1. F-246
Treatment of Clostridium difficile Infection using SQ641, a Capuramycin Analogue, Increases Survival, Decreases Relapse and Improves Clinical Measures of Disease in a Murine Model
Moore, JH; Van Opstal, EJ; Kolling, GL; Bogatcheva E; Nikonenko B; Einck L; Guerrant RL; Protopopova M;
Warren, CA
University of Virginia
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Introduction
Results
Results
Clostridium difficile infection (CDI) is a major cause of nosocomial diarrhea, with relapse rates following initial treatment of up to 25% and succeeding treatment of up to 60%. SQ641 is an analogue of capuramycin, a nucleoside-based compound produced by the bacterium Streptomyces griseus. It inhibits phospho-MurNAc-pentapeptide translocase (translocase I, MraY), the enzyme responsible for the first step in peptidoglycan synthesis which facilitates the transfer of the phospho-MurNAc-pentapeptide to undecaprenyl-phosphate in gram-positive bacteria. SQ641 has shown in vitro efficacy against C. difficle while having a minimal effect on other potentially beneficial commensal flora. Additionally, SQ641 has very poor systemic bioavailability and maintains high concentrations in the colon (where C. difficile grows and shows pathology) on oral administration. Our aim was to determine the efficacy of SQ641 in an established murine model of CDI, and compare it with other commercially available treatments, such as vancomycin.
Combined weight change of C. difficile infected animals over 6 experiments. Animals in the infected control group had significantly decreased weight compared with SQ641 dosages mg/kg days 2-4 post-infection (p<0.05, two-way ANOVA with Bonferroni correction post-test). Vancomycin treated animals had significantly decreased weight day 10 post –infection compared with SQ641 groups receiving between mg/kg dosage (p<0.05)
Animals receiving SQ641 at 0.01 and 0.1 mg/kg did not have significant differences in weight across the 14 days of observation post-infection. Animals receiving 10 mg/kg SQ641 had higher bodyweight day 3 post infection than either 0.01 or 0.1 mg/kg SQ641 day 3 post infection (p<0.01, two-way ANOVA with Bonferroni correction post-test).
Methods
Six separate animal experiments were performed following the same schedule as below:
C. difficile stool shedding confirmed by Quantitative Real-Time PCR (qPCR); samples were run in duplicate from the 5 experiments we analyzed. C. difficile toxin B gene (tcdB) primers were used to quantify C. difficile shedding in stool. C. difficile shedding in stool was significantly inhibited with SQ641 drug treatment at doses mg/kg or with vancomycin treatment compared with the vehicle control on samples taken on day 2 or 3 post-infection (p<0.001, one-way ANOVA with Bonferroni correction post-test). Stool taken from animals pre-infection confirmed toxogenic C. difficile bacteria was not present (not shown). Bacterial shedding was significantly inhibited in SQ641 groups receiving 300 and 100 mg/kg and vancomycin compared with infected control in samples collected day 7-8 post-infection (p<0.05). There was no significant difference in shedding between groups in samples collected at day post-infection.
All animals used for this experiment were 8 week old (on arrival) C57BL/6 male mice from Jackson Laboratories. All experiments were performed according to approved protocols from the University of Virginia Animal Care and Use Committee. Clostridium difficile strain VPI was used in all infections. SQ641 was administered via oral gavage at either 300, 100, 10, 1, 0.1, or 0.01 mg/kg/day in either a 10% dimethyl sulfoxide (DMSO)/1% methylcellulose (MC) solution or in a 10% d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS) solution, depending on experiment, beginning 1 day after infection. Vancomycin was administered at 20 mg/kg/day in the same drug vehicle, depending on the experiment. Infected controls were given drug vehicle for 5 days; either DMSO/MC or TPGS depending on the experiment. Stool samples were collected at select time points to compare bacterial burden via qPCR and C. difficile toxins A/B production via ELISA. Clinical scoring of mice was based on defined criteria including mouse activity, posture, coat appearance, diarrhea, eye appearance (squinting or closed), and weight loss on a combined scale of 0 to 20. Mice found dead were given a score of 20. Moribund mice or mice with a clinical score of more than 14 were humanely euthanized. Histopathology was performed by fixing colon and cecum specimens in Bouin’s fixative for 24 hours and stored in 70% ETOH until specimens were submitted to the University of Virginia Research Histology core for hemotoxin and eosin staining. Scoring of histopathological specimens was based on criteria such as inflammation, mucosa thickness, exudate, erosion, and submucosal edema on a scale of Samples were scored in a blinded fashion and for both colon and cecum tissues.
Significance across all experiments is defined as p< GraphPad Prism 5.0 was used to generate graphs and perform statistical analysis (Mantel-Cox Log-Rank test, one-way and two-way ANOVAs with Bonferroni correction post-test).
