Preclinical Characterization of Therapeutic Candidates for Inflammatory Disease and Chemical Injury

Abstract

Herein, I report the use of a cytotoxicity assay to rapidly evaluate the safety and biological activity of newly synthesized small molecules. Using cultured mammalian cells, overt toxicity was measured by quantification of ATP as a viability biomarker. The preceding data provided an efficient early assessment of compound-induced cytotoxicity, enabling initial prioritization for animal studies. Advancing compounds were evaluated with murine safety studies to detect early signs of adverse central nervous system (CNS) effects and systemic toxicity. Using open-field and rotarod tests, locomotor activity was quantified and the motor coordination of mice after compound administration. The rotarod test is a reliable and sensitive in vivo sensorimotor assay that provides insight into compound-related sedation, ataxia, impaired coordination or other adverse effects.1The open field test quantified changes in volunteer movement between the vehicle and the compound-treated mice. Various drug candidates were evaluated supporting the selection process for lead candidates. Pharmacokinetic (PK) studies were conducted to define the compound’s absorption, distribution, metabolism, and elimination. This work provided information to select dosing for subsequent pharmacodynamic studies. Furthermore, quantitative toxicology studies using escalating-dose and repeated-doses were conducted. In combination, these efforts helped to define the maximum tolerated dose (MTD), daily exposure limits, and potential for drug accumulation. Different compound formulations were then evaluated with the goal of enhancing solubility, stability, and sustained release, which included the microencapsulation of a select compound (Compound 1). Next, Compound 1 was investigated across complementary murine models of atopic dermatitis (AD), asthma, and inflammatory bowel disease (IBD). Compound 1 and Compound 2 was evaluated in a vitamin D3 analog (MC903)-induced atopic dermatitis (AD) model. Followed by quantification of key inflammatory and disease-related biomarkers. Further validation of Compound 1 was conducted with an ovalbumin-sensitized asthma model and quantification of lung Thymic stromal lymphopoietin (TSLP) levels. Analogs of Compound 1 were investigated for their effects on airway hyperresponsiveness (AHR) using methacholine-challenged A/J mice.2 Finally, treatments options for dextran sulfate sodium (DSS)-induced colitis using both C57 and CFW mice, we investigated for potential changes of in-life and post mortem indices of IBD. These models were chosen due to their common features of epithelial barrier and comparable inflammation across barrier tissues, allowing to evaluate peripheral gamma-aminobutyric A (GABAA) receptor modulation as a strategy to improve clinical outcome. Building on prior efficacy studies, Compound 1 was investigated as a medical countermeasure for chlorine gas-induced lung injury. This work addressed a critical unmet need for potentiating injury following accidental exposure or chemical warfare. Compound 1 was designed to activate non-neuronal GABAA receptors to suppress calcium-dependent inflammatory signaling. Extensive preclinical testing demonstrated a favorable safety profile, rapid onset of action, temperature stability suitable for stockpiling, and potent anti-inflammatory effects across multiple pulmonary and inflammatory in vivo models. Two study designs were developed to assess survival and recovery after chlorine exposure, providing temporal measures of injury and physiological improvement needed to evaluate therapeutic efficacy. Finally, recombinant full-length and truncated human FIS1 proteins were expressed in E. coli, purified, and evaluated for their ability to bind fluorescently labeled peptide PEP213. Milligram amounts of each protein were obtained from liter cultures of E. coli at sufficient concentrations of about 3 mg/ml.