SYN-006, a Novel Carbapenemase, Intended to Protect the Gut Microbiome from Antibiotic-Mediated Damage May Also Reduce Propagation of Carbapenem-Resistant Pathogens

May Also Reduce Propagation of Carbapenem-Resistant Pathogens Michael Kaleko1, Christian Furlan Freguia1, Nur A. Hasan2, Poorani Subramanian2, Rita R. Colwell2, Sheila Connelly1 1Synthetic Biologics, Inc., Rockville, MD, 2CosmosID, Inc., Rockville, MD • SYN-006 has a broad antibiotic degradation profile including penicillins, cephalosporins, and carbapenems • In dogs, oral SYN-006 resulted in degradation of intestinal meropenem and did not affect meropenem serum levels • A pig model of ertapenem-mediated dysbiosis was established • Ertapenem exposure rapidly resulted in the emergence of antibioticresistance genes, including a rare carbapenemase gene, IMP-27 • An enteric-coated formulation of SYN-006 is currently in production and is expected to be tested in the pig dysbiosis model SYN-006 has the potential protect the gut microbiome from certain IV beta-lactam antibiotics including carbapenems and to reduce emegence of antibiotic resistance Many IV beta-lactam antibiotics are excreted via the bile into the intestine where they can disrupt the intestinal microbiota and potentially lead to the outgrowth of pathogens like Clostridium difficile. Ribaxamase is a clinical stage, oral beta-lactamase enzyme therapy for use with IV beta-lactam antibiotics to preserve the gut microbiome by degrading residual antibiotics in the intestine. A phase 2b study met its primary endpoint of significantly reducing C. difficile infection (CDI) in patients treated with ceftriaxone and ribaxamase. While SYN-004 degrades penicillins and cephalosporins, it does not inactivate carbapenems [1]. To expand this prophylactic approach to all classes of betalactams, we are developing SYN-006, a broad spectrum carbapenemase. Extended Spectrum b-Lactamase Genes Chyme Meropenem and SYN-006 Jejunally-fistulated dogs (n=6) received meropenem (30 mg/kg, IV) alone or with a liquid formulation of SYN-006 (1 mg/kg, PO). Meropenem levels in the chyme of animals treated with meropenem alone were ~3.0 ug/g. In the presence of SYN-006, meropenem was undectable in two animals, or 2.0 ug/g in one animal. SYN-006 chyme levels were extremely variable, ranging from 80 U/g to 0.2 U/g. This variability is likely due to SYN-006’s sensitivity to acid, as the enzyme was delivered orally in a non-enteric-coated liquid formulation. SYN-006 had no effect on systemic meropenem levels. Therefore, when present in the chyme at levels of 0.5 U/g or higher, SYN-006 completely degraded the meropenem in the dog GI tract without affecting meropenem serum levels. Heatmap analyses compared bacterial species present in microbiomes of pigs prior to and after ertapenem treatment and reveal that the antibiotic caused the depletion of some species (yellow boxes) and the overgrowth of others (white boxes). The likelihood ratio test compared the microbiomes prior to ertapenem treatment (Day -4) to the microbiomes after antibiotic exposure. Microbiomes after antibiotic treatment were significantly different from pre-exposure microbiomes. A pig model of ertapenem-mediated dysbiosis was successfully established. To evaluate SYN-006 efficacy in protecting the microbiome from beta-lactam antibiotics, including carbapenems, a pig model of ertapenem-mediated dysbiosis was established. Normal pigs (20 kg, n=5) were treated with IV ertapenem (30 mg/kg, IV, SID) for 7 consecutive days. Feces were collected at two pre-antibiotic time points (Days -7 and -4), during treatment (Day 4), and after antibiotics were stopped (Days 8 and 9). Fecal DNA was subjected to whole genome shotgun sequence analyses. Heat map analyses of the fecal microbial community was based on species relative abundance. Each square represents a bacterial species present in individual animal microbiomes. The yellow and white boxes display changes in species diversity caused by ertapenem treatment. The Dirichlet-Multinomial model likelihood ratio test was used to compare microbiome populations prior to and after antibiotic treatment. Ertapenem Rapidly Disrupts the Pig Gut Microbiome Purified beta-lactamase enzymes were assessed for antibiotic hydrolysis potency with a microtiter plate activity assay using E. coli growth as the read-out for antibiotic inactivation. A total of 10, 100, or 1000 ug/ml of each antibiotic was mixed with 100 ng/ml of ribaxamase or SYN-006. E. coli was added and growth quantified. The graph displays the highest antibiotic concentration at which bacterial growth was observed, indicating antibiotic inactivation. Antibiotic Degradation Profile SYN-006 displayed a broader antibiotic degradation profile that included the carbapenems (MEM, IPM, ERT, DOR), compared to ribaxamase. SYN-006 showed good activity against the penicillins (AMP, PIP) and the cephalosporins (CRO, CTX, CFZ, CXM, CFP, CDR, LEX, CAZ) and was active in the presence of beta-lactamase inhibitors. SYN-006 Degrades Meropenem in the Dog GI Tract Treatment (n=3) Dog SYN006 (U/g) Meropenem (ug/g) Meropenem Alone 1 NA 3.0 2 NA 3.2 3 NA 3.0 Meropenem + SYN-006 4 80 0 5 0.5 0 6 0.2 2.0 SYN-006 1. Kaleko, M., et al. (2016) Development of SYN-004, an Oral Beta-Lactamase Treatment to Protect the Gut Microbiome from Antibiotic-Mediated Damage and Prevent Clostridium difficile infection. Anaerobe 41:58-67. doi: 10.1016/j.anaerobe.2016.05.015 2. Mollenkopf, D.F., et al. (2017) Carbapenemase-Producing Enterobacteriaceae Recovered from the Environment of a Swine Farrow-to-Finish Operation in the United States. Antimicrob Agents Chemother 61. doi: 10.1111/zph.122993 Treatment Group Chi squared P value