Novel, non-colonizing, single-strain live biotherapeutic product ADS024 protects against Clostridioides difficile infection challenge in vivo.

BACKGROUND: The Centers for Disease Control and Prevention estimate that Clostridioides difficile (C. difficile) causes half a million infections (CDI) annually and is a major cause of total infectious disease death in the United States, causing inflammation of the colon and potentially deadly diarrhea. We recently reported the isolation of ADS024, a Bacillus velezensis (B. velezensis) strain, which demonstrated direct in vitro bactericidal activity against C. difficile, with minimal collateral impact on other members of the gut microbiota. In this study, we hypothesized that in vitro activities of ADS024 will translate in vivo to protect against CDI challenge in mouse models. AIM: To investigate the in vivo efficacy of B. velezensis ADS024 in protecting against CDI challenge in mouse models. METHODS: To mimic disruption of the gut microbiota, the mice were exposed to vancomycin prior to dosing with ADS024. For the mouse single-dose study, the recovery of ADS024 was assessed via microbiological analysis of intestinal and fecal samples at 4 h, 8 h, and 24 h after a single oral dose of 5 × 108 colony-forming units (CFU)/mouse of freshly grown ADS024. The single-dose study in miniature swine included groups that had been pre-dosed with vancomycin and that had been exposed to a dose range of ADS024, and a group that was not pre-dosed with vancomycin and received a single dose of ADS024. The ADS024 colonies [assessed by quantitative polymerase chain reaction (qPCR) using ADS024-specific primers] were counted on agar plates. For the 28-d miniature swine study, qPCR was used to measure ADS024 levels from fecal samples after oral administration of ADS024 capsules containing 5 × 109 CFU for 28 consecutive days, followed by MiSeq compositional sequencing and bioinformatic analyses to measure the impact of ADS024 on microbiota. Two studies were performed to determine the efficacy of ADS024 in a mouse model of CDI: Study 1 to determine the effects of fresh ADS024 culture and ADS024 spore preparations on the clinical manifestations of CDI in mice, and Study 2 to compare the efficacy of single daily doses vs dosing 3 times per day with fresh ADS024. C. difficile challenge was performed 24 h after the start of ADS024 exposure. To model the human distal colon, an anerobic fecal fermentation system was used. MiSeq compositional sequencing and bioinformatic analyses were performed to measure microbiota diversity changes following ADS024 treatment. To assess the potential of ADS024 to be a source of antibiotic resistance, its susceptibility to 18 different antibiotics was tested. RESULTS: In a mouse model of CDI challenge, single daily doses of ADS024 were as efficacious as multiple daily doses in protecting against subsequent challenge by C. difficile pathogen-induced disease. ADS024 showed no evidence of colonization based on the observation that the ADS024 colonies were not recovered 24 h after single doses in mice or 72 h after single doses in miniature swine. In a 28-d repeat-dose study in miniature swine, ADS024 was not detected in fecal samples using plating and qPCR methods. Phylogenetic analysis performed in the human distal colon model showed that ADS024 had a selective impact on the healthy human colonic microbiota, similarly to the in vivo studies performed in miniature swine. Safety assessments indicated that ADS024 was susceptible to all the antibiotics tested, while in silico testing revealed a low potential for off-target activity or virulence and antibiotic-resistance mechanisms. CONCLUSION: Our findings, demonstrating in vivo efficacy of ADS024 in protecting against CDI challenge in mouse models, support the use of ADS024 in preventing recurrent CDI following standard antibiotic treatment.

Development of ADS051, an oral, gut-restricted, small molecule neutrophil modulator for the treatment of neutrophil-mediated inflammatory diseases.

