Comparative Efficacy of Therapeutics for Clostridioides difficile Infection Biotherapeutics Inc (BTI) is an emerging biotech company that synergistically combines the power of advanced computational modeling with translational experimentation to accelerate the development of novel precision immunology therapeutics. Clostridioides difficile infection (CDI) is a gastrointestinal (GI) infection, established upon disruption of gut microbiome commonly following antibiotic administration. Almost half a million people in the United States are affected by CDI every year, causing an economic burden of over $6 billion.. Despite the status of CDI as an antibiotic-associated infection, the standard of care (SOC) for CDI is dominated by antimicrobials, reporting high infection recurrence rates. Novel antimicrobial-free strategies have been developed to address this dichotomy, including fecal microbiome transplantation and administration of monoclonal antibodies against C. difficile toxin B (TcdB). However, safety concerns and high costs, create the need for more effective, antimicrobial-free therapeutics with novel mechanisms of action. BTI has developed a novel, oral, once-daily, first-in-class, immunoregulatory therapeutic being evaluated in clinical development for inflammatory bowel disease (IBD) that provides therapeutic efficacy in acute and recurrent CDI, in an antimicrobial-free, microbiome-preserving manner. This SBIR aims to accelerate the development of this novel therapeutic through a head-to-head comparison of the efficacy of our product candidate versus traditional and recently approved therapeutics for the treatment of CDI. The specific aims of this project are to: AIM 1. Evaluate the comparative efficacy of our new product candidate versus FDA-approved therapeutics for CDI in two recurrent mouse models. We will assess weight loss, disease activity, histopathological lesions, and colitis through flow cytometry immunophenotype and gene expression profile. AIM 2. Compare the effects of our new product candidate versus current therapeutics in modulating gut microbiome dynamics during CDI. In a recurrent model of CDI, we will assess gut microbiome composition and diversity, plus abundance of C. difficile-resistant strains through 16S sequencing and metabolomic analysis. Expected outcomes: i) ≥ 30% decrease of histopathological scores; ii) ≥ 40% decrease of Th17 infiltration; and iii) ≥ 60% increase of alpha diversity compared to SOC. The SBIR Phase II will validate the therapeutic efficacy in hamster and pig models of CDI. finalize IND-enabling studies and file an IND for treating CDI. Commercial application: The long-term goal of this project is to develop a new line of host-centered small molecule therapeutics that are safer and more effective than standard of care (SOC) treatments for CDI, with reduced disease severity and recurrence rates, and initial clinical testing expected by 2024.