Myxopyronin B (MyxB) binds to the switch region of RNA polymerase (RNAP) and inhibits transcriptional initiation. To evaluate the potential development of MyxB as a novel class of antibiotic, we characterized the antimicrobial activity of MyxB against Staphylococcus aureus. Spontaneous MyxB resistance in S. aureus occurred at a frequency of 8 × 10(-8) , similar to that of rifampin. The MyxB-resistant mutants were found to be altered in single amino acid residues in the RNAP subunits that form the MyxB-binding site. In the presence of human serum albumin, the MyxB minimum inhibitory concentration against S. aureus increased drastically (≥128-fold) and 99.5% of MyxB was protein bound. Because of the high serum protein binding and resistance rate, we conclude that MyxB is not a viable starting point for antibiotic development.

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.

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.

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 μg/ml, whereas the highest MIC of rifalazil was 0.125 μg/ml. Derivatives of rifalazil (new chemical entities; NCEs) showed from 2∼4 fold lower MICs, as well as 2∼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.

ABSTRACT 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 μg/ml, respectively; the corresponding concentrations of rifampin were 0.016, 0.8, and 3.6 μg/ml, respectively. After a single intraperitoneal dose of 12.5 mg/kg of body weight, the peak concentration in cage fluid was 1.13 μg/ml of ABI-0043 and 0.98 μg/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 log10 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 log10 and 1.57 ± 0.53 log10 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.

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 13C urea breath testing (UBT) (n=55). An increase of >2.4 parts per thousand 13CO2 at 15 min compared to baseline was considered a positive test. Total cumulative urease activity (μmol) 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 13CO2 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.

We describe novel rifamycins that have improved activity, compared with rifampin, against clinical isolates of staphylococci and streptococci, with MIC90s of 0.008 and 0.0005 μg/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.

Rifalazil is a novel rifamycin that, like other members of this class, inhibits bacterial transcription by targeting the β 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-Log 10 over either drug used alone and delayed the appearance of rifamycin-resistant mutants, 3) rifalazil and vancomycin in combination killed stationary phase cultures by 3∼4 Log 10 by 48 hours.

ABSTRACT Novel rifamycins (new chemical entities [NCEs]) having MICs of 0.002 to 0.03 μg/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.

ABSTRACT 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 μg/ml, in contrast to MICs of rifampin and rifalazil, which were 512 μg/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.

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(10)CFU).

Rifalazil is a potent second-generation ansamycin that kills bacterial cells by inhibiting the β 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.

