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Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.

BackgroundIntestinal colonization by antimicrobial resistant (AMR) pathogens is a known health and infection risk, and is common among individuals with recurrent Clostridioides difficile infections (rCDI). Accordingly, therapeutic approaches that decolonize the gut of AMR pathogens could be valuable to patients to reduce risk of associated illnesses. Herein, we assessed gut colonization with AMR bacteria before and after treatment with RBX2660—a microbiota-based investigational live biotherapeutic—in the PUNCH CD3 Phase 3 trial for reducing CDI recurrence.MethodsrCDI participants enrolled in PUNCH CD3 received a blinded single dose of RBX2660 or placebo within 24 to 72 hours after completing antibiotic treatment for the most recent rCDI episode. Clinical response was the absence of CDI recurrence at eight weeks after treatment, and participants were asked to submit stool samples prior to RBX2660 or placebo treatment (baseline) and 1, 4 and 8 weeks, 3 and 6 months after study treatment. Samples were extracted and sequenced using a shallow shotgun method. The presence and number of AMR genes was determined for 175 participant samples and 116 RBX2660 samples using 90% K-mer sequence coverage based on the MEGARes database. AMR gene count data were collapsed into count columns to adjust for sparseness in the matrices and were analyzed using a Generalized Linear Mixed Model.ResultsClinically, RBX2660 demonstrated superior efficacy versus placebo (70.4% and 58.1%, respectively), and the total AMR genes per participant decreased significantly from before to after treatment in RBX2660-treated responders (p< .05, Figure 1) and remained low to at least 6 months. Among genes that decreased in RBX2660 responders were clinically important extended-spectrum beta-lactamase (blaTEM, blaSHV, blaCTX-M), vancomycin resistance (vanA, vanB), and fluoroquinolone resistance genes (gyrA, parC). Total AMR genes per PUNCH CD3 participant among RBX2660-treated responders at the indicated time points and in the RBX2660 investigational product. The red lines indicate timepoint group medians.ConclusionIn the PUNCH CD3 Phase 3 trial of RBX2660 for rCDI, AMR gene content decreased after RBX2660 treatment and remained low to at least 6 months, consistent with prior RBX2660 trials. This underscores the potential of microbiota-based biotherapeutics for decolonizing AMR bacteria from gut microbiota and thereby reducing AMR infection risks.Disclosures Heidi Hau, PhD, Rebiotix Inc. (Employee) Dana M. Walsh, PhD, Rebiotix (Employee) Ken Blount, PhD, Rebiotix Inc., a Ferring Company (Employee)

Approximately 50 million US patients undergo ambulatory surgery annually. Postoperative opioid overprescribing is problematic, yet many patients report inadequate pain relief. In major inpatient surgery, intraoperative single-dose methadone produces better analgesia and reduces opioid use compared with conventional repeated dosing of short-duration opioids. This investigation tested the hypothesis that in same-day ambulatory surgery, intraoperative methadone, compared with short-duration opioids, reduces opioid consumption and pain, and determined an effective intraoperative induction dose of methadone for same-day ambulatory surgery. A double-blind, dose-escalation protocol randomized 60 patients (2:1) to intraoperative single-dose intravenous methadone (initially 0.1 then 0.15 mg/kg ideal body weight) or conventional as-needed dosing of short-duration opioids (eg, fentanyl, hydromorphone; controls). Intraoperative and postoperative opioid consumption, pain, and opioid side effects were assessed before discharge. Patient home diaries recorded pain, opioid use, and opioid side effects daily for 30 days postoperatively. Primary outcome was in-hospital (intraoperative and postoperative) opioid use. Secondary outcomes were 30 days opioid consumption, pain intensity, and opioid side effects. Median (interquartile range) methadone doses were 6 (5-6) and 9 (8-9) mg in the 0.1 and 0.15 mg/kg methadone groups, respectively. Total opioid consumption (morphine equivalents) in the postanesthesia care unit was significantly less compared with controls (9.3 mg, 1.3-11.0) in subjects receiving 0.15 mg/kg methadone (0.1 mg, 0.1-3.3; P < .001) but not 0.1 mg/kg methadone (5.0 mg, 3.3-8.1; P = .60). Dose-escalation ended at 0.15 mg/kg methadone. Total in-hospital nonmethadone opioid use after short-duration opioid, 0.1 mg/kg methadone, and 0.15 mg/kg methadone was 35.3 (25.0-44.0), 7.1 (3.7-10.0), and 3.3 (0.1-5.8) mg morphine equivalents, respectively (P < .001 for both versus control). In-hospital pain scores and side effects were not different between groups. In the 30 days after discharge, patients who received methadone 0.15 mg/kg had less pain at rest (P = .02) and used fewer opioid pills than controls (P < .0001), whereas patients who received 0.1 mg/kg had no difference in pain at rest (P = .69) and opioid use compared to controls (P = .08). In same-day discharge surgery, this pilot study identified a single intraoperative dose of methadone (0.15 mg/kg ideal body weight), which decreased intraoperative and postoperative opioid requirements and postoperative pain, compared with conventional intermittent short-duration opioids, with similar side effects.