Neutrophils are an essential component of the innate immune system; however, uncontrolled neutrophil activity can lead to inflammation and tissue damage in acute and chronic diseases. Despite inclusion of neutrophil presence and activity in clinical evaluations of inflammatory diseases, the neutrophil has been an overlooked therapeutic target. The goal of this program was to design a small molecule regulator of neutrophil trafficking and activity that fulfilled the following criteria: (a) modulates neutrophil epithelial transmigration and activation, (b) lacks systemic exposure, (c) preserves protective host immunity, and (d) is administered orally. The result of this discovery program was ADS051 (also known as BT051), a low permeability, small molecule modulator of neutrophil trafficking and activity via blockade of multidrug resistance protein 2 (MRP2)- and formyl peptide receptor 1 (FPR1)-mediated mechanisms. ADS051, based on a modified scaffold derived from cyclosporine A (CsA), was designed to have reduced affinity for calcineurin with low cell permeability and, thus, a greatly reduced ability to inhibit T-cell function. In cell-based assays, ADS051 did not inhibit cytokine secretion from activated human T cells. Furthermore, in preclinical models, ADS051 showed limited systemic absorption (<1% of total dose) after oral administration, and assessment of ADS051 in human, cell-based systems demonstrated inhibition of neutrophil epithelial transmigration. In addition, preclinical toxicology studies in rats and monkeys receiving daily oral doses of ADS051 for 28 days did not reveal safety risks or ADS051-related toxicity. Our results to date support the clinical development of ADS051 in patients with neutrophil-mediated inflammatory diseases.

A novel dual NLRP1 and NLRP3 inflammasome inhibitor for the treatment of inflammatory diseases.

Objectives: Inflammasomes induce maturation of the inflammatory cytokines IL-1ß and IL-18, whose activity is associated with the pathophysiology of a wide range of infectious and inflammatory diseases. As validated therapeutic targets for the treatment of acute and chronic inflammatory diseases, there has been intense interest in developing small-molecule inhibitors to target inflammasome activity and reduce disease-associated inflammatory burden. Methods: We examined the therapeutic potential of a novel small-molecule inhibitor, and associated derivatives, termed ADS032 to target and reduce inflammasome-mediated inflammation in vivo. In vitro, we characterised ADS032 function, target engagement and specificity. Results: We describe ADS032 as the first dual NLRP1 and NLRP3 inhibitor. ADS032 is a rapid, reversible and stable inflammasome inhibitor that directly binds both NLRP1 and NLRP3, reducing secretion and maturation of IL-1ß in human-derived macrophages and bronchial epithelial cells in response to the activation of NLPR1 and NLRP3. ADS032 also reduced NLRP3-induced ASC speck formation, indicative of targeting inflammasome formation. In vivo, ADS032 reduced IL-1ß and TNF-α levels in the serum of mice challenged i.p. with LPS and reduced pulmonary inflammation in an acute model of lung silicosis. Critically, ADS032 protected mice from lethal influenza A virus challenge, displayed increased survival and reduced pulmonary inflammation. Conclusion: ADS032 is the first described dual inflammasome inhibitor and a potential therapeutic to treat both NLRP1- and NLRP3-associated inflammatory diseases and also constitutes a novel tool that allows examination of the role of NLRP1 in human disease.

Rifalazil and derivative compounds show potent efficacy in a mouse model of H. pylori colonization.

The rifamycin rifalazil (RFZ), and derivatives (NCEs) were efficacious in a mouse model of Helicobacter pylori colonization. Select NCEs were more active in vitro and showed greater efficacy than RFZ. A systemic component contributes to efficacy.

Rifalazil retains activity against rifampin-resistant mutants of Chlamydia pneumoniae.

Rifampin-resistant mutants of the obligate intracellular pathogen Chlamydia pneumoniae were isolated and characterized, including strains that contained multiple mutations in the rpoB gene encoding the rifampin binding site. The highest MIC of rifampin against a mutant strain exceeded 100 microg/ml, whereas the highest MIC of rifalazil was 0.125 microg/ml. Derivatives of rifalazil (new chemical entities; NCEs) showed from 2 approximately 4 fold lower MICs, as well as 2 approximately 8 fold lower bactericidal concentrations against both wild type and mutant strains when compared with rifalazil. These results suggest that rifalazil and NCEs are appropriate therapeutic agents for the treatment of C. pneumoniae infections from the point of view of potency and resistance development.

