SAFETY OUTCOMES AND CONCOMITANT MEDICATION CHANGES IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE BEING TREATED WITH RBX2660 FOR RECURRENT <i>CLOSTRIDIOIDES DIFFICILE</i> INFECTION

Background: Recurrent Clostridioides difficile infection (rCDI) is a debilitating disease associated with significant healthcare burden and low quality of life. The greatest risk factor for rCDI is a history of recurrence, although older age (≥65 years) and comorbidities also increase risk. Recurrent CDI occurs in 20-36% of patients with 1st recurrence and ≥40% in those with ≥2 recurrences. In a Phase 3 randomized controlled trial (ECOSPOR III), the investigational oral microbiome therapeutic SER-109 was superior to placebo in reducing risk of rCDI at 8 weeks in patients with ≥2 recurrences (12% vs 40%) and this response was durable through 24 weeks [Feuerstadt NEJM 2022; Cohen JAMA 2022]. Here we present safety and efficacy data from a Phase 3 open-label trial (ECOSPOR IV), including durability of response in patients with 1^st and ≥2 recurrences.

Methods: Adults with rCDI were enrolled at 72 North American sites in 2 cohorts: 1) rollover subjects with rCDI in ECOSPOR III, diagnosed by toxin EIA) and 2) subjects with ≥1 CDI recurrence (diagnosed by PCR or toxin EIA, inclusive of the current episode). After standard-of-care antibiotics, subjects received SER-109 (4 capsules daily x 3 days). Efficacy endpoints, including the proportion of subjects with rCDI (toxin+ diarrhea requiring treatment), were evaluated through Week 8; safety and durability of response were assessed through Week 24.

Results: Of 351 subjects screened, 263 were enrolled (Cohort 1: N=29; Cohort 2: N=234; 68% female; mean age 64 years). Approximately 1/3 of subjects enrolled with their 1st recurrence. Comorbidities included cardiac disorders (31%), neoplasms (21%), Type 2 diabetes (11%), COPD (10%), chronic kidney disease (9%), and hepatobiliary disorders (9%). Overall, 141 subjects (54%) had treatment-emergent adverse events (TEAEs), the majority of which were mild to moderate and gastrointestinal. There were 8 deaths (3%) and 33 subjects (13%) with serious TEAEs; none were treatment-related (Table 1).

Overall, 240 subjects (91.3%) had clinical response (ie, no CDI recurrence) at 8 weeks. Of these, 227 subjects (94.6%) maintained a durable response through Week 24. Among the 240 responders at Week 8, 72 subjects (30%) had a history of 1st recurrence; the remainder had a history of ≥2 CDI recurrences. The proportion of Week 8 responders who maintained durable response through Week 24 was similar between those with a history of 1st recurrence vs ≥2 recurrences (94.4% vs 94.6%, respectively; Figure 1).

Conclusions: SER-109, a potential first-in-class oral investigational microbiome therapeutic, was well-tolerated in this population of patients with multiple comorbidities. High and durable clinical response rates were observed through Week 24, regardless of the number of prior CDI recurrences, supporting the potential benefit of microbiome repair following antibiotics to treat rCDI.

Background: Antibiotics used to treat CDI perturb the gut microbiome, increasing susceptibility to recurrent CDI (rCDI). Fecal microbiota transplants and donor-derived treatments promote the establishment of a gut environment resistant to CDI, but the composition and quality attributes of these are inherently variable; they are difficult to scale up, and have resulted in transfer of harmful pathogens. In contrast, VE303 is a rationally defined consortium that is manufactured from clonal cell banks, obviating the need for donors and overcoming the limitations of donor-derived treatments. In the Phase 2 CONSORTIUM Study, the VE303 high dose arm had an acceptable safety profile and significantly reduced the risk of rCDI compared with placebo. The high dose also induced superior VE303 strain colonization at 14 days, achieved long-term engraftment, and promoted early restoration of the microbiota and beneficial metabolites.

