Abstract
E-4767 {(−)-7-[3-(R)-amino-2-(S)-methyl-1-azetidinyl]-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4- oxo-3-quinolinecarboxylic acid} and E-5065 [(−)-7-(3-amino-1-azetidinyl)-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid] are two new chlorofluoroquinolones with an azetidine moiety at position 7. Their in vitro activities were evaluated in comparison with those of ciprofloxacin, ofloxacin, fleroxacin, and tosufloxacin, while ciprofloxacin was used as a reference for in vivo studies. Against gram-positive organisms, E-4767 and E-5065 were, in general, eight- and fourfold more active than tosufloxacin, which is the most potent of the reference compounds. E-4767 and E-5065 were also more potent than the reference compounds against all species of enteric bacteria tested. The MICs of E-4767 and E-5065 at which 90% of the isolates tested were inhibited (MIC90s) were 0.007 to 0.5 μg/ml and 0.03 to 2 μg/ml, respectively, for gram-positive organisms and ≤0.003 to 0.06 μg/ml and 0.007 to 0.12 μg/ml, respectively, for members of the family Enterobacteriaceae except Serratia marcescens and Providencia spp. (MIC90s of E-4767 and E-5065 for these species were ≤0.5 μg/ml and ≤2 μg/ml, respectively). For Pseudomonas aeruginosa both compounds had a MIC90 of 0.5 μg/ml. E-4767 and E-5065 were 356- and 32-fold more potent than ciprofloxacin against Bacteroides spp., and their MIC90s for Clostridium spp. were 0.25 and 0.5 μg/ml, respectively. Both products showed a remarkable reduction of activity when the pH was below 4.8 and, in general, were less active in the presence of 5 or 10 mM Mg2+. The presence of horse serum or human urine (pH 7.2) decreased the activity of E-4767 and E-5065 only two- to fourfold more than the activity observed in broth. After an oral dose of 50 mg/kg of body weight, the maximum levels in serum (the maximum concentration of drug in serum was reached 30 min postadministration) of E-4767 and E-5065 were approximately threefold higher than that of ciprofloxacin. The area under the concentration-time curve from 0 to 4 h for ciprofloxacin was about two- and fourfold lower than that for E-4767 and E-5065, respectively. These two new chlorofluoroquinolones were as effective as or more effective than ciprofloxacin against all experimental infections evaluated, not only against gram-negative bacteria, such as Escherichia coli or P. aeruginosa, but also against gram-positive pathogens, such as Staphylococcus aureus or Streptococcus pneumoniae. E-4767 was the most effective compound, with a 50% effective dose (ED50) of ≤17 mg/kg for all strains tested except ciprofloxacin-resistant S. aureus strains. The ED50 of E-4767 for these strains was ≤47.5 mg/kg. Against gram-positive experimental infections, the ED50 values of E-4767 were 3- to 14-fold lower than those of E-5065 and up to 25 times lower than those of ciprofloxacin.
Since the discovery of nalidixic and oxolinic acids (10, 13), many modifications have been introduced in the chemical structure of the quinolonic ring, yielding new drugs with improved antibacterial efficacy against gram-positive and anaerobic bacteria and with enhanced bioavailability, such as norfloxacin (11), ofloxacin (21), ciprofloxacin (25), and tosufloxacin (23). In a recent review, Domagala (4) established a model in which every junction of a substituent has a specific property in addition to that of the substituent itself. Substituents at position 7 influence antimicrobial potency, the spectrum of activity, and the pharmacokinetic characteristics. Pyrrolidines at this position offer great potency against gram-positive bacteria, and piperazines introduce excellent activity against gram-negative bacteria. Furthermore, among the variations described at position 7 (1, 2, 7, 12, 16), it has been proven that the presence of an azetidine moiety (3, 5, 6, 8, 9) yields a broader spectrum and enhanced activity against gram-positive bacteria. On the other hand, the substituent at position 8 controls in vivo efficacy and is largely responsible for the activity against anaerobic bacteria, with halogens being one of the optimal substitution groups at this site. Among the quinolones with a Cl at position 8, DU-6859 (20), BAY-Y-3118 (24), and clinafloxacin (19) have been the most active.
E-4767 and E-5065 (Fig. 1) are structurally characterized by the presence of an azetidine ring with different C-2′ and C-3′ substitutions at position 7 of the molecule. E-4767 has a 2-methyl-3-aminoazetidin-1-yl substituent at position 7, while E-5065 is characterized by a 3-aminoazetidin-1-yl group in the same position. Both compounds possess a Cl as R8.
FIG. 1.
Chemical structures of E-4767 and E-5065.
In this work, the in vitro activities of E-4767 and E-5065 are compared with those of ciprofloxacin, fleroxacin, ofloxacin, and tosufloxacin against groups of pathogenic bacterial clinical isolates. The effects of various assay conditions on in vitro activity, such as pH, magnesium ion concentration, and the presence of serum and urine, were investigated. We also studied the pharmacokinetic properties and the in vivo protective efficacy of these compounds against lethal systemic infections in mice. Ciprofloxacin was selected as the reference compound for in vivo studies.
MATERIALS AND METHODS
Antibacterial agents.
E-4767, E-5065, ofloxacin, and tosufloxacin were synthesized at Laboratorios Esteve S.A. (Barcelona, Spain). Ciprofloxacin (Bayer A.G., Wuppertal, Germany) and fleroxacin (Roche S.A., Madrid, Spain) were obtained as standard powders of known potency. Solutions were prepared immediately before use. For determination of MICs, a stock solution was prepared in 0.1 N NaOH and diluted in broth medium to the appropriate concentration. For in vivo tests, all antibacterial agents were dissolved in 0.1 N NaOH, appropriately diluted with sterile water, and finally mixed in 0.1% carboxymethyl cellulose.
Organisms.