Efficacy of superoxide dismutase mimetic M40403 in attenuating radiation-induced oral mucositis in hamsters.

PURPOSE: M40403 is a small-molecule superoxide dismutase mimetic that has shown efficacy in animal model disease states in which superoxide anions are thought to play a key role. Radiation treatment and chemotherapy for cancer generate free oxygen radicals that are hypothesized to trigger unwanted side effects in healthy tissue. For some patients undergoing these antineoplastic treatments, one of the most prevalent side effects is oral mucositis, which is a painful, often dose-limiting condition. Preclinical and clinical studies of this condition have shown the positive effect of treatment with compounds that decrease free oxygen radicals. This study investigated the efficacy M40403 in a clinically relevant hamster model of acute, radiation-induced oral mucositis. METHODS: Oral mucositis was induced in hamsters by irradiation of the cheek pouch. The ability of i.p. administered M40403 to decrease the duration and severity of oral mucositis was assessed after treatment at different doses and dosing schedules. Oral mucositis was scored using the WHO grading scale. RESULTS: Compared with placebo-treated animals, those irradiated on day 0 and treated twice daily with 30 mg/kg M40403 had significantly less severe and shorter duration mucositis over a range of treatment schedules, including from days -1 to 3, day 0 to 3, and day 0 alone. Similar efficacy was achieved at doses of 10 and 3 mg/kg twice daily on days -1 to 3. CONCLUSIONS: These results implicate free oxygen radicals in the onset of oral mucositis and also provide the basis for further development of M40403 in the prevention of this condition in at-risk cancer patients.

In vitro activity of novel rifamycins against gram-positive clinical isolates.

We describe novel rifamycins that have improved activity, compared with rifampin, against clinical isolates of staphylococci and streptococci, with MIC(90)s of 0.008 and 0.0005 microg/ml, respectively. This enhanced antibacterial activity, along with their potential lack of drug-drug interactions, are considerations that suggest the potential of these novel rifamycins in combination therapy to treat serious Gram-positive infections.

Efficacy of a novel rifamycin derivative, ABI-0043, against Staphylococcus aureus in an experimental model of foreign-body infection.

We compared the efficacy of a novel rifamycin derivative, ABI-0043, with that of rifampin, alone and in combination with levofloxacin, against methicillin-susceptible Staphylococcus aureus ATCC 29213 in a guinea pig tissue-cage infection model. The MIC, logarithmic-growth-phase minimal bactericidal concentration, and stationary-growth-phase minimal bactericidal concentration of ABI-0043 were 0.001, 0.008, and 0.25 microg/ml, respectively; the corresponding concentrations of rifampin were 0.016, 0.8, and 3.6 microg/ml, respectively. After a single intraperitoneal dose of 12.5 mg/kg of body weight, the peak concentration in cage fluid was 1.13 micarog/ml of ABI-0043 and 0.98 microg/ml of rifampin. Five days after completion of treatment, levofloxacin administered alone (5 mg/kg/12 h) resulted in bacterial counts in cage fluid that were similar to those for untreated controls (>8.0 log(10) CFU/ml), whereas rifampin and ABI-0043 administered alone (12.5 mg/kg/12 h) decreased the mean titers of bacteria +/- standard deviations to 1.43 +/- 0.28 log(10) and 1.57 +/- 0.53 log(10) CFU/ml, respectively, in cage fluid. In combination with levofloxacin, both rifamycins cleared bacteria from the cage fluid. The cure rates of cage-associated infections with rifampin and ABI-0043 administered alone were 46% and 58%, respectively, and increased to 88% and 92% in combination with levofloxacin. Emergence of rifamycin resistance was observed in 42% of cages after ABI-0043 therapy and in 38% of cages after rifampin therapy; no emergence of resistance occurred with combination treatment with levofloxacin. In conclusion, ABI-0043 had cure rates comparable to that of rifampin. ABI-0043 in combination with a quinolone has the potential for treatment of implant-associated infections caused by susceptible strains of S. aureus, potentially without drug-drug interactions.

