Microbiome-based therapeutics are increasingly evaluated as a strategy to reduce recurrent Clostridioides difficile infection (rCDI), with proposed mechanisms including restoration of the microbiota and microbiota-mediated functions, such as bile acid (BA) metabolism. This study reports a quantitative and sensitive assay for targeted metabolomic assessment, and the application of the assay to profile BA composition in a Phase 2 trial of the investigational microbiota-based live biotherapeutic RBX2660 for reduction of rCDI. A liquid chromatography tandem mass spectrometry method was developed to extract and quantify 35 BAs from 113 participant stool samples from 27 RBX2660-treated rCDI participants in the double-blinded, placebo-controlled clinical trial. The results demonstrate a high-confidence assay as represented by sensitivity, linearity, accuracy, and precision. Furthermore, the assay enabled the observation of primary BAs as the dominant BA species at baseline in stool samples from clinical trial participants, consistent with the expected loss of commensals after broad-spectrum antibiotic treatment. After RBX2660 administration, there was a significant drop in primary BAs concurrent with increased secondary BAs that sustained through 24 months post-RBX2660. Taken together, we describe a robust assay that demonstrates altered BA metabolism in rCDI patients treated with RBX2660 administration.

Intestinal microbiome disruption is associated with recurrent Clostridioides difficile infection (rCDI), which poses a high risk of morbidity and mortality. Microbiome-based therapeutics are increasingly evaluated as a strategy to reduce rCDI, and their proposed mechanisms include restoration of the microbiota and microbiota-mediated functions, including bile acid (BA) metabolism. This study reports the development of a highly quantitative and sensitive assay for targeted metabolomic assessment of bile acids, and the application of the assay to profile bile acid composition in a Phase 2 trial of the investigational microbiota-based live biotherapeutic RBX2660 for reduction of rCDI (PUNCH CD2; NCT02299570). Participants were asked to provide stool samples before and up to 24 months after treatment. A liquid chromatography tandem mass spectrometry method was developed to extract and quantify 35 BAs from a total of 113 participant stool samples from 27 rCDI participants treated with RBX2660 in the double-blinded, placebo-controlled clinical trial. The results demonstrate a high-confidence assay as represented by sensitivity, linearity, accuracy, and precision of the output measurements of BAs. When the assay was utilized to assess stool samples from the clinical trial participants, primary BAs were the dominant BA species at baseline, consistent with the expected loss of commensals after broad-spectrum antibiotic treatment. As early as 7 days after RBX2660 administration, there was a significant drop in primary BAs concurrent with increased secondary BAs, and this profile was sustained through 24 months after RBX2660 administration. Taken together, we describe a robust assay that demonstrates altered BA metabolism associated with RBX2660 administration, shifting towards a profile that is consistent with a healthier BA profile and clinical response.