Effective treatment options for recurrent C. difficile infection (rCDI) are limited, with high recurrence rates associated with the current standard of care. RBX2660, a standardized microbiota-based drug, was efficacious for preventing rCDI in a double-blinded Phase 2 clinical trial. Herein we report results from a subsequent open-label trial to evaluate the safety, efficacy, and microbiome altering-activity of RBX2660 for preventing rCDI. This prospective, multicenter, open-label Phase 2 study enrolled subjects who had experienced either ≥2 recurrences of CDI following standard-of-care antibiotic therapy or ≥2 episodes of severe CDI requiring hospitalization. Participants received up to 2 doses of RBX2660 delivered via enema with doses 7 days apart. Efficacy was defined as absence of CDI at 8 weeks from the last dose and was compared with the 8-week CDI-free rate of subjects who received antibiotic treatment alone in a matched historical control arm. 16s rRNA analysis was conducted on stool samples that were voluntarily submitted from RBX2660-treated subjects at baseline, 7, 30, and 60 days after treatment, and operational taxonomic units were compared longitudinally. 132 RBX2660 and 110 historical control subjects were included from 31 and 4 centers, respectively, in the USA and Canada. RBX2660’s efficacy in preventing rCDI (78.8%) was higher than CDI-free rates in the Historical Control group (51.8%, P < 0.001). A total of 476 adverse events (AEs) were reported by 97 RBX2660 subjects (73.5%) compared with 691 AEs among 69 historical controls (62.7%). Following treatment, the diversity of the RBX2660 treated subjects’ microbiomes significantly increased and the relative proportion of Bacteroidia and Clostridia, while decreasing the relative proportion of Gammaproteobacteria. The resulting RBX2660-treated subject microbiota more closely resembled healthy donors than at study entry. This open-label Phase 2 study demonstrates RBX2660’s efficacy in preventing rCDI compared with an antibiotic-treated historical control group. The RBX2660 safety profile is consistent with results from previous clinical trials and microbiota analysis suggests that RBX2660 may rehabilitate patient microbiota. This analysis was funded by Rebiotix Inc., Roseville, MN. R. Orenstein, Rebiotix, Inc: Scientific Advisor, Consulting fee; E. R. Dubberke, Rebiotix, Inc.: Scientific Advisor, Consulting fee; S. Khanna, Rebiotix, Inc.: Scientific Advisor, Consulting fee; G. Hecht, Rebiotix, Inc.: Scientific Advisor, Consulting fee; H. Dupont, Rebiotix, Inc.: Investigator, Research support; C. Lee, Rebiotix, Inc.: Scientific Advisor, Consulting fee; K. Blount, Rebiotix, Inc.: Employee, Salary; S. Carter, Rebiotix, Inc.: Research Contractor, Consulting fee

Cardiac and vascular diseases represent one of the most substantial medical areas for the applications of regenerative medicine. Despite advances in endovascular repair, surgical intervention, and disease management, atherosclerosis and heart failure continue to be prominent health problems. This report analyzes the regenerative medicine treatment opportunities in both cardiovascular and peripheral vascular repair, examining the treatment opportunities for tissue-engineered vascular grafts as well as cell-based therapies. U.S. hospital discharge data were used to generate a detailed estimate of the relative target populations for cardiac and vascular disease. Gap analyses were performed for vascular access, small caliber vascular grafts, and cell-based therapies for revascularization and heart failure. The analysis compared current alternatives, gaps in medical need, and what a tissue-engineered or regenerative alternative should achieve for optimum medical and commercial feasibility. Although the number of coronary bypass grafts vastly outnumbered peripheral grafts, a detailed consideration of re-grafts and the success of first grafts combined with gap analysis (GAP) leads us to conclude that peripheral vascular disease is the more commercially feasible and attractive target opportunity for engineered small caliber grafts for the foreseeable future. Cardiac bypass would need substantial long-term clinical experience, which could be a significant hurdle. Vascular access, often regarded as a first-in-man indication, is an excellent opportunity for an engineered graft as an alternative to arteriovenous fistula that could overcome complications associated with a prosthetic graft. The GAP also suggests that for heart failure, cellular therapies should link near-term changes in repair, such as improvement in cardiac output and reduced scarring with limiting progression of the disease, reducing the need for complex pharmacologic management, and reducing rates of hospitalization. Naturally, researchers must determine where their technology and know-how can be applied most effectively, but it is clear from our analysis that an astute strategy in the use of science and technology will be important to successful translation in this space.