Quantifying gastric Helicobacter pylori infection: a comparison of quantitative culture, urease breath testing, and histology.

Although there are several methods to detect Helicobacter pylori infection, there is no simple validated test to quantify the density of infection, which is believed to play a major role in the pathogenesis of H. pylori-associated gastritis and peptic ulceration. The aim of this study was to evaluate and compare noninvasive and invasive tests for assessing the level of H. pylori infection so as to facilitate the development and clinical testing of new antibiotic treatments. Healthy volunteers (n=323) were screened for H. pylori infection by serology and, if positive (n=86), invited to undergo (13)C urea breath testing (UBT) (n=55). An increase of >2.4 parts per thousand (13)CO(2) at 15 min compared to baseline was considered a positive test. Total cumulative urease activity (mumol) at 60 min was also calculated. UBT-positive subjects underwent endoscopy and nine biopsies were obtained from defined sites for quantitative culture and histological grading using the modified Sydney System. A total of 19 subjects were studied, 4 of whom underwent repeat testing. All subjects were positive for H. pylori by serology, UBT, culture, and histology. The increase in (13)CO(2) at 30 min correlated with the total cumulative urease activity at 60 min (r (2)=0.92, P< 0.0001). Bacterial counts (log cfu/biopsy; mean+/-SD) were 3.9+/-0.5, 3.9+/-0.4, and 3.9+/-0.6 at the lesser curve antrum, greater curve antrum, and corpus, respectively. There was no significant correlation between UBT results and bacterial counts at any biopsy site. Nor was there any significant correlation between the histology grading and either the UBT or the bacterial counts at any site. This study indicates that there is little correlation among the three methods used to measure bacterial burden in H. pylori infection. Thus, decrements in bacterial numbers during single-agent therapy cannot be measured reliably by UBT and therefore cannot be used to evaluate the potential efficacy of novel agents to treat gastric H. pylori infection.

Enhanced activity of rifalazil in combination with levofloxacin, linezolid, or mupirocin against Staphylococcus aureus in vitro.

Rifalazil is a potent second-generation ansamycin that kills bacterial cells by inhibiting the beta subunit of RNA polymerase. Rifalazil has several improved properties compared with rifampicin, but retains rifampicin's propensity to develop resistant mutants at high frequency. To explore strategies to overcome resistance development, we studied the effects of rifalazil in combination with several different antibiotics in an in vitro time-kill model, against both log phase and stationary phase Staphylococcus aureus cells. Experiments were carried out at high initial cell density so that the frequency and proliferation of resistant mutants could be monitored. We found that each combination was advantageous in terms of enhanced killing and the suppression of mutants, compared with each drug used alone. None of the three combinations was effective against stationary phase cells.

Efficacy of novel rifamycin derivatives against rifamycin-sensitive and -resistant Staphylococcus aureus isolates in murine models of infection.

Novel rifamycins (new chemical entities [NCEs]) having MICs of 0.002 to 0.03 microg/ml against Staphylococcus aureus and retaining some activity against rifampin-resistant mutants were tested for in vivo efficacy against susceptible and rifampin-resistant strains of S. aureus. Rifalazil and rifampin had a 50% effective dose (ED50) of 0.06 mg/kg of body weight when administered as a single intravenous (i.v.) dose in a murine septicemia model against a susceptible strain of S. aureus. The majority of NCEs showed efficacy at a lower i.v. dose (0.003 to 0.06 mg/kg). In addition, half of the NCEs tested for oral efficacy had ED50s in the range of 0.015 to 0.13 mg/kg, i.e., lower or equivalent to the oral ED50s of rifampin and rifalazil. NCEs were also tested in the septicemia model against a rifampin-resistant strain of S. aureus. Twenty-four of 169 NCEs were efficacious when administered as a single oral dose of 80 mg/kg. These NCEs were examined in the murine thigh infection model against a susceptible strain of S. aureus. Several NCEs dosed by intraperitoneal injection at 0.06 mg/kg caused a significant difference in bacterial titer compared with placebo-treated animals. No NCEs showed efficacy in the thigh model against a highly rifampin-resistant strain. However, several NCEs showed an effect when tested against a partially rifampin-resistant strain. The NCEs having a 25-hydroxyl moiety were more effective as a group than their 25-O-acetyl counterparts. These model systems defined candidate NCEs as components of potential combination therapies to treat systemic infections or as monotherapeutic agents for topical applications.

