John F. Kokai-Kun

Antibody against the carboxyl terminus of intimin alpha reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization.

ABSTRACT The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin α.

Development of SYN-004, an oral beta-lactamase treatment to protect the gut microbiome from antibiotic-mediated damage and prevent Clostridium difficile infection

The gut microbiome, composed of the microflora that inhabit the gastrointestinal tract and their genomes, make up a complex ecosystem that can be disrupted by antibiotic use. The ensuing dysbiosis is conducive to the emergence of opportunistic pathogens such as Clostridium difficile. A novel approach to protect the microbiome from antibiotic-mediated dysbiosis is the use of beta-lactamase enzymes to degrade residual antibiotics in the gastrointestinal tract before the microflora are harmed. Here we present the preclinical development and early clinical studies of the beta-lactamase enzymes, P3A, currently referred to as SYN-004, and its precursor, P1A. Both P1A and SYN-004 were designed as orally-delivered, non-systemically available therapeutics for use with intravenous beta-lactam antibiotics. SYN-004 was engineered from P1A, a beta-lactamase isolated from Bacillus licheniformis, to broaden its antibiotic degradation profile. SYN-004 efficiently hydrolyses penicillins and cephalosporins, the most widely used IV beta-lactam antibiotics. In animal studies, SYN-004 degraded ceftriaxone in the GI tract of dogs and protected the microbiome of pigs from ceftriaxone-induced changes. Phase I clinical studies demonstrated SYN-004 safety and tolerability. Phase 2 studies are in progress to assess the utility of SYN-004 for the prevention of antibiotic-associated diarrhea and Clostridium difficile disease.

The Oral β-Lactamase SYN-004 (Ribaxamase) Degrades Ceftriaxone Excreted into the Intestine in Phase 2a Clinical Studies

ABSTRACT SYN-004 (ribaxamase) is a β-lactamase designed to be orally administered concurrently with intravenous β-lactam antibiotics, including most penicillins and cephalosporins. Ribaxamases anticipated mechanism of action is to degrade excess β-lactam antibiotic that is excreted into the small intestine. This enzymatic inactivation of excreted antibiotic is expected to protect the gut microbiome from disruption and thus prevent undesirable side effects, including secondary infections such as Clostridium difficile infections, as well as other antibiotic-associated diarrheas. In phase 1 clinical studies, ribaxamase was well tolerated compared to a placebo group and displayed negligible systemic absorption. The two phase 2a clinical studies described here were performed to confirm the mechanism of action of ribaxamase, degradation of β-lactam antibiotics in the human intestine, and were therefore conducted in subjects with functioning ileostomies to allow serial sampling of their intestinal chyme. Ribaxamase fully degraded ceftriaxone to below the level of quantitation in the intestines of all subjects in both studies. Coadministration of oral ribaxamase with intravenous ceftriaxone was also well tolerated, and the plasma pharmacokinetics of ceftriaxone were unchanged by ribaxamase administration. Since ribaxamase is formulated as a pH-dependent, delayed-release formulation, the activity of ribaxamase in the presence of the proton pump inhibitor esomeprazole was examined in the second study; coadministration of these drugs did not adversely affect ribaxamases ability to degrade ceftriaxone excreted into the intestine. These studies have confirmed the in vivo mechanism of action of ribaxamase, degradation of β-lactam antibiotics in the human intestine (registered at ClinicalTrials.gov under NCT02419001 and NCT02473640).

Nonclinical Safety Assessment of SYN-004 An Oral β-lactamase for the Protection of the Gut Microbiome From Disruption by Biliary-Excreted, Intravenously Administered Antibiotics

SYN-004 is a first in class, recombinant β-lactamase that degrades β-lactam antibiotics and has been formulated to be administered orally to patients receiving intravenous β-lactam antibiotics including cephalosporins. SYN-004 is intended to degrade unmetabolized antibiotics excreted into the intestines and thus has the potential to protect the gut microbiome from disruption by these antibiotics. Protection of the gut microbiome is expected to protect against opportunistic enteric infections such as Clostridium difficile infection as well as antibiotic-associated diarrhea. In order to demonstrate that oral SYN-004 is safe for human clinical trials, 2 Good Laboratory Practice-compliant toxicity studies were conducted in Beagle dogs. In both studies, SYN-004 was administered orally 3 times per day up to the maximum tolerated dose of the formulation. In the first study, doses of SYN-004 administered over 28 days were safe and well tolerated in dogs with the no-observed-adverse-effect level at the high dose of 57 mg/kg/day. Systemic absorption of SYN-004 was minimal and sporadic and showed no accumulation during the study. In the second study, doses up to 57 mg/kg/day were administered to dogs in combination with an intravenous dose of ceftriaxone (300 mg/kg) given once per day for 14 days. Coadministration of oral SYN-004 with intravenous ceftriaxone was safe and well tolerated, with SYN-004 having no noticeable effect on the plasma pharmacokinetics of ceftriaxone. These preclinical studies demonstrate that SYN-004 is well tolerated and, when coadministered with ceftriaxone, does not interfere with its systemic pharmacokinetics. These data supported advancing SYN-004 into human clinical trials.

