Auphos, a novel drug that improves colitis by microbiome induced metabolic changes

Abstract BACKGROUND Dysbiotic intestinal microbiomes dominated by facultative anaerobes are strongly associated with inflammatory bowel disease (IBD). More recently, data from Baumler and colleagues support a model where mitochondrial dysfunction causes disease-associated dysbiosis by increasing oxygen (O2) availability to the microbiome. We posit that the restoration of epithelial mitochondrial respiration contributes to the restoration of a healthy microbiome dominated by facultative anaerobes such as Firmicutes [that produce short chain fatty acids (SCFA)]. Here, we tested a novel compound (AuPhos) that increases an O2 consumption in intestinal epithelial cells (IECs) thereby reducing O2 availability to the microbiome and promotes a healthy anaerobic environment (and blooms of firmicutes). METHOD Inbred C57BL/6 mice (n=3) were given oral vehicle (0.5% DMSO/Tween-80) or AuPhos (2.5 or 25mg/Kg;q3d) for two weeks, and colon and stool samples were extracted at day-15 sacrifice. Microbial DNA was isolated from stool samples using PoweFecal kit followed by 16S rRNA metagenomic sequencing. Linear Discriminant Analysis (LDA) and PICRUST2 were performed to reveal differentially abundant bacterial species and functional potentials of bacterial communities, respectively. Similar microbial profiling was performed on AuPhos-fed acute colitis mice (2% DSS-7d; Recovery-16d). Effect of AuPhos on microbial metabolism was determined by 1H NMR for detecting SCFA synthesis by gut microbiota in stool samples (n=3) collected after 3 consecutive doses. AuPhos-induced hypoxia in IECs was assessed by Hypoxyprobe-1 kit. RESULT Metagenomic analysis showed reduction in relative abundance of (O2 consuming) Proteobacteria and facultatively anaerobic Enterobacteriaceae in the gut, in AuPhos-fed mice. Conversely, AuPhos treatment dose- dependently increased the relative abundance of signature anaerobic bacteria e.g. Firmicutes including Clostridia (Faecalibacterium prausnitzii, Roseburia sp.), Bifidobacterium, etc. PICRUST2 and LDA revealed that AuPhos decreased bacterial LPS biosynthetic pathway and increased overall fatty acid biosynthesis pathways. AuPhos increased butyrate and propionate levels by >35% and acetate by 60% (n=3) compared to stools from vehicle-treated mice. In DSS-colitis mice, AuPhos reduced the relative abundance of inflammatory Proteobacteria, which includes facultative anaerobic Enterobacteriaceae family and increased Firmicutes over time (p<0.05). Interestingly, hypoxia staining showed AuPhos-induced O2 utilization in colonic surface IECs, facilitating obligate anaerobe-promoting environment in the gut. CONCLUSION These finding suggest that AuPhos is a “first-in-class” oral therapeutic that has a potential to correct microbial dysbiosis in IBD by reducing epithelial oxygenation and thereby promoting a healthy microbiome dominated by obligate anaerobes.