Clinical isolates for in vitro activity evaluation were randomly collected from various hospitals in Spain. Selected strains (Citrobacter freundii HSP-73, Enterobacter aerogenes HSP-145, Escherichia coli HM-42, Klebsiella pneumoniae HSP-30, Proteus vulgaris HSP-99, Salmonella enterica serovar Enteritidis HSP-928/F, Pseudomonas aeruginosa HSP-116, Staphylococcus aureus HS-93, coagulase-negative Staphylococcus sp. HSP-10, and Enterococcus faecalis HSP-30) were used to evaluate the effects of different conditions upon the in vitro activity of the new chlorofluoroquinolones, and additional strains (ciprofloxacin-resistant [Cipr] P. aeruginosa B-464, Cipr S. aureus 73777, Cipr S. aureus 73940, Streptococcus pneumoniae 1625, S. pneumoniae 84551, and S. pneumoniae 1752) were used in previous studies for in vivo efficacy evaluation (6, 8, 9).
Five additional strains (S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, Clostridium perfringens ATCC 13124, and Bacteroides fragilis ATCC 25285) obtained from the American Type Culture Collection (Rockville, Md.) were used as susceptibility test controls for in vitro studies (data not shown). Bacillus subtilis ATCC 6633 was used as indicator strain in pharmacokinetic bioassay studies.
All strains were stored frozen at −70°C in our laboratories until used.
Determination of MICs and MBCs.
For aerobic and facultatively anaerobic organisms, MICs were determined in Mueller-Hinton (MH) broth (Oxoid Ltd., Basingstoke, England) by a microtiter twofold dilution method using a Quick Spense II system (Dynatech AG, Denkendorf, Germany) as recommended by the National Committee for Clinical Laboratory Standards (17). For Streptococcus species, brain heart infusion broth medium (Oxoid Ltd.) was used. The inoculum was approximately 5 × 105 CFU/ml. MICs, defined as the lowest concentrations of antibacterial agent that inhibited development of growth, were recorded after 18 h of incubation at 37°C. Minimum bactericidal concentrations (MBCs), defined as the lowest compound concentrations that killed ≥99.9% of the initial inoculum, were in turn determined by subculturing 10 μl of broth from the drug-free control well, the first well containing growth, and each clear well on MH agar plates.
Anaerobic bacteria were tested by the agar dilution method as recommended by the National Committee for Clinical Laboratory Standards (18) on Wilkins-Chalgren agar medium (Oxoid Ltd.) supplemented with 5% horse blood and a final inoculum of 104 CFU per spot. The plates were inoculated with a Steers-type multipoint inoculator (22). Anaerobic incubation was carried out at 35°C for 48 h in anaerobic GasPak jars (BBL Microbiology Systems, Cockeysville, Md.). Two plates of test medium without antibacterial agent were also incubated. One was incubated anaerobically to serve as a growth control, and the other was incubated aerobically to detect possible aerobic contamination. The MIC on solid medium was defined as the lowest antibacterial agent concentration that inhibited development of visible growth on agar.
Factors affecting in vitro activity.
The effects of medium pH, magnesium ion concentration, and the presence of serum and urine on the in vitro activities of E-4767 and E-5065 against C. freundii HSP-73, E. aerogenes HSP-145, E. coli HM-42, K. pneumoniae HSP-30, P. vulgaris HSP-99, S. enterica serovar Enteritidis HSP-928/F, P. aeruginosa HSP-116, S. aureus HS-93, coagulase-negative S. aureus HSP-10, and E. faecalis HSP-30 were determined as described above for aerobic and facultatively anaerobic organisms in MH broth supplemented with the different tested factors. Unsupplemented MH broth, MH broth adjusted to different pH values, and magnesium-, serum-, or urine-supplemented wells without antibacterial agents were used as control media.
(i) Medium pH.
The effects of pH were determined in MH broth adjusted to pH 4.8, 5.8, 6.8, 7.8, and 8.8.
(ii) Mg2+ concentration.
Magnesium (as MgCl2 · 6H2O) was added to MH broth at 1, 5, and 10 mM.
(iii) Serum.
Horse serum inactivated at 56°C for 30 min was added to MH broth to a final concentration of 20 or 70% (vol/vol) with the pH adjusted to 7.2.
(iv) Urine.
The urine samples used were early morning pooled and obtained from healthy human male volunteers. The pH was adjusted to 5.5 or 7.2, followed by sterilization by filtering through a 0.22-μm-pore-size membrane filter (Millipore Corp., Bedford, Mass.).
Pharmacokinetics in mice.
Pharmacokinetic assays were performed using male Swiss mice (Charles River, Cleon, France) weighing approximately 30 g. Each compound (E-4767, E-5065, and ciprofloxacin) was administered once by gavage at a dose of 50 mg/kg of body weight after fasting for 4 h. Samples of blood were collected from groups of six mice at 30, 60, 120, and 240 min postadministration. The blood samples were immediately chilled and centrifuged at 1,000 × g for 15 min. Prior to analysis, the standards (initially solubilized in 0.1 N NaOH) and the plasma specimens obtained were suitably diluted with 0.07 M phosphate buffer (pH 6.0).
Concentrations of E-4767, E-5065, or ciprofloxacin in plasma were determined by the agar diffusion method under standard conditions using 7-mm-diameter Oxford cylinders. B. subtilis ATCC 6633 was used as an indicator organism. Plates were incubated at 37°C for approximately 18 h. The lower limit of detection was <0.05 μg/ml. The areas under the concentration-time curve from 0 to 4 h (AUC0–4) were calculated from the mean concentrations by the trapezoidal method. Results obtained with the agar diffusion method were validated by high-pressure liquid chromatography analysis.
Mouse protection tests on experimental systemic infections.
Male HC:CFLP mice weighing approximately 25 g (Interfauna U.K. Ltd., Huntingdon, England) were used in protection tests on systemic infections. Mouse protection tests were performed against the strains E. coli HM-42, P. aeruginosa HSP-116, Cipr P. aeruginosa B-464, S. aureus HS-93, Cipr S. aureus 73777, Cipr S. aureus 73940, S. pneumoniae 1625, S. pneumoniae 84551, and S. pneumoniae 1752.
