Date on Master's Thesis/Doctoral Dissertation

5-2023

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Matoba, Nobuyuki

Committee Co-Chair (if applicable)

Siskind, Leah

Committee Member

Siskind, Leah

Committee Member

Yaddanapudi, Kavitha

Committee Member

Hong, Kyung

Committee Member

Dryden, Gerald

Author's Keywords

Epicertin; mucosal healing; ulcerative colitis

Abstract

This dissertation describes the development of a biotherapeutic agent, EPICERTIN, that fulfills an unmet treatment need for ulcerative colitis (UC): mucosal healing. EPICERTIN is a recombinant cholera toxin B subunit (CTB) variant with a KDEL endoplasmic reticulum (ER) retention motif that is crucial for its wound healing capabilities. Previous studies have demonstrated that EPICERTIN’s mucosal healing activity is due, in part, to its prolonged residence in the ER which initiates an unfolded protein response (UPR) and subsequent TGF-β signaling and barrier repair in colonic epithelial cells. This activity was demonstrated in a dextran sulfate sodium (DSS) colitis model as well as in human colon explants from inflammatory bowel disease (IBD) patients. Epithelial barrier repair has been implicated as a key step to achieving mucosal healing, which is predictive of remission and improved quality of life for UC patients. No currently approved therapeutic indicated for UC aims to target this highly prognostic treatment goal. As such, a pre-IND package was submitted to the FDA to inform necessary studies prior to IND submission and first-in-human trials. Herein, Chapter 1 discusses what is known about EPICERTIN’s mechanism of action thus far and provides an overview of the biopharmaceutical development process in the United States. The methodology of the present studies is detailed in Chapter 2. As CTB is a known potent immunomodulator, Chapter 3 explores the impact of the immune system on the therapeutic activity of EPICERTIN. The immune-mediated oxazolone (OXA)-induced colitis model was utilized to corroborate the therapeutic efficacy of EPICERTIN observed previously in the DSS colitis studies. We found that two doses of 3 μg EPICERTIN three days apart (Q3D) after the onset of OXA-induced colitis is effective in mitigating disease severity. This Chapter also describes a preliminary investigation into the impact of EPICERTIN on innate and adaptive immune cell populations of the healthy colon lamina propria in mice compared to CTB to identify cell types with a potential role in EPICERIN’s healing activity. Under healthy conditions, the tested dose conditions were not sufficient to alter these populations in the colon lamina propria, despite the robust production of anti-EPICERTIN IgG and IgA antibodies. Chapter 4 steps away from mechanistic investigation and describes stability, pharmacokinetic (PK), and toxicological assessments of EPICERTIN, which are crucial aspects of a future IND submission. EPICERTIN drug substance was found to remain stable following storage at 4°C and 25°C for 24 months. A slight increase in the protein concentration and potency of EPICERTIN stored at 25°C was noted starting at 12 months, which is speculated to be a result of evaporation from the containment closure system due to the fact that purity and identity parameter specifications were met at all timepoints. PK profiles in male and female mice and rats were established for an enema formulation of EPICERTIN with a

theoretical target clinical dose of 0.1 μM. It was revealed that EPICERTIN was not absorbed into the systemic circulation upon intrarectal (ir) administration of 50 or 100 times the target clinical dose for healthy and colitic mice or healthy rats, respectively. A single ascending dose-range finding toxicity study demonstrated that the maximum tolerated dose (MTD) and no observed adverse effect level (NOAEL) in rats is at least 50 times the target clinical dose. These results provide encouraging implications regarding the safety of EPICERTIN in humans. Chapter 5 summarizes the results of the previous chapters as well as implicates future directions for the project. In summary, EPICERTIN is able to mitigate disease in an immune-mediated colitis model. However, this dose was not sufficient to alter innate or adaptive immune cell populations of the colon lamina propria in healthy mice. Regarding preclinical development, EPICERTIN is stable for up to 24 months upon storage at 4°C or 25°C. EPICERTIN is also not systemically absorbed upon ir administration and does not exhibit any overt toxic effects at a dose of at least 50 times the target clinical dose. Taken together, these results support the continued development of EPICERTIN as a first-in-class mucosal healing agent for UC.

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