Methods: The CONSORTIUM Study was a randomized, double-blind, placebo-controlled, dose-finding study in individuals at high risk of rCDI. After completing a course of antibiotics for a lab-confirmed CDI episode, subjects were randomized 1:1:1 to low-dose VE303 (1.6 x 10⁸ CFU), high-dose VE303 (8 x 10⁸ CFU), or placebo orally once daily for 14 days. Subjects were followed for 24 weeks to monitor safety, rCDI episodes, and gut microbiota composition. Fecal samples were collected during dosing and at weeks 4 and 8; metagenomic sequencing and metabolomics analysis was performed to identify associations between VE303 strain engraftment, resident microbes, stool metabolites, and clinical safety and efficacy.

Results: Among 378 analyzed species, VE303-08, -02 and -01 were the only bacterial species found to be significantly associated with nonrecurrence (panel A, p-adjust<0.25, linear mixed effects [LME] model). Table 1 shows taxa associated with clinical response and VE303 at species and higher taxonomic levels, with greater Clostridia in nonrecurrent subjects and Proteobacteria in recurrent subjects. VE303 was negatively correlated (p-adjust<0.05, LME) with recurrence-associated taxa Veillonella and Kluyvera, and positively correlated with response-associated taxa. Also, multiple VE303 strains were positively correlated with gut metabolites (panel B, p-adjust<0.05, LME) including the short-chain fatty acids valerate, acetate and butyrate, and the 2° bile acid UDCA, which confers colonization resistance against C. difficile and inhibits spore germination.

Conclusion: In subjects at high risk of rCDI, VE303 species were positively correlated with beneficial Clostridia, short-chain fatty acids, and UDCA, and negatively correlated with recurrence-associated Proteobacteria. VE303 may protect against rCDI through a combination of direct exposure to the consortium strains and VE303-mediated recovery of the endogenous microbiota and metabolites.

Background: The prevalence of primary and recurrent Clostridioides difficile infection (CDI) has increased since 2000, but contributing factors remain unclear. Biofilm formation decreases C. difficile antibiotic susceptibility and can contribute to increased fitness in the gut. The aim of this study was to determine if patients with CDI are more likely to have colonic mucosal colonization with C. difficile.
Methods: We identified 52 patients with primary episode of CDI and a matched group of 84 patients without CDI for whom archived colonic biopsies (formalin fixed, paraffin embedded) were collected within one year prior (range 7-363; median 134). We assessed presence or absence of C. difficile biofilm in biopsies by looking for overlap of universal bacterial 16S rRNA (EUB338 and C. difficile-specific 16S rRNA fluorescent in-situ hybridization (FISH) probe) and C. difficile biofilm polysaccharide II (PSII; immunohistochemistry). To identify factors contributing to CDI we used univariate and multivariate generalized logistic regression analysis (GLM; R stats V 3.6.2 package). To quantify C. difficile biofilm in colonic mucosa, signals from DAPI and MUC2 channels identified background host tissue and a custom script contoured the host tissue. The same mask was applied to images in PSII, EUB338, and C. difficile 16S FISH channels. The overlap of pixels detected in EUB338, C. difficile 16S FISH and PSII was considered positive for C. difficile biofilm. Since biofilms may position around C. difficile cells in unpredictable locations, the PSII detection range extended to 10µm around the cell to allow identification of biofilm matrix. Biofilm detection algorithm was tested on 10 biofilm-positive or 10 negative samples examined and sorted manually. There was statistically significant higher pixel count in biofilm-positive samples. A pixel count cutoff of 100 separated biofilm-positive samples from biofilm-negative samples.
Results: There were no significant differences in age, sex, BMI, or inflammatory bowel disease among CDI and non-CDI groups (Table 1). C. difficile biofilm was identified in 30/51 (58.8%) patients prior to primary episode of CDI and 5/84 (5.6%) patients without CDI. Univariate analysis showed patients with CDI were more likely to have antibiotic use, hospitalization, corticosteroid use (trend), and colonic mucosal colonization as evidenced by C. difficile biofilm (Table 2). A multivariate analysis showed patients with CDI were more likely to have hospitalization, corticosteroid use (trend) and C. difficile biofilm (Table 2).
Conclusion: Our findings suggest asymptomatic individuals can harbor C. difficile in the colonic mucosa and such colonization may increase risk of future CDI. Larger prospective studies are needed to confirm the role of C. difficile biofilm formation in primary and recurrent CDI.