Test organisms were grown overnight on MH agar plates, except S. pneumoniae strains, which were grown on MH agar supplemented with 5% horse blood and incubated in a candle jar.
Mice were inoculated intraperitoneally with 0.5 ml of a bacterial suspension adjusted to the appropriate concentration (five times the minimum lethal dose) with physiological saline solution, with the exception of the Cipr strains, which were adjusted with 5% hog gastric mucin (ICN Biomedicals, Columbus, Ohio). The challenge inoculum was sufficient to kill 100% of untreated control mice within 2 days postinfection, with the exception of mice experimentally infected with S. aureus and S. pneumoniae strains, some of which died within 4 days after the challenge. Immediately after the challenge, the mice were subjected to a single oral administration of the test compound, with the exception of those experimentally infected with Cipr strains (which received an additional dose at 6 h postinfection) and those infected with S. pneumoniae strains (which received additional doses at 6, 12, and 24 h postinfection). Four groups of 10 mice each were treated with different doses of each antibacterial agent.
ED50 (50% effective dose) and 95% confidence intervals were calculated by probit analysis (15) and the method of Litchfield and Wilcoxon (14), respectively, 7 days after infection. The ED50 values for Cipr and S. pneumoniae strains were determined for the total drug dose given.
RESULTS
Susceptibilities in vitro.
The susceptibilities of clinical isolates to E-4767 and E-5065 were compared with their susceptibilities to ciprofloxacin, fleroxacin, ofloxacin, and tosufloxacin (Table 1). The two new compounds were highly active against gram-positive cocci, including Staphylococcus spp. (methicillin-resistant [Metr] S. aureus, Cipr S. aureus, coagulase-negative methicillin-susceptible [Mets] Staphylococcus spp., coagulase-negative Metr Staphylococcus spp., and coagulase-negative Cipr Staphylococcus spp.), S. pneumoniae, Streptococcus viridans, and E. faecalis. E-4767 and E-5065 were the most effective compounds against all species of gram-positive organisms tested. The MIC90s (MICs at which 90% of the strains tested were inhibited) of E-4767 and E-5065 for Mets Staphylococcus spp., including S. aureus and coagulase-negative Staphylococcus spp., were 0.007 and 0.06 μg/ml, respectively. Against these groups of isolates, E-4767 and E-5065 were 16- and 2-fold more potent than tosufloxacin, which was the most active of the reference compounds tested. Against MetR Staphylococcus spp. isolates (including S. aureus and coagulase-negative Staphylococcus spp.), E-4767 and E-5065 were four- to eightfold more active than any standard reference compound tested. In addition, E-4767 and E-5065 exhibited significant activity against Cipr Staphylococcus sp. strains. For Cipr S. aureus isolates, the MIC90s of E-4767 and E-5065 were 0.25 and 2 μg/ml, respectively, versus 0.25 and 1 μg/ml, respectively, for coagulase-negative Cipr Staphylococcus sp. organisms. In turn, the MIC90s of the four antibacterial agents used as reference controls against Cipr Staphylococcus spp. isolates were higher than 4 μg/ml in all cases. The MIC90 of E-4767 for S. pneumoniae isolates was 0.03 μg/ml, i.e., 8 times lower than that of tosufloxacin and 64 times lower than that of either ciprofloxacin or ofloxacin. Against S. pneumoniae isolates, E-5065 was eightfold more active than ciprofloxacin and ofloxacin. For S. viridans and E. faecalis, the MIC90s of E-4767 and E-5065 were 0.5 and 1 μg/ml, respectively. Against these organisms, the activity of all marketed compounds evaluated was, in general, eight times lower than that of E-4767, and four times lower than that of E-5065.
TABLE 1.
In vitro activities of E-4767, E-5065, and reference quinolones against clinical isolates