Combined clinical scoring of C. difficile infected animals over 6 experiments with SQ641 treatment range of mg/kg and vancomycin. There was significantly increased clinical scoring in the infected control group compared with SQ641 dosages mg/kg and vancomycin days 2-3 post-infection (p<0.05, two-way ANOVA with Bonferroni correction post-test), while vancomycin treated mice had significantly higher clinical scores than SQ641 receiving groups days 9-11 (p<0.05).
Combined clinical scoring of C. difficile infected animals over 6 experiments with SQ641 treatment range of mg/kg and infected control. SQ641 doses of 1 and 10 mg/kg had significantly lower clinical scores than doses 0.01 and 0.1 mg/kg (p<0.01, two-way ANOVA with Bonferroni correction post-test).
Results
Combined survival of C. difficile infected animals over our 6 experiments. There was significantly higher survival in each of the treatment groups receiving mg/kg SQ641 compared with the drug vehicle control (least significant p-value = , log-rank test) and significantly higher survival in groups receiving mg/kg SQ641 compared with vancomycin treatment (least significant p-value = , log-rank test).
C. difficile toxin A/B ELISA from stools collected is shown above. Each data point represents a separate sample; 4 experiments were analyzed for toxin levels. There were not significant differences between groups days on days 1 or 12 post-infection. Toxin A/B in stool was significantly less in the 300 mg/kg, 100 mg/kg SQ641 and vancomycin receiving groups compared with the 10 or 1 mg/kg SQ641 receiving groups on days 2-3 and 7-8 post-infection (p<0.05, one-way ANOVA with Bonferroni correction post-test).. Doses of SQ641 at 100 and 300 mg/kg/day (and vancomycin) inhibited toxin A/B production in samples collected on day 7 post-infection; however this effect was lost by day 12 (7 days after ceasing administration of SQ641 or vancomycin). A B
Representative histology of hematoxylin and eosin stained colons of mice undergoing separate treatments as follows: A – Uninfected, B- Infected with Vancomycin (terminal, day 11 post-infection), C – Infected Control (terminal, day 3 post-infection), D – Infected with 10 mg/kg SQ641 (day 14 post-infection). SQ641 administration at 100 mg/kg did not appear to cause intestinal damage without infection (not shown). C. difficile infection with drug vehicle treatment or vancomycin treatment caused increased epithelial erosion, exudate into the luminal space, and submucosal edema. SQ641 treatment at 10 mg/kg with infection prevented erosion, exudate, and submucosal edema.
Conclusion
Compound SQ641 performed extremely well in comparison with vancomycin or drug vehicle alone in the treatment of C. difficile infection in our animal model. SQ641 doses tested between mg/kg had significantly greater survival than our drug vehicle group, while SQ641 doses between 1 mg/kg and 100 mg/kg had significantly greater long-term survival than vancomycin treatment (where animals died due to relapse following cessation of drug). SQ641 at select doses significantly inhibited C. difficile shedding and toxin production in stool on days 2-3 and 7-8 post infection.
Interestingly, decreasing dosages of SQ641 (to an effective minimum of 1 mg/kg) increased survival of animals, even though the lowest effective doses (at 10 mg/kg and 1 mg/kg) did not appear to inhibit C. difficile toxins A/B production to the extent that higher doses (300 and 100 mg/kg) or vancomycin did. This may be due to larger doses of SQ641 having an increased impact on protective members of the resident microbiome; as systemic toxicity studies performed by Sequella in healthy mice in doses up to 500 mg/kg have not resulted in mortality. We hope to continue to examine SQ641 in mice to determine how resident flora are impacted in vivo and other potential mechanisms of action. SQ641 appears to be a very promising candidate for C. difficile treatment in humans pending based on our findings in a murine model of infection.
SQ641 is property of Sequella, Inc.
Research supported by NIH grant R01 AI094458 C D
Combined survival of C. difficile infected animals showing drug efficacy floor. Doses of SQ641 at mg/kg did not have significantly different survival than the infected control; while doses of 1-10 mg/kg had significantly higher survival than either 0.01 or 0.1 mg/kg (least significant p-value , log-rank test). Overall there appears to be an inverse dose-response relationship with SQ641 in CDI treatment, with larger doses decreasing survival due to relapse, with a minimum efficacious dose of 1 mg/kg.
Combined and averaged cecum and colon histopathology scoring of C. difficile infected animals over 4 experiments. Cecum and colon specimens were taken either from euthanized survivors at the end of the experiment or from moribund animals that had to be euthanized during the course of infection. There was significantly lower combined histopathological scoring (p<0.05, one-way ANOVA with Bonferroni correction post-test) in the SQ641 groups receiving 1, 10, or 100 mg/kg compared with either drug vehicle or vancomycin (p<0.05).