Introduction: Live biotherapeutic products (LBPs) have shown efficacy in reducing recurrent Clostridioides difficile infection (rCDI). While not fully understood, their mechanism of action involves shifting the gut microbiome composition and diversity toward those found in healthy populations, wherein the microbiota help suppress C. difficile outgrowth and CDI recurrence. These shifts are likely facilitated by clonal engraftment of microbiota from LBPs in recipients. Using a single nucleotide variant method, we report clonal engraftment of bacterial populations from RBX2660, the first FDA approved, single-dose, microbiota-based LBP, in participants of the PUNCH CD3 phase 3 trial (NCT03244644).

Methods: Participants enrolled in PUNCH CD3 were ≥18 years old with ≥1 rCDI episode. After completing SOC antibiotic therapy for the enrolling CDI episode, participants received a single blinded dose of RBX2660 or placebo. Treatment success was defined as remaining free of CDI recurrence for 8 weeks after treatment. As an exploratory analysis, participants were asked to provide stool samples before (baseline) and at several time points after assigned study treatment. Samples were extracted and sequenced using deep (>20 MM reads) shotgun methods (Diversigen, MN). Species-level profiling was based on a metagenomic species (MGS) approach. Polymorphic positions (PMPs) were used to determine whether identified MGS populations were the same using a threshold of >50 overlapping PMPs. Microbiome restoration at the taxonomic class level was determined based on MGS assignments. As a control analysis, all MGS from a randomly selected RBX2660 dose were paired with each placebo-treated participant sample and overlapping PMPs were compared to assess clonal engraftment.

Results: RBX2660-treated participants who achieved 8-week treatment success showed restoration of gut microbiota to a composition and diversity similar to those of healthy individuals as soon as 1 week after treatment. Successful outcomes were significantly associated with greater levels of clonal engraftment from RBX2660 (p<0.001). Among RBX2660-treated participants, a median of ≥10 instances of clonal engraftment per participant were detected at 1 week after treatment, and this persisted through the 6-month follow up. MGS found within the Bacteroidia and Clostridia taxonomic classes showed the highest clonal engraftment. The control analysis showed no significant clonal engraftment in placebo-treated participants.

Discussion: A clonally-resolved MGS-based method is ideal for assessing engraftment of LBP bacterial populations. In the PUNCH CD3 phase 3 trial, clinical response to RBX2660 was associated with clonal engraftment of RBX2660 microbiota into trial participants. Bacteroidia- and Clostridia-class bacteria were the most effective engrafters.