| Organism (no. of isolates tested) and antibacterial agent | MIC (μg/ml)
|
||
|---|---|---|---|
| Range | 50% | 90% | |
| Enterococcus faecalis (27) | |||
| E-4767 | 0.03–0.5 | 0.06 | 0.5 |
| E-5065 | 0.12–1 | 0.25 | 1 |
| Ciprofloxacin | 0.5–4 | 1 | 4 |
| Fleroxacin | >4 | >4 | >4 |
| Ofloxacin | 1–4 | 2 | 4 |
| Tosufloxacin | 1–4 | 0.5 | 2 |
| Staphylococcus aureus (28) | |||
| E-4767 | ≤0.03–0.12 | 0.007 | 0.007 |
| E-5065 | 0.015–0.12 | 0.03 | 0.03 |
| Ciprofloxacin | 0.12–4 | 0.25 | 0.5 |
| Fleroxacin | 0.25–4 | 0.5 | 1 |
| Ofloxacin | 0.12–2 | 0.25 | 0.5 |
| Tosufloxacin | 0.015–0.5 | 0.03 | 0.12 |
| CiprStaphylococcus aureus (12) | |||
| E-4767 | 0.12–0.5 | 0.12 | 0.25 |
| E-5065 | 0.5–4 | 1 | 2 |
| Ciprofloxacin | >4 | >4 | >4 |
| Fleroxacin | >4 | >4 | >4 |
| Ofloxacin | >4 | >4 | >4 |
| Tosufloxacin | >4 | >4 | >4 |
| MetrStaphylococcus aureus (14) | |||
| E-4767 | 0.06–0.12 | 0.12 | 0.12 |
| E-5065 | 0.5 | 0.5 | 0.5 |
| Ciprofloxacin | >4 | >4 | >4 |
| Fleroxacin | >4 | >4 | >4 |
| Ofloxacin | >4 | >4 | >4 |
| Tosufloxacin | 2–4 | 2 | 4 |
| Coagulase-negative CiprStaphylococcus spp. (20) | |||
| E-4767 | 0.06–0.25 | 0.12 | 0.25 |
| E-5065 | 0.5–1 | 1 | 1 |
| Ciprofloxacin | >4 | >4 | >4 |
| Fleroxacin | >4 | >4 | >4 |
| Ofloxacin | >4 | >4 | >4 |
| Tosufloxacin | >4 | >4 | >4 |
| Coagulase-negative MetrStaphylococcus spp. (12) | |||
| E-4767 | ≤0.003–0.5 | 0.007 | 0.5 |
| E-5065 | 0.015–1 | 0.12 | 1 |
| Ciprofloxacin | 0.06–>4 | 1 | 4 |
| Fleroxacin | 0.25–>4 | 4 | >4 |
| Ofloxacin | 0.12–>4 | 1 | 4 |
| Tosufloxacin | 0.03–>4 | 0.25 | 4 |
| Coagulase-negative MetsStaphylococcus spp. (19) | |||
| E-4767 | ≤0.003–0.007 | 0.007 | 0.007 |
| E-5065 | 0.015–0.06 | 0.03 | 0.06 |
| Ciprofloxacin | 0.03–1 | 0.12 | 0.12 |
| Fleroxacin | 0.25–>4 | 0.5 | 4 |
| Ofloxacin | 0.12–1 | 0.25 | 1 |
| Tosufloxacin | 0.03–0.5 | 0.06 | 0.12 |
| Streptococcus pneumoniae (28) | |||
| E-4767 | ≤0.003–0.03 | 0.015 | 0.03 |
| E-5065 | 0.03–0.25 | 0.12 | 0.25 |
| Ciprofloxacin | 0.12–2 | 0.5 | 2 |
| Fleroxacin | 4–>4 | 4 | >4 |
| Ofloxacin | 0.05–2 | 1 | 2 |
| Tosufloxacin | 0.06–0.25 | 0.12 | 0.25 |
| Streptococcus viridans (9) | |||
| E-4767 | ≤0.003–0.5 | 0.03 | 0.5 |
| E-5065 | 0.03–1 | 0.25 | 1 |
| Ciprofloxacin | 0.25–4 | 2 | 4 |
| Fleroxacin | 0.5–>4 | >4 | >4 |
| Ofloxacin | 0.25–>4 | 2 | 4 |
| Tosufloxacin | 0.03–>4 | 0.5 | 4 |
| Citrobacter freundii (29) | |||
| E-4767 | ≤0.003–0.06 | 0.007 | 0.015 |
| E-5065 | 0.007–0.06 | 0.015 | 0.03 |
| Ciprofloxacin | 0.007–0.12 | 0.015 | 0.06 |
| Fleroxacin | 0.06–1 | 0.12 | 0.5 |
| Ofloxacin | 0.06–1 | 0.12 | 0.25 |
| Tosufloxacin | 0.007–0.5 | 0.03 | 0.25 |
| Enterobacter aerogenes (12) | |||
| E-4767 | ≤0.003–0.015 | 0.007 | 0.007 |
| E-5065 | 0.007–0.03 | 0.015 | 0.03 |
| Ciprofloxacin | 0.015–0.06 | 0.015 | 0.03 |
| Fleroxacin | 0.12–0.5 | 0.12 | 0.25 |
| Ofloxacin | 0.06–0.25 | 0.12 | 0.25 |
| Tosufloxacin | 0.015–0.06 | 0.03 | 0.06 |
| Enterobacter agglomerans (12) | |||
| E-4767 | ≤0.003–0.007 | ≤0.003 | ≤0.003 |
| E-5065 | ≤0.003–0.03 | 0.007 | 0.015 |
| Ciprofloxacin | ≤0.003–0.015 | 0.015 | 0.015 |
| Fleroxacin | 0.03–0.25 | 0.06 | 0.25 |
| Ofloxacin | 0.03–0.12 | 0.06 | 0.06 |
| Tosufloxacin | 0.015–0.12 | 0.03 | 0.06 |
| Enterobacter cloacae (24) | |||
| E-4767 | ≤0.003–0.25 | 0.007 | 0.06 |
| E-5065 | 0.007–1 | 0.015 | 0.12 |
| Ciprofloxacin | 0.007–1 | 0.03 | 1 |
| Fleroxacin | 0.12–4 | 0.25 | 1 |
| Ofloxacin | 0.06–4 | 0.12 | 1 |
| Tosufloxacin | 0.015–4 | 0.03 | 1 |
| Escherichia coli (29) | |||
| E-4767 | ≤0.003–0.007 | ≤0.003 | 0.007 |
| E-5065 | ≤0.003–0.03 | 0.007 | 0.007 |
| Ciprofloxacin | ≤0.003–0.06 | 0.007 | 0.015 |
| Fleroxacin | 0.03–0.25 | 0.06 | 0.12 |
| Ofloxacin | 0.03–0.12 | 0.06 | 0.12 |
| Tosufloxacin | 0.007–0.06 | 0.015 | 0.06 |
| Klebsiella oxytoca (18) | |||
| E-4767 | ≤0.003–0.03 | ≤0.003 | 0.015 |
| E-5065 | 0.007–0.06 | 0.007 | 0.06 |
| Ciprofloxacin | 0.007–0.12 | 0.015 | 0.12 |