Serious gram-positive infections are frequently caused by Staphylococcus aureus or Streptococcus pyogenes. The rifamycin rifampin (RIF) is often used as part of the treatment for such infections, because it has potent bactericidal activity against both free-living and biofilm-associated gram-positive bacteria (13) and has favorable pharmacokinetics (2). Despite this, RIF's use as monotherapy has been limited because of treatment failure due to the emergence of rifamycin-resistant organisms (3, 4, 7, 8). Resistance results from amino acid substitutions within the conserved rifamycin resistance-determining region (RRDR) of the RpoB subunit of RNA polymerase, the target of rifamycins. Because of resistance emergence, RIF is used mainly as part of a multidrug treatment regimen in which the partner drug(s) is able to prevent outgrowth of resistant subpopulations. A previous study described the activity of the benzoxazinorifamycins rifalazil (RFZ) and related new chemical entities (NCEs) against RIF-resistant S. aureus (10). The maximum MICs of the most potent NCEs were 2 to 4 μg/ml against the most rifamycin-resistant S. aureus strains. RIF and RFZ have MICs of >512 μg/ml against these same strains (10). Considering that a large proportion of serious gram-positive infections are caused by streptococci, we sought to determine in the present study whether NCEs had improved activity against rifamycin-resistant strains of S. pyogenes. The two mutations conferring RIF resistance in clinical isolates of S. pyogenes (4, 7) and several mutations in other streptococci (1, 5, 6, 9, 12) all reside in the RRDR of RpoB in these organisms. S. pyogenes ATCC 19615 mutants with decreased susceptibility to RIF were selected for and characterized as described previously (10). The resistance frequencies of S. pyogenes for RIF were 7.0 × 10−8 (at 0.24 μg/ml), 6.0 × 10−8 (at 0.12 μg/ml), and 2.9 × 10−8 (at 0.06 μg/ml). Thirty-three colonies with decreased susceptibility to RIF were isolated, and oligonucleotide primers SPY-F and SPY-R (5′-AACCGTCGTATCCGTGCCGTTGGT-3′ and 5′-TGCCGTCGCAACAGCAACTACCTG-3′, respectively) were used to amplify and DNA sequence their RRDR regions, encoded by base pairs 1228 to 1884 (S. pyogenes MGAS315 coordinates; GenBank accession number NC 004070). Seventeen discrete amino acid changes at 10 different positions within the S. pyogenes RRDR were identified (Table ​(Table1).1). Changes at H486, R489, and S491 correspond to positions that, when altered, confer the high levels of resistance to rifamycins in other bacteria (10). The H486N substitution was previously identified in a RIF-resistant clinical strain of S. pyogenes (7). TABLE 1. Susceptibility of S. pyogenes RIF-resistant mutants to RIF, RFZ, and select NCEs The MICs of RIF, RFZ, and NCEs for S. pyogenes ATCC 19615 and its rifamycin-resistant derivatives were determined using standard methods (11). S. pyogenes ATCC 19615 was 16- to 32-fold more susceptible to RFZ and NCEs than was S. aureus ATCC 29213 (Table ​(Table1)1) (10). MICs of the most potent NCEs (e.g., ABI-0043) against rifamycin-resistant S. pyogenes strains ranged from 0.00025 μg/ml to 0.25 μg/ml for the most resistant strain (Table ​(Table1)1) and 0.016 μg/ml to 4 μg/ml for the most rifamycin-resistant S. aureus strains (10). In fact, the MICs of NCEs against S. pyogenes mutants (Table ​(Table1)1) were consistently ≥8-fold lower than those for S. aureus mutants containing mutations in the corresponding codon of the rpoB gene (10). It is anticipated that NCEs having in vivo efficacy against rifamycin-resistant S. aureus strains would be effective against the most resistant S. pyogenes mutants. Experimental infection models using resistant strains from both of these species should verify this assumption.

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.

La presente invention a trait a des procedes pour le criblage d'agents antimicrobiens mettant en oeuvre des polypeptides microbiens mutes. Etant donne que ces polypeptides presentent une activite biologique et entrainent une resistance accrue aux medicaments antimicrobiens, des composes candidats de ciblage specifique desdits polypeptides fournissent des agents therapeutiques ou des tetes de serie therapeutiques pour le traitement d'infections provoquees par des agents pathogenes microbiens resistants aux medicaments.

Purpose: To quantify, compare, and correlate non-invasive and invasive diagnostic assays for assessing degrees of H. pylori infection for use in antibiotic development. Methods: Volunteers (N = 152, 100%) were screened for circulating IgG antibodies to H pylori using the QuickVue H. pylori gII test and, if positive (N = 42,27.6%), further screened with13 C urease breath test (UBT) (N = 20, 13.2%). UBT data were obtained at baseline, 15, 30, 45, and 60 min. post po administration of 13C; fifteen patients were positive (9.9%). Urease activity was calculated as umolesof 13CO2 formed/minute at time points (15, 30,45, 60 min). Urease activity was highest at 30 min., consistent with previously published reports. Volunteers with positive results for both screening tests underwent endoscopy (N = 4, 2.6%). Six biopsy samples were obtained, two each from the lesser and greater curvatures of the antrum (GA, LA, 2 cm and 4 cm from pylorus), and two samples from the greater curvature of the corpus (GC, 3 and 5 cmproximal to angulus). Within 2 hours, biopsy samples were homogenized, serially diluted and plated on on Skirrow's agar for H. pylori colony forming units (cfu). H. pylori colonies were counted on Day 3 after plating and cfu/biopsy site was calculated. Correlation analysis was used to relate cfu obtained per biopsy site to urease activity. Results: We report here the preliminary findings of this study which demonstrate good correlation (r2 = 0.93, p = 0.023) between log cfu at biopsy location GA-4cm and 30 minute urease activity rate. However, no correlation with the 30 minute urease activity rate was found with other biopsy sites. Conclusions: A correlation was observed between log cfu and urease activity at 30 minutes on UBT at one of six biopsy sites in this limited sample. We speculate that either inadequate sample size or uneven distribution of H. pylori infection may explain the lack of correlation at other sites. This may be useful in antibiotic therapy development by providing an early indicator of bacterial kill, which could guide the dosing and sequence of component administration in combination therapies. This warrants further study.