Background and aims: We evaluated a microbiota-modifying drug, CSA13, that inhibited Clostridioides difficile infection (CDI) in mice. Metabolomics analysis indicated that four fecal metabolites (3-aminobutyric acid, citrulline, retinol, and ursodeoxycholate) are protective against CDI. We hypothesize that citrulline is the best metabolite for reducing inflammation, protecting mucosa, and maintaining survival.
Methods: Fresh human colonic tissues from CDI patients, primary human colonic epithelial cells from CDI patients, and CDI serum exosome-conditioned primary human macrophages were used to determine metabolite-regulated cytokine secretion. C. difficile (VPI10463)-infected mice and hypervirulent ribotype 027-infected hamsters were used to study the metabolite efficacy.
Results: Compared to other tested CSA13-dependent metabolites, 10mg/kg/day of oral citrulline treatment was the best in protecting survival and preventing recurrence in C. difficile-infected mice and hamsters. Citrulline inhibited toxin A-mediated macrophage inflammatory protein one alpha (MIP-1alpha) secretion, promoted toxin B-mediated interleukin 10 (IL-10) mRNA expression, and protected against toxin-mediated tissue injury in CDI patient-derived fresh colonic tissues. Citrulline dephosphorylated glycogen synthase kinase 3 (GSK3alpha/beta) and inhibited toxin A-mediated MIP-1alpha secretion in human colonic epithelial cells. This anti-inflammatory effect was reversed by insulin-like growth factor 1 (IGF-1) because IGF-1 dephosphorylated GSK3. Citrulline also activated heat shock protein 27 (HSP27) phosphorylation and secretion and promoted IL-10 secretion in toxin B-treated primary human macrophages, which was reversed by HSP27 inhibitors. Oral citrulline treatment ameliorated colitis with reduced colonic MIP-1alpha expression in infected mice, but this protective effect was abolished by an intraperitoneal injection of mouse MIP-1alpha. Intraperitoneal injection of mouse IL-10 did not protect the mice against CDI. In the infected hamsters, oral citrulline treatment ameliorated cecitis with reduced cecal MIP-1alpha expression and increased cecal IL-10 expression. Intraperitoneal injection of either MIP-1alpha or anti-IL-10 neutralizing antibodies abolished the survival protection of citrulline treatment in the infected hamsters. Oral citrulline and vancomycin treatment enhanced the cecal abundance of Bacteroides and prevented vancomycin-associated CDI recurrence in hamsters. Cecal microbiota transplantation from citrulline- and vancomycin-treated donors conferred survival protection to vancomycin-treated recipient hamsters.
Conclusion: Depending on animal models, citrulline ameliorated primary CDI via MIP-1alpha inhibition (mice and hamsters) and IL-10 expression (hamsters only). Citrulline-driven microbiota modification might protect against CDI recurrence in the long term.

Introduction: RBX2660 is the first FDA approved, single-dose, microbiota-based live biotherapeutic used to prevent recurrence of Clostridioides difficile infection (rCDI) in adults. Underlying inflammatory bowel disease (IBD) is a known risk factor for rCDI, yet patients with IBD are often excluded from prospective clinical trials. We report the safety outcomes and changes in IBD-related medications after RBX2660 treatment, in adult patients categorized by IBD comorbidity subgroups in the ongoing phase 3 PUNCH CD3-OLS trial and a retrospective study of use under enforcement discretion.

Methods: An ad hoc analysis of the pooled safety populations in the PUNCH CD3-OLS trial and retrospective study was conducted to identify IBD subgroups, specifically Crohn’s disease (CD) and ulcerative colitis (UC), based on medical history. Patients were monitored for treatment-emergent adverse events (TEAEs) for at least 6 months after treatment. The number of IBD-related medication(s) taken when RBX2660 was administered and/or during the 8-week observational baseline period were compared with those 8 weeks after RBX2660 administration.

Results: In the pooled safety population (N=547), 18 patients were identified as having CD only and 38 had UC only. Of the 56 patients evaluated, 39 patients (69.6%) received IBD-related medication at the time of RBX2660 administration. IBD-related medications included 5-ASA (mesalazine or balsalazide), corticosteroids (budesonide or prednisone), interleukin inhibitors (ustekinumab), tumor necrosis factor α (TNFα) inhibitors (infliximab or adalimumab), and other immunosuppressants (vedolizumab or tofacitinib citrate). Twenty-six of 38 patients with UC received IBD-related concomitant medications at the time of RBX2660 treatment with the top three classes being 5-ASA (n=18), other immunosuppressants (n=8), and TNFα inhibitors (n=8). Thirteen of 18 patients with CD received IBD-related concomitant medications at the time of RBX2660 treatment. The top drug classes were corticosteroids (n=8), 5-ASA (n=5), and TNFα inhibitors (n=3). The majority (n=32/39 [82.1%]) did not experience a drug change after 8 weeks of RBX2660 treatment. With regards to overall safety, TEAEs did not differ between IBD patients and non-IBD patients (57.1% vs 63.1%). TEAEs were mostly gastrointestinal in nature (predominantly diarrhea and abdominal pain) in both patients with and without IBD.

Discussion: RBX2660 treatment did not result in more adverse events in patients with IBD, and concomitant IBD-related medications remained the same after RBX2660 treatment in most patients. The results of this analysis suggest that RBX2660 is a safe therapeutic option for patients with rCDI and IBD.