HUMAN FECAL BILE ACID ANALYSIS AFTER INVESTIGATIONAL MICROBIOTA-BASED LIVE BIOTHERAPEUTIC DELIVERY FOR RECURRENT CLOSTRIDIOIDES DIFFICILE INFECTION | medRxiv
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][1]). 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. ### Competing Interest Statement RP, NF, KS, and BCF are employees of Ferring Pharmaceuticals. KB is an employee of Rebiotix Inc., a Ferring company. CG and BS are employed by BioRankings, LLC, which received fees for this analysis from Rebiotix Inc., a Ferring company. ### Clinical Trial NCT02299570 ### Funding Statement The work was supported by Rebiotix Inc., a Ferring company, and the Ferring Research Institute of Ferring Pharmaceuticals. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The institutional review board/research ethics board (IRB/REB) at each participating center approved the study protocol and informed consent form (Quorum Review, University of Chicago Biological Sciences Division/University of Chicago Medical Center; Western IRB; Louis Stokes Cleveland Department of Veterans Affairs Medical Center IRB; Henry Ford Health System IRB; University of Pennsylvania Institutional Review Board; St. Vincents Hospital and Health Care Center, Inc IRB; Loyola University Chicago Health Sciences Division Institutional Review Board for the Protection of Human Subjects; Mayo Clinic Institutional Review Boards; Weill Cornell Medical College IRB; IRB of St. Ritas Health Partners; HealthPartners Institute for Education and Research IRB; Hamilton Integrated Research Ethics Board; University of British Columbia Clinical Research Ethics Board; Washington University Human Research Protection Office). All study participants provided written informed consent. An independent medical monitor provided safety oversight. All methods were carried out in accordance with relevant guidelines and regulations. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The datasets presented in this article are not readily available because they are restricted by an ongoing commercialization process. Requests to access the datasets should be directed to KB. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02299570&atom=%2Fmedrxiv%2Fearly%2F2022%2F09%2F01%2F2022.08.30.22278604.atom