| Fleroxacin | 0.06–1 | 0.06 | 0.5 |
| Ofloxacin | 0.06–1 | 0.06 | 0.5 |
| Tosufloxacin | 0.015–0.25 | 0.03 | 0.06 |
| Klebsiella pneumoniae (30) | |||
| E-4767 | ≤0.003–0.06 | 0.007 | 0.06 |
| E-5065 | ≤0.003–0.12 | 0.015 | 0.12 |
| Ciprofloxacin | 0.007–0.5 | 0.03 | 0.25 |
| Fleroxacin | 0.06–2 | 0.12 | 1 |
| Ofloxacin | 0.03–2 | 0.12 | 1 |
| Tosufloxacin | 0.007–0.5 | 0.06 | 0.5 |
| Proteus mirabilis (30) | |||
| E-4767 | ≤0.003–0.03 | 0.015 | 0.03 |
| E-5065 | 0.007–0.06 | 0.03 | 0.03 |
| Ciprofloxacin | 0.015–0.12 | 0.03 | 0.06 |
| Fleroxacin | 0.06–0.5 | 0.12 | 0.25 |
| Ofloxacin | 0.03–0.5 | 0.12 | 0.25 |
| Tosufloxacin | 0.06–0.5 | 0.12 | 0.25 |
| Proteus vulgaris (13) | |||
| E-4767 | 0.007–0.015 | 0.007 | 0.015 |
| E-5065 | 0.015–0.03 | 0.015 | 0.015 |
| Ciprofloxacin | 0.007–0.06 | 0.015 | 0.03 |
| Fleroxacin | 0.06–0.25 | 0.06 | 0.12 |
| Ofloxacin | 0.06–0.25 | 0.06 | 0.25 |
| Tosufloxacin | 0.06–0.25 | 0.12 | 0.25 |
| Providencia rettgeri (13) | |||
| E-4767 | ≤0.003–0.25 | 0.007 | 0.25 |
| E-5065 | 0.007–0.5 | 0.015 | 0.5 |
| Ciprofloxacin | 0.007–1 | 0.03 | 0.5 |
| Fleroxacin | 0.03–4 | 0.12 | 2 |
| Ofloxacin | 0.06–4 | 0.25 | 2 |
| Tosufloxacin | 0.03–2 | 0.12 | 1 |
| Providencia stuartii (15) | |||
| E-4767 | 0.03–1 | 0.5 | 0.5 |
| E-5065 | 0.06–4 | 1 | 2 |
| Ciprofloxacin | 0.25–>4 | 4 | >4 |
| Fleroxacin | 0.5–>4 | >4 | >4 |
| Ofloxacin | 1–>4 | >4 | >4 |
| Tosufloxacin | 0.12–>4 | 4 | >4 |
| Salmonella enterica serovar Typhi (27) | |||
| E-4767 | ≤0.003–0.007 | ≤0.003 | 0.007 |
| E-5065 | ≤0.003–0.015 | 0.007 | 0.007 |
| Ciprofloxacin | ≤0.003–0.12 | 0.015 | 0.03 |
| Fleroxacin | 0.03–0.12 | 0.06 | 0.12 |
| Ofloxacin | 0.015–0.12 | 0.03 | 0.12 |
| Tosufloxacin | 0.007–0.06 | 0.03 | 0.03 |
| Serratia marcescens (26) | |||
| E-4767 | ≤0.003–0.25 | 0.03 | 0.25 |
| E-5065 | 0.015–0.25 | 0.06 | 0.25 |
| Ciprofloxacin | 0.015–1 | 0.06 | 1 |
| Fleroxacin | 0.06–4 | 0.25 | 2 |
| Ofloxacin | 0.12–4 | 0.25 | 2 |
| Tosufloxacin | 0.03–4 | 0.25 | 2 |
| Pseudomonas aeruginosa (38) | |||
| E-4767 | ≤0.003–0.5 | 0.12 | 0.5 |
| E-5065 | ≤0.003–1 | 0.25 | 0.5 |
| Ciprofloxacin | ≤0.003–2 | 0.12 | 0.5 |
| Fleroxacin | 0.03–>4 | 2 | 4 |
| Ofloxacin | 0.03–>4 | 1 | 4 |
| Tosufloxacin | 0.007–2 | 0.5 | 2 |
| Bacteroides spp. (14) | |||
| E-4767 | 0.12–0.25 | 0.12 | 0.12 |
| E-5065 | 0.5–1 | 1 | 1 |
| Ciprofloxacin | 4–32 | 16 | 32 |
| Clostridium spp. (14) | |||
| E-4767 | 0.06–0.25 | 0.25 | 0.25 |
| E-5065 | 0.12–0.5 | 0.25 | 0.5 |
| Ciprofloxacin | 0.12–1 | 0.5 | 0.5 |
E-4767 and E-5065 were more potent than ciprofloxacin, fleroxacin, ofloxacin, and tosufloxacin against Enterobacteriaceae. For the enteric bacteria tested, the MIC90 of E-4767 was ≤0.06 μg/ml versus a MIC90 of ≤0.12 μg/ml for E-5065, with the exception of Serratia marcescens and Providencia spp. In all cases, MIC90s of the two new chlorofluoroquinolones were lower than those of standard reference compounds.
Regarding activity against S. marcescens isolates, E-4767 and E-5065 (MIC90, 0.25 μg/ml) were fourfold more potent than ciprofloxacin and eightfold more active than fleroxacin, ofloxacin, or tosufloxacin. The MIC90s of E-4767 and E-5065 for Providencia spp., including Providencia rettgeri and Providencia stuartii, were 0.5 and 2 μg/ml, respectively. In this case, the MIC90s of ciprofloxacin, fleroxacin, ofloxacin, and tosufloxacin for P. rettgeri were 0.5, 2, 2, and 1 μg/ml, respectively. On the other hand, the activities of marketed compounds against P. stuartii were even lower, with MIC90s of >4 μg/ml in all cases.
E-4767 and E-5065 showed potent activities against P. aeruginosa. The antibacterial activities of the two new chlorofluoroquinolones (MIC90, 0.5 μg/ml) were comparable to that of ciprofloxacin (MIC90, 0.5 μg/ml), and both compounds were more potent than fleroxacin, ofloxacin, and tosufloxacin (MIC90s of 4, 4, and 2 μg/ml, respectively).