Development potential of rifalazil and other benzoxazinorifamycins.

Rifalazil and other benzoxazinorifamycins (new chemical entities [NCEs]) are rifamycins that contain a distinct planar benzoxazine ring. Rifalazil has excellent antibacterial activity, high intracellular levels and high tissue penetration, which are attributes that favour its use in treating diseases caused by the obligate intracellular pathogens of the genus Chlamydia. Recent studies have shown that rifalazil has efficacy in the treatment of human sexually transmitted disease caused by Chlamydia trachomatis. The extraordinary potency of rifalazil and other NCEs, such as ABI-0043, extends to the related microorganism, C. pneumoniae, a respiratory pathogen that can disseminate and persist chronically in the vasculature, resulting in increased plaque formation in animal studies. A pivotal clinical trial with rifalazil has been initiated for the treatment of peripheral arterial disease. Other opportunities include gastric ulcer disease caused by Helicobacter pylori and antibiotic-associated colitis caused by infection with Clostridium difficile in the colon. The NCEs could prove to be valuable as follow-on compounds in these indications, as rifampin replacements in antibacterial combination therapy or as stand-alone topical antibacterials (e.g., to treat acne). Neither rifalazil nor NCEs appear to induce the cytochrome P450 3A4, an attribute of rifampin that can result in adverse events due to drug-drug interactions.

In vitro time-kill activities of rifalazil, alone and in combination with vancomycin, against logarithmic and stationary cultures of Staphylococcus aureus.

Rifalazil is a novel rifamycin that, like other members of this class, inhibits bacterial transcription by targeting the beta subunit of prokaryotic DNA-dependent RNA polymerase. To address the high-frequency resistance seen with rifamycins, we assessed the ability of rifalazil, alone and in combination with vancomycin, to both kill cells and to suppress the appearance of resistant mutants in log and stationary phase Staphylococcus aureus cultures, using high cell densities in an in vitro kill curve model. We found that (1) rifalazil alone killed log-phase cultures more rapidly than rifampicin, but both drugs quickly selected for resistant mutants, (2) co-treatment of log phase cultures with rifalazil and vancomycin increased bacterial killing by about 3-Log10 over either drug used alone and delayed the appearance of rifamycin-resistant mutants, (3) rifalazil and vancomycin in combination killed stationary phase cultures

In vitro activity of novel rifamycins against rifamycin-resistant Staphylococcus aureus.

We describe novel rifamycin derivatives (new chemical entities [NCEs]) that retain significant activity against a comprehensive collection of Staphylococcus aureus strains that are resistant to rifamycins. This collection of resistant strains contains 21 of the 26 known single-amino-acid alterations in RpoB, the target of rifamycins. Some NCEs also demonstrated a lower frequency of resistance development than rifampin and rifalazil in S. aureus as measured in a resistance emergence test. When assayed for activity against the strongest rifamycin-resistant mutants, several NCEs had MICs of 2 microg/ml, in contrast to MICs of rifampin and rifalazil, which were 512 microg/ml for the same strains. The properties of these NCEs therefore demonstrate a significant improvement over those of earlier rifamycins, which have been limited primarily to combination therapy due to resistance development, and suggest a potential use of these NCEs for monotherapy in several clinical indications.

Bactericidal efficacy of ABI-0043, a novel rifamycin, in a murine pneumococcal pneumonia model.

Novel rifamycins such as ABI-0043, ABI-0369, and ABI-0699 had comparable in vivo bactericidal activity with ceftriaxone against a penicillin-G-resistant, mefA-positive Streptococcus pneumoniae in a murine pneumonia model. ABI-0043 demonstrated a dose-dependent response with a high correlation to bacterial kill (+0.1 to -3.7 log 10CFU).