDL) particles. The BET inhibitor apabetalone reduced endothelial and microglial activation in preclinical models of neuroinflammation. Apabetalone is a small molecule administered orally. It is metabolized by the liver and exhibits dose-proportional pharmacokinetics for single and multiple doses. Food increases its bioavailability; the pharmacokinetics are not affected by renal compromise. The half-life of apabetalone is 11 hours within the relevant dose range. In phase 2 studies apabetalone showed a reduction in broad-based CVD events of 44% which was most pronounced in patients with diabetes or with metabolic inflammation as defined by a high sensitive C-reactive protein (hsCRP) >2mg/L. Apabetalone lowers ALP geneexpression and serum ALP in a dose-response manner which is seen as a proxy for the multiple pathways that are regulated towards normal profiles, including inflammation, acute phase reactants, complement and coagulation. Sporadic elevated transaminases (>3x normal) occur in 7-8% of those exposed to apabetalone. After apabetalone treatment in more than 2000 patients for up to 3.5 years no combined bilirubin and ALT elevations have been observed indicating benign nature of the transaminase elevations. Apabetalone is being assessed in a Phase 3 multicenter double blind, parallel group, placebocontrolled trial in post-acute coronary syndrome patients with type 2 diabetes, low levels of HDL-C, to determine whether BET inhibition increases the time to major adverse cardiovascular events (MACE). The primary outcome of the BETonMACE study is time to a composite event of any of cardiovascular death, nonfatal myocardial infarction, or stroke. A pre-specified secondary analysis of BETonMACE will examine the effects of apabetalone on cognitive function using the Montreal Cognitive Assessment (MoCA) in patients 70 and older at randomization. In BETonMACE, MoCA was performed at baseline in 19% (n=470) of the population across 195 centers and 13 countries. Of those, approximately 52% (n=246) had a baseline MoCA score , suggesting potentially compromised cognition, and approximately 18% (n=84) had MoCA score <21 suggesting dementia. Significant contributors to a lower MoCA score came from domains of language and memory (both p A low MoCA score was associated with Caucasian race, history of hypertension, and previous percutaneous coronary intervention. At baseline, a lower MoCA score was associated with higher serum ALP. Exploration of the effects of apabetalone on MoCA scores and effects on quality of life (QoL, EQ-5D) will provide preliminary insight into the potential benefits of BET modulation on cognition and effects on QoL. A variety of biomarkers are being collected as secondary outcomes in the trial including ALP, hsCRP, fibrinogen ApoA-I, ApoB, LDL-C, HDL-C, triglycerides, HbA1c, fasting glucose, fasting insulin, transcription factor change in whole blood, and proteomic profiles. As pre-specified, provided a favorable signal of apabetalone treatment on MOCA in this diabetes population archive plasma samples are available. Archive samples would be used for assessing apabetalone treatment effects in population with neurodegenerative pathology and AD burden. Depending on results apabetalone would be expanded to neurodegenerative indications. Interrogation of the relationship between changes in biomarkers and drug-placebo differences on the MoCA will inform understanding of the biology of observed differences.

Poster presentations

P192- EPIGENETIC MODULATOR APABETALONE I N H I B I T S M O N O C Y T E A D H E S I O N T O B R A I N ENDOTHELIAL CELLS BY DOWNREGULATING KEY NEUROINFLAMMATION MARKERS IN VITRO AND IN VIVO. S. Wasiak1 , E. Daze1 , L.M. Tsujikawa1 , S. Das1 , L. Fu1 , D. Gilham1 , B.D. Rakai1 , S.C. Stotz1 , C.D. Sarsons1 , D. Studer2 , K.D. Rinker2 , R. Jahagirdar1 , N.C.W. Wong1 , M. Sweeney3 , J.O. Johansson3 , E. Kulikowski1 ((1) Resverlogix Corp - Calgary (Canada), (2) University of Calgary - Calgary (Canada), (3) Resverlogix Corp - San Francisco (United States))

Background: Circulating cytokines induce inflammatory changes in brain vascular endothelial cells that promote monocyte adhesion and transmigration across the blood brain barrier. This process contributes to the initiation and exacerbation of neuroinflammation, which ultimately leads to neuronal injury and neurodegeneration. Bromodomain and extraterminal domain (BET) proteins are histone acetylation readers that activate cytokine-dependent transcription in monocytes and endothelial cells in chronic vascular inflammation models. Targeting BETs with epigenetic therapies may reduce endothelial activation during neuroinflammation. Objectives: To evaluate the anti-inflammatory properties of apabetalone, a clinical stage small molecule that inhibits the transcriptional activity of BET proteins, in cellular models of brain inflammation. Methods: THP-1 monocyte gene expression was examined in response to TNFα +/- apabetalone by real time PCR (rtPCR) after 4-24h of exposure. Primary human brain microvascular endothelial cells (HBMVECs) or the brain endothelial hCMEC/D3 cell line were stimulated with IL-1β, TNFα and/or IFNγ +/- apabetalone for 4-24h, and assayed for gene expression (rtPCR), cytokine secretion (ELISA, MILLIPLEX® Multiplex Assays) and/or surface cell adhesion protein abundance (flow cytometry). THP-1 cell adhesion to monolayers of HBMVECs was measured in laminar flow conditions. In vivo neuroinflammation was assessed in C57BL/6 male mice pretreated with apabetalone for 7 days (150 mg/ kg b.i.d.), and then injected with 10 mg lipopolysaccharide (LPS) intraperitoneally. mRNA from homogenized brain tissue was analyzed 24h post LPS injection. Results: In THP-1 monocytes, apabetalone suppressed the expression of genes induced by TNFα, including IL-1β, the chemokine MCP1, chemokine receptors CCR1 and CCR2, and the adhesion molecule VLA-4 (40% to 90% reduction, p<0.05)). In hCMEC/ D3 endothelial cells, cytokine stimulated secretion of key inflammatory chemokines involved in monocyte attraction and vascular inflammation was reduced by apabetalone, including granulocyte-macrophage colony-stimulating factor, fractalkine, MCP-3, IP-10, and IL-6 (40% to 90% reduction, p<0.05). In TNFα and IFNγ stimulated HBMVECs, apabetalone inhibited the mRNA levels and the surface abundance of the cell adhesion proteins VCAM-1 (80% reduction) and E-selectin (50% reduction). In agreement with this inflammatory marker downregulation, apabetalone treatment countered THP-1 adhesion to HBMVECs in laminar flow assays. In mice, apabetalone treatment attenuated the LPS-induced mRNA expression of inflammation markers in the brain including E-selectin, ICAM, CCR2, and CD68. Conclusions: Apabetalone treatment decreased brain endothelial cell activation and monocyte interactions. By reducing immune cell transmigration in pro-inflammatory conditions, apabetalone may impact brain inflammation and potentially reduce cognitive decline. The effect of apabetalone treatment on the cognition of diabetic patients ≥70 years old with acute coronary syndrome is being evaluated through repeat MoCA testing in the phase 3 BETonMACE trial (results expected in H2 2019).