Tolerability and Pharmacokinetics of SYN-004, an Orally Administered β-Lactamase for the Prevention of Clostridium difficile-Associated Disease and Antibiotic-Associated Diarrhea, in Two Phase 1 Studies

BackgroundSYN-004 is an orally administered β-lactamase enzyme, designed to be given concurrently with certain intravenous β-lactam antibiotics like cephalosporins. SYN-004 is intended to degrade residual antibiotics excreted into the intestine as a result of hepatobiliary excretion and to prevent the disruption of the gut microbiome by these excess antibiotics. Preserving the gut microbiome is expected to prevent secondary infections by pathogens like Clostridium difficile and protect against other antibiotic-associated diarrheas.MethodsTwo, randomized, double blind, placebo-controlled Phase 1 clinical studies were conducted in normal healthy adult volunteers to assess the tolerability and systemic absorption of single and multiple doses of SYN-004. A single-ascending dose study investigated single oral doses of 75–750xa0mg SYN-004 and was conducted in 40 subjects (five cohorts of six active and two placebo subjects). A multiple-ascending dose study investigated doses of 75–300xa0mg SYN-004, administered every 6xa0h for 7xa0days and was conducted in 24 subjects (three cohorts of six active and two placebo subjects). The safety and tolerability of SYN-004 was assessed and serial plasma and serum samples were collected to assess the pharmacokinetics and potential immunogenicity of SYN-004.ResultsMinimal and mild adverse events were reported in ~30xa0% of the subjects who received active drug and placebo and no antidrug antibodies were detected in any subject. Analysis of serial plasma samples demonstrated negligible systemic bioavailability of SYN-004 with most plasma concentrations being below the lower limit of quantitation (0.8xa0ng/mL) for the assay. SYN-004 was well tolerated in the 48 subjects who received active drug, and adverse events in those subjects were comparable to the 16 subjects who received placebo, up to the maximum doses administered in each study.ConclusionSYN-004 was well tolerated up to a singlexa0oral dose of 750xa0mg and multiple doses of 300xa0mg every 6xa0h for 7xa0days. The pharmacokinetic results support that SYN-004 remained localized in the intestine.

Lovastatin lactone may improve irritable bowel syndrome with constipation (IBS-C) by inhibiting enzymes in the archaeal methanogenesis pathway.

Methane produced by the methanoarchaeon Methanobrevibacter smithii ( M. smithii) has been linked to constipation, irritable bowel syndrome with constipation (IBS-C), and obesity. Lovastatin, which demonstrates a cholesterol-lowering effect by the inhibition of HMG-CoA reductase, may also have an anti-methanogenesis effect through direct inhibition of enzymes in the archaeal methanogenesis pathway. We conducted protein-ligand docking experiments to evaluate this possibility. Results are consistent with recent clinical findings. METHODS: F420-dependent methylenetetrahydromethanopterin dehydrogenase ( mtd), a key methanogenesis enzyme was modeled for two different methanogenic archaea: M. smithii and Methanopyrus kandleri. Once protein models were developed, ligand-binding sites were identified. Multiple ligands and their respective protonation, isomeric and tautomeric representations were docked into each site, including F420-coenzyme (natural ligand), lactone and β-hydroxyacid forms of lovastatin and simvastatin, and other co-complexed ligands found in related crystal structures. RESULTS: 1) Generally, for each modeled site the lactone form of the statins had more favorable site interactions compared to F420; 2) The statin lactone forms generally had the most favorable docking scores, even relative to the native template PDB ligands; and 3) The statin β-hydroxyacid forms had less favorable docking scores, typically scoring in the middle with some of the F420 tautomeric forms. Consistent with these computational results were those from a recent phase II clinical trial ( NCT02495623) with a proprietary, modified-release lovastatin-lactone (SYN-010) in patients with IBS-C, which showed a reduction in symptoms and breath methane levels, compared to placebo. CONCLUSION: The lactone form of lovastatin exhibits preferential binding over the native-F420 coenzyme ligand in silico and thus could inhibit the activity of the key M. smithii methanogenesis enzyme mtd in vivo. Statin lactones may thus exert a methane-reducing effect that is distinct from cholesterol lowering activity, which requires HMGR inhibition by statin β-hydroxyacid forms.

Development of a Modified Release Formulation of Lovastatin Targeted to Intestinal Methanogens Implicated in Irritable Bowel Syndrome with Constipation

There is growing evidence that methane production, predominantly by Methanobrevibacter smithii, in the intestines is a cause of constipation, pain, and bloating in irritable bowel syndrome with constipation (IBS-C). M smithii resides primarily in the large intestine but can also colonize the small intestine. Inxa0vitro studies found that the prodrug lactone form of lovastatin, found in cholesterol-lowering drugs, inhibited methane production in stool samples from patients with IBS-C. However, the cholesterol-lowering lovastatin β-hydroxyacid was ineffective at inhibiting methane production in this system. A considerable amount of lovastatin is converted to hydroxyacid in the stomach and is absorbed. It was hypothesized that galenic innovations could protect lovastatin from the stomach and allow release in 2 strategic locations, the duodenum and the ileocecal region, to reach M smithii. The desired release profile was achieved by developing an oral dosage form containing lovastatin and coated with 2 different enteric polymers that enabled a pH-dependent dual pulse drug release. Combinations of the 2 coated tablets were encapsulated together to deliver the desired amount of lovastatin to the targeted intestinal locations. The capsules have been tested inxa0vitro and inxa0vivo and show promise in treating IBS-C.

Characterization of Clostridium difficile Isolates Collected during a Phase 2b Clinical Study with SYN-004 (ribaxamase) for the Prevention of C. difficile Infection.

During a Phase 2b study with SYN-004 (ribaxamase) for prevention of Clostridium difficile infection (CDI) conducted in North America and Eastern Europe, 45 C. difficile isolates from subjects with laboratory-confirmed CDI and or colonized with C.xa0difficile were collected and characterized. Several C.xa0difficile PCR ribotypes, including 027 and 198, were identified.