Finally, the activities of E-4767 and E-5065 against anaerobic organisms, such as Clostridium spp. and Bacteroides spp., were compared with that of ciprofloxacin. For Clostridium spp., the MIC90s of E-4767 and E-5065 were 0.25 and 0.5 μg/ml, respectively. These activities were twofold higher than (for E-4767) and equivalent to (for E-5065) that of ciprofloxacin (MIC90, 0.5 μg/ml). However, against Bacteroides spp., the activity of ciprofloxacin (MIC90, 32 μg/ml) was as much as 256 times lower than that of E-4767 and 32 times lower than the activity of E-5065.
Factors affecting in vitro activities.
Tables 2 to 4 show the effects of medium pH, increasing magnesium concentration, and the presence of serum or urine at different pH values on the MICs of E-4767 and E-5065.
TABLE 2.
Effects of medium pH on the activity of E-4767 and E-5065
| Organism | Antibacterial agent | Value (μg/ml) at a pH of:
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 4.8
|
5.8
|
6.8
|
7.8
|
8.8
|
|||||||
| MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | ||
| Citrobacter freundii HSP-73 | E-4767 | 0.06 | 0.12 | 0.03 | 0.03 | 0.007 | 0.007 | 0.007 | 0.015 | 0.03 | 0.03 |
| E-5065 | 0.25 | 0.25 | 0.06 | 0.06 | 0.015 | 0.015 | 0.03 | 0.03 | 0.06 | 0.06 | |
| Enterobacter aerogenes HSP-145 | E-4767 | 0.12 | 0.12 | 0.06 | 0.06 | 0.015 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
| E-5065 | 0.5 | 0.5 | 0.06 | 0.06 | 0.03 | 0.03 | 0.03 | 0.06 | 0.06 | 0.06 | |
| Escherichia coli HM-42 | E-4767 | 0.06 | 0.06 | 0.03 | 0.03 | 0.003 | 0.007 | 0.003 | 0.007 | 0.003 | 0.015 |
| E-5065 | 0.12 | 0.12 | 0.06 | 0.06 | 0.015 | 0.015 | 0.03 | 0.03 | 0.06 | 0.06 | |
| Klebsiella pneumoniae HSP-30 | E-4767 | 0.12 | 0.12 | 0.06 | 0.06 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 | 0.06 |
| E-5065 | 1 | 1 | 0.12 | 0.12 | 0.03 | 0.03 | 0.06 | 0.06 | 0.06 | 0.06 | |
| Proteus vulgaris HSP-99 | E-4767 | 0.06 | 0.06 | 0.03 | 0.06 | 0.003 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
| E-5065 | 0.12 | 0.12 | 0.015 | 0.03 | 0.03 | 0.03 | 0.03 | 0.06 | |||
| Salmonella enterica serovar Enteritidis HSP-928/F | E-4767 | 0.06 | 0.06 | 0.03 | 0.03 | 0.007 | 0.007 | 0.015 | 0.015 | 0.015 | 0.03 |
| E-5065 | 0.12 | 0.12 | 0.06 | 0.06 | 0.015 | 0.015 | 0.03 | 0.06 | 0.06 | 0.06 | |
| Pseudomonas aeruginosa HS-116 | E-4767 | 0.5 | 1 | 0.5 | 0.5 | 0.25 | 0.25 | 0.25 | 0.25 | 0.5 | 1 |
| E-5065 | 0.5 | 0.5 | 0.5 | 0.5 | 0.25 | 0.25 | 0.5 | 1 | 1 | 1 | |
| Staphylococcus aureus HS-93 | E-4767 | 0.06 | 0.06 | 0.015 | 0.015 | 0.003 | 0.007 | 0.003 | 0.007 | 0.03 | 0.03 |
| E-5065 | 0.12 | 0.12 | 0.03 | 0.03 | 0.015 | 0.015 | 0.03 | 0.06 | 0.06 | 0.12 | |
| Coagulase-negative Staphylococcus sp. HSP-10 | E-4767 | 0.12 | 0.12 | 0.03 | 0.03 | 0.015 | 0.015 | 0.015 | 0.03 | 0.015 | 0.015 |
| E-5065 | 0.06 | 0.12 | 0.03 | 0.06 | 0.03 | 0.03 | 0.06 | 0.12 | |||
| Enterococcus faecalis HSP-30 | E-4767 | 0.5 | 0.5 | 0.06 | 0.06 | 0.06 | 0.06 | 0.12 | 0.12 | 0.25 | 0.25 |
| E-5065 | 2 | 2 | 0.5 | 0.5 | 0.25 | 0.25 | 0.5 | 0.5 | 0.5 | 0.5 | |
TABLE 4.
Effects of serum and urine on the activity of E-4767 and E-5065
| Organism | Antibacterial agent | Value (μg/ml) in:
|
|||||||
|---|---|---|---|---|---|---|---|---|---|
| Serum at the following % of medium:
|
Urine at a pH of:
|
||||||||
| 20
|
70
|
5.5
|
7.2
|
||||||
| MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | ||
| Citrobacter freundii HSP-73 | E-4767 | 0.015 | 0.015 | 0.03 | 0.06 | 0.12 | 0.12 | 0.03 | 0.06 |
| E-5065 | 0.03 | 0.03 | 0.03 | 0.03 | 0.25 | 0.25 | 0.06 | 0.12 | |
| Enterobacter aerogenes HSP-145 | E-4767 | 0.03 | 0.03 | 0.06 | 0.12 | 0.25 | 0.25 | 0.12 | 0.12 |
| E-5065 | 0.06 | 0.06 | 0.06 | 0.25 | 0.5 | 0.5 | 0.12 | 0.12 | |
| Escherichia coli HM-42 | E-4767 | 0.007 | 0.007 | 0.015 | 0.03 | 0.12 | 0.12 | 0.03 | 0.03 |
| E-5065 | 0.015 | 0.015 | 0.03 | 0.03 | 0.25 | 0.25 | 0.06 | 0.06 | |
| Klebsiella pneumoniae HSP-30 | E-4767 | 0.03 | 0.03 | 0.12 | 0.12 | 0.5 | 1 | 0.06 | 0.06 |
| E-5065 | 0.06 | 0.06 | 0.06 | 0.12 | 0.25 | 0.5 | 0.06 | 0.12 | |
| Proteus vulgaris HSP-99 | E-4767 | 0.03 | 0.03 | 0.03 | 0.03 | >8 | >8 | ||
| E-5065 | 0.03 | 0.03 | 0.03 | 0.03 | >8 | >8 | |||
| Salmonella enterica serovar Enteritidis HSP-928/F | E-4767 | 0.015 | 0.015 | 0.03 | 0.03 | 0.12 | 0.12 | 0.03 | 0.03 |
| E-5065 | 0.03 | 0.03 | 0.03 | 0.06 | 0.25 | 0.25 | 0.06 | 0.06 | |
| Pseudomonas aeruginosa HS-116 | E-4767 | 0.25 | 0.5 | 1 | 1 | 2 | 2 | 1 | 1 |
| E-5065 | 0.5 | 1 | 1 | 2 | 1 | 4 | 0.5 | 2 | |
| Staphylococcus aureus HS-93 | E-4767 | 0.015 | 0.015 | 0.015 | 0.03 | 0.06 | 0.06 | ||
| E-5065 | 0.03 | 0.06 | 0.06 | 0.12 | 0.12 | 0.25 | |||
| Coagulase-negative Staphylococcus sp. HSP-10 | E-4767 | 0.03 | 0.03 | 0.03 | 0.06 | 0.03 | 0.03 | 0.03 | 0.06 |
| E-5065 | 0.06 | 0.06 | 0.12 | 0.25 | 0.06 | 0.12 | 0.12 | 0.12 | |
| Enterococcus faecalis HSP-30 | E-4767 | 0.06 | 0.06 | 0.06 | 0.25 | 0.25 | 0.5 | 0.12 | 0.25 |
| E-5065 | 0.25 | 0.25 | 1 | 1 | 1 | 1 | 0.5 | 0.5 | |
(i) Effects of medium pH.
The effects of pH on the activities of E-4767 and E-5065 are shown in Table 2. At pH 4.8, the MICs of E-4767 for the isolates tested were 4- to 16-fold higher than at pH 6.8, with the exception of the MIC for P. aeruginosa HS-116, which increased only 2-fold. At pH 5.8, 7.8, and 8.8, the MICs of E-4767 were in general two- to fourfold higher than at pH 6.8. Compared with the MIC of E-4767 at pH 6.8, the MICs at pH 5.8, 7.8, and 8.8 for P. vulgaris HSP-99 and the MIC at pH 5.8 for E. coli HM-42 increased eightfold. The MICs of E-5065 for the strains tested were generally between two- and eightfold higher at pH 5.8 and two- to fourfold higher at pH 7.8 and 8.8 than the MICs at pH 6.8. At pH 4.8, the MICs of E-5065 increased 8- to 16-fold, with the exception of coagulase-negative Staphylococcus sp. HSP-10, for which the increase was only 2-fold, and K. pneumoniae HSP-30, for which the MIC at pH 5.8 was more than 128-fold higher than at pH 6.8.
(ii) Effects of magnesium ion concentration.
Table 3 shows the effects of increasing the concentration of Mg2+ upon the MICs of E-4767 and E-5065. MICs of both compounds in MH broth supplemented with 1 mM Mg2+ remained unchanged, with the exception of the E-4767 MICs for P. vulgaris HSP-99 and P. aeruginosa HS-116 and the E-5065 MICs for C. freundii HSP-73 and coagulase-negative Staphylococcus sp. HSP-10. In the case of medium supplemented with 5 or 10 mM Mg2+, MICs increased two- to eightfold. In general, there was no difference between MICs and MBCs.
TABLE 3.
Effects of Mg2+ on the activity of E-4767 and E-5065
| Organism | Antibacterial agent | Value (μg/ml) in:
|
|||||||
|---|---|---|---|---|---|---|---|---|---|
| MH broth alone
|
MH broth containing Mg2+ at a concn (mM) of:
|
||||||||
| 1
|
5
|
10
|
|||||||
| MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | ||
| Citrobacter freundii HSP-73 | E-4767 | 0.007 | 0.007 | 0.007 | 0.007 | 0.03 | 0.03 | 0.015 | 0.015 |
| E-5065 | 0.015 | 0.015 | 0.03 | 0.03 | 0.06 | 0.06 | 0.06 | 0.06 | |
| Enterobacter aerogenes HSP-145 | E-4767 | 0.015 | 0.03 | 0.007 | 0.015 | 0.06 | 0.06 | 0.06 | 0.06 |
| E-5065 | 0.03 | 0.03 | 0.03 | 0.03 | 0.12 | 0.12 | 0.06 | 0.06 | |
| Escherichia coli HM-42 | E-4767 | 0.003 | 0.007 | 0.003 | 0.007 | 0.015 | 0.015 | 0.007 | 0.007 |
| E-5065 | 0.015 | 0.015 | 0.015 | 0.015 | 0.03 | 0.06 | 0.03 | 0.03 | |
| Klebsiella pneumoniae HSP-30 | E-4767 | 0.03 | 0.03 | 0.007 | 0.015 | 0.03 | 0.03 | 0.015 | 0.03 |
| E-5065 | 0.03 | 0.03 | 0.03 | 0.03 | 0.06 | 0.06 | 0.03 | 0.06 | |
| Proteus vulgaris HSP-99 | E-4767 | 0.003 | 0.03 | 0.015 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
| E-5065 | 0.015 | 0.03 | 0.015 | 0.015 | 0.03 | 0.06 | 0.03 | 0.06 | |
| Salmonella enterica serovar Enteritidis HSP-928/F | E-4767 | 0.007 | 0.007 | 0.007 | 0.007 | 0.03 | 0.03 | 0.015 | 0.03 |
| E-5065 | 0.015 | 0.015 | 0.015 | 0.015 | 0.06 | 0.06 | 0.03 | 0.03 | |
| Pseudomonas aeruginosa HS-116 | E-4767 | 0.25 | 0.25 | 0.12 | 0.5 | 0.5 | 1 | 0.25 | 1 |
| E-5065 | 0.25 | 0.25 | 0.25 | 0.5 | 0.5 | 1 | 0.5 | 0.5 | |
| Staphylococcus aureus HS-93 | E-4767 | 0.003 | 0.007 | 0.003 | 0.007 | 0.015 | 0.015 | 0.007 | 0.015 |
| E-5065 | 0.015 | 0.015 | 0.007 | 0.015 | 0.06 | 0.06 | 0.015 | 0.03 | |
| Coagulase-negative Staphylococcus sp. HSP-10 | E-4767 | 0.015 | 0.015 | 0.015 | 0.015 | 0.03 | 0.03 | 0.03 | 0.03 |
| E-5065 | 0.03 | 0.03 | 0.06 | 0.06 | 0.12 | 0.12 | 0.06 | 0.06 | |
| Enterococcus faecalis HSP-30 | E-4767 | 0.06 | 0.06 | 0.06 | 0.06 | 0.12 | 0.25 | 0.06 | 0.06 |
| E-5065 | 0.12 | 0.12 | 0.12 | 0.12 | 0.5 | 0.5 | 0.12 | 0.12 | |
(iii) Effects of serum.
