Identification, Characterization, and Formulation of a Novel Carbapenemase Intended to Prevent Antibiotic-Mediated Gut Dysbiosis

Abstract

Antibiotics can damage the gut microbiome leading to opportunistic infections and the emergence of antibiotic resistance. Microbiome protection via antibiotic inactivation in the gastrointestinal (GI) tract represents a strategy to limit antibiotic exposure of the colonic microbiota. Proof of concept for this approach was achieved with an orally-administered beta-lactamase enzyme, SYN-004 (ribaxamase), that was demonstrated to degrade ceftriaxone excreted into the GI tract and protect the gut microbiome from antibiotic-mediated dysbiosis. Ribaxamase efficiently degrades penicillin and cephalosporin beta-lactam antibiotics, but is not active against carbapenems. To expand this microbiome protection strategy to include all classes of beta-lactams, three distinct carbapenemases were evaluated for manufacturability, antibiotic degradation spectrum, and stability in human intestinal fluid. E. coli production strains were generated for P2A, a novel metallo-enzyme isolated from B. cereus, New Delhi metallo-beta-lactamase (NDM), and Klebsiella pneumoniae carbapenemase (KPC). While all three enzymes effectively inactivated a broad range of antibiotics, including penicillins, most cephalosporins, and carbapenems in vitro, only P2A retained biological activity when incubated with human chyme. As functional stability in the intestinal tract is a key requirement for an orally-delivered enzyme, P2A was chosen as a potential clinical candidate. An enteric formulation of P2A was developed, called SYN-006, that was inert under high acid conditions, with enzyme dissolution occurring at pH > 5.5. SYN-006 has the potential to expand microbiome protection via antibiotic inactivation to include all classes of beta-lactam antibiotics.

Oral Metallo-Beta-Lactamase Protects the Gut Microbiome From Carbapenem-Mediated Damage and Reduces Propagation of Antibiotic Resistance in Pigs

“…SYN-006 is the oral formulation of P2A (targeted recombinant beta-lactamase 2), a Class B1 metallo-beta-lactamase isolated from B. cereus (Stiefel et al, 2005). P2A was manufactured in E. coli and formulated for oral delivery by incorporation into Eudragit ® -coated sucrose pellets designed for release of active enzyme at pH 5.5 or greater (Connelly et al, 2019). Formulation of P2A was similar to that of ribaxamase (Bristol et al, 2017).…”

“…To expand microbiome protection to all classes of beta-lactams, a novel metallo-beta-lactamase, P2A, isolated from Bacillus cereus (previously named targeted recombinant beta-lactamase 2) (Stiefel et al, 2005), was characterized. P2A demonstrated inactivation of a broad spectrum of beta-lactams including penicillins, cephalosporins, carbapenems, as well as antibiotic/beta-lactamase inhibitor combinations in vitro (Stiefel et al, 2005; Connelly et al, 2019). Further in vitro characterization revealed that P2A lost function at low pH (≤5.5), while retaining biological activity in the presence of human intestinal fluid, a key requirement for an enzyme intended for function in the GI tract (Connelly et al, 2019).…”

“…P2A demonstrated inactivation of a broad spectrum of beta-lactams including penicillins, cephalosporins, carbapenems, as well as antibiotic/beta-lactamase inhibitor combinations in vitro (Stiefel et al, 2005; Connelly et al, 2019). Further in vitro characterization revealed that P2A lost function at low pH (≤5.5), while retaining biological activity in the presence of human intestinal fluid, a key requirement for an enzyme intended for function in the GI tract (Connelly et al, 2019). Despite pH sensitivity, orally-administered P2A was demonstrated to preserve colonization resistance in mice treated with the antibiotic-beta-lactamase inhibitor combination, piperacillin-tazobactam (Stiefel et al, 2005).…”

“…Despite pH sensitivity, orally-administered P2A was demonstrated to preserve colonization resistance in mice treated with the antibiotic-beta-lactamase inhibitor combination, piperacillin-tazobactam (Stiefel et al, 2005). Therefore, to optimize enzyme function in vivo , P2A was formulated for oral delivery similarly to ribaxamase (Bristol et al, 2017) by use of an enteric coating intended to protect the enzyme from high acid conditions of the stomach, with enzyme dissolution occurring in the upper small intestine, at pH ≥ 5.5 (Connelly et al, 2019). Formulated P2A was named SYN-006 (Connelly et al, 2019).…”

“…Therefore, to optimize enzyme function in vivo , P2A was formulated for oral delivery similarly to ribaxamase (Bristol et al, 2017) by use of an enteric coating intended to protect the enzyme from high acid conditions of the stomach, with enzyme dissolution occurring in the upper small intestine, at pH ≥ 5.5 (Connelly et al, 2019). Formulated P2A was named SYN-006 (Connelly et al, 2019).…”