Initiation Of Mucosal Inflammation Research Articles

NANOPARTICLE-ENCAPSULATED BROMODOMAIN-CONTAINING PROTEIN 4 INHIBITORS FOR THERAPEUTICS OF INFLAMMATORY BOWEL DISEASE

Abstract Ulcerative Colitis (UC) and Crohn’s disease (CD), the two major types of inflammatory bowel disease (IBD), are chronic diseases with recurrent symptoms and significant morbidity. Long-term persistence of chronic inflammation in IBD is among the major factors contributing to neoplastic transformation and the development of colitis-associated colorectal cancer. There exists a lack of efficient medications for IBD, primarily due to either limited efficacy or side effects. Targeting bromodomain-containing protein 4 (BRD4) represents a novel therapeutic strategy for IBD. Recently, we have successfully identified proprietary, highly potent, and specific BRD4 inhibitors which significantly suppressed the initiation of mucosal inflammation and chronicity in proof-of-concept studies in several animal models of IBD. One of the most pressing challenges in current IBD research is to develop technologies to enable mucosal targeted drug delivery systems that enhance efficacy and decrease side effects. Intriguingly, we found that local delivery of our first-generation BRD4 inhibitors encapsulated in nanoparticles can achieve significantly higher in vivo efficacy at much lower doses than that attained by systemic administration. Local delivery of nanoparticle-encapsulated BRD4 inhibitors by designing nanoparticles that bind to inflamed epithelium would offer superior pharmacotherapy for IBD with higher efficacy, specific delivery, long-lasting release, and a better therapeutic and safety window. We have successfully encapsulated our BRD4 inhibitors in biodegradable nanoparticles with excellent encapsulation efficiency and favorable particle size. Our new generation of BRD4 inhibitors ZL0513, ZL0742, and ZL0591 were encapsulated by a modified solvent displacement method using a polymeric matrix of PEGylated poly (lactic-co-glycolic acid) (PLGA). To evaluate their potential cytotoxic effects, we incubated human colonic epithelial cells (HCECs) and peripheral blood mononuclear cells (PBMCs) with PEGylated-PLGA-ZL0513, ZL0591, and ZL0742 overnight. No apparent increase in cell death was detected in HCECs or PBMCs even at 40 μM. Furthermore, oral administration of our nano-encapsulated BRD4 inhibitors at the dosage of 2 mg/kg effectively block colonic inflammation in both IBD animal models of dextran sulfate sodium (DSS)-induced colitis and oxazolone (OXA)-induced colitis. Collectively, our compelling in vivo efficacy data support that our nano-encapsulated BRD4 inhibitors effectively block colonic inflammation in animal models of IBD. Local delivery of nanoparticle-encapsulated BRD4 inhibitors may offer superior pharmacotherapy for IBD patients. Funding support: Crohn’s & Colitis Foundation Entrepreneurial Investing (EI) Initiative award and Litwin IBD Pioneers Program award from the Crohn’s & Colitis Foundation of America.

6 NOVEL BRD4 INHIBITORS BLOCK THE PATHOLOGICAL ACTIVATION OF BRD4-NFκB SIGNALING AND SUPPRESS COLONIC INFLAMMATION IN IBD MOUSE MODELS

Abstract Ulcerative Colitis (UC) and Crohn’s Disease (CD) are two major types of inflammatory bowel disease (IBD), with recurrent symptoms and significant morbidity. Long-term persistence of chronic inflammation in IBD is among the major factors contributing to neoplastic transformation and development of colitis-associated colorectal cancer. There is a lack of efficient medications for IBD, due to either limited efficacy or side effects. Antibodies against TNFα are effective therapies; however, they are expensive, and up to 40% of patients are non-responders. Thus, improved therapy with higher efficacy, enhanced safety and better patient affordability is urgently needed. The master regulator of innate immune response NFκB plays a critical role in the initiation and chronicity of IBD mucosal inflammation. We demonstrated that bromodomain-containing protein 4 (BRD4), an epigenetic reader, is required for stabilization of NFκB binding on the promoters of inflammatory genes, activation of RNA polymerase II, and histone H3 Lys122 acetylation (H3K122ac) to permit high levels of inflammatory gene expressions by sentinel epithelial cells and stromal fibroblasts. Our data using human IBD patient samples suggest that BRD4 activation is critical to the initiation of colonic inflammation. We have successfully identified highly potent and specific BRD4 inhibitors, demonstrating that inhibition of BRD4 suppresses expression of inflammatory cytokines TNFα, IL-6, IL-17A, and IL-8. Administration of our lead inhibitors (ZL0454 & ZL0420) significantly reduces initiation of the mucosal inflammation in both dextran sulfate sodium (DSS)-induced and Cbir1 T cell transfer colitis models in vivo, but with low toxicity and much safer than the generic NFκB/IKK inhibitor BMS345541. The levels of NFkB and BRD4 activation were also compared using immunofluorescence staining of NFkB/RelA translocation, phospho-Ser276 RelA formation, and induction of the BRD4 marker H3K122Ac. The BRD4 inhibitors reduced DSS-induced NFkB-BRD4 activation in colon tissue, demonstrating the target specificity in vivo. Our data suggest that BRD4 activation is critical to the initiation of the colonic inflammation during IBD. BRD4 inhibition disrupts its interactions with acetylated histone lysine residues, thereby blocking the pathological activation of the BRD4-NFκB signaling and suppressing colonic inflammation. Therefore, inhibition of the BRD4 activation is an attractive target for the development of novel IBD therapy and in prevention of CAC. Using our novel BRD4 inhibitors, we will further seek to evaluate the effect of long-term BRD4 inhibition on UC-induced neoplastic transformation and development of CAC. Funding support: Crohn’s & Colitis Foundation Entrepreneurial Investing (EI) Initiative award and a research fellowship award from the Crohn’s & Colitis Foundation of America.