In medium containing 20 or 70% horse serum, E-4767 and E-5065 were two to four times less active than in broth against most species tested (Table 4). However, for P. vulgaris HSP-99, the MIC of E-4767 increased eightfold in the presence of 20% serum. On the other hand, for K. pneumoniae HSP-30, P. aeruginosa HS-116, and E. faecalis HSP-30, the MICs of E-4767 were not affected by the presence of 20% serum. Finally, the MICs of E-5065 for E. coli HM-42 and E. faecalis HSP-30 were likewise not affected by the presence of 20% serum.
(iv) Effects of urine.
The effects of urine on the in vitro activity of E-4767 and E-5065 are shown in Table 4. In general, the activity of E-4767 decreased two- to eightfold when tested in fresh urine at pH 7.2, with the exception of E. faecalis HSP-30, for which the MIC of E-5065 was more than 16-fold higher than in MH broth. Against P. vulgaris HSP-99, E-4767 and E-5065 were not active in fresh urine at pH 7.2. At pH 5.5, E-4767 and E-5065 were 8 to 32 times less active than in broth, except against P. vulgaris HSP-99 and coagulase-negative Staphylococcus sp. HSP-10. The MIC of E-4767 for E. faecalis HSP-30 showed a twofold increase compared to that obtained in urine with a pH of 7.2.
Pharmacokinetics in mice.
The time courses of drug levels in serum for E-4767, E-5065, and ciprofloxacin in mice after oral administration (dose, 50 mg/kg) are shown in Table 5.
TABLE 5.
Pharmacokinetics of E-4767, E-5065, and ciprofloxacin in mice
| Antibacterial agenta | Concn (μg/ml) in serum atb:
|
AUC (μg · h/ml)c | |||
|---|---|---|---|---|---|
| 30 min | 60 min | 120 min | 240 min | ||
| E-4767 | 5.7 ± 1.7 | 2.0 ± 1.2 | 0.4 ± 0.1 | 0.3 ± 0.1 | 3.8 |
| E-5065 | 6.2 ± 3.0 | 3.8 ± 2.3 | 1.3 ± 0.7 | 0.6 ± 0.1 | 8.5 |
| Ciprofloxacin | 2.3 ± 2.0 | 1.2 ± 0.1 | 0.5 ± 0.3 | ND | 2.3 |
Compounds were administered at a dose of 50 mg/kg of body weight once orally.
Values are expressed as means ± standard deviations. ND, not detectable (<0.05 μg/ml).
AUC was determined from mean concentrations.
Absorption of E-4767 and E-5065 was very rapid, with peak levels in serum of 5.7 ± 2 and 6.2 ± 3 μg/ml, respectively, reached within 30 min. The concentration of ciprofloxacin at this time was 2.3 ± 2 μg/ml. The AUC0–4 of ciprofloxacin was two times lower than that of E-4767 and four times lower than that of E-5065.
Antibacterial efficacy in vivo.
Table 6 shows the therapeutic efficacies of E-4767 and E-5065 compared with that of ciprofloxacin against lethal systemic infections in mice caused by selected gram-positive and -negative pathogens, such as E. coli HM-42, P. aeruginosa HSP-116, Cipr P. aeruginosa B-464, S. aureus HS-93, Cipr S. aureus 73777, Cipr S. aureus 73940, S. pneumoniae 1625, S. pneumoniae 84551, and S. pneumoniae 1752. As can be seen, several Cipr strains have been included in the in vivo evaluation panel to assess the ability of the two new chlorofluoroquinolones to overcome resistance mechanisms.
TABLE 6.
In vivo activities of E-4767, E-5065, and ciprofloxacin against systemic infections in mice
| Organism | Challenge dose (CFU/mouse)a | Test compoundb | MIC (μg/ml) | MBC (μg/ml) | ED50 (mg/kg)c | 95% confidence intervald |
|---|---|---|---|---|---|---|
| Escherichia coli HM-42 | 3.3 × 108 | E-4767 | 0.004 | 0.008 | 3.34 | 3.12–3.57 |
| E-5065 | 0.008 | 0.015 | 2.72 | 2.17–3.14 | ||
| Ciprofloxacin | 0.004 | 0.008 | 7.49 | 6.51–8.56 | ||
| Pseudomonas aeruginosa HS-116 | 9.0 × 108 | E-4767 | 0.12 | 0.12 | 31.49 | 21.36–45.39 |
| E-5065 | 0.5 | 0.5 | 83.8 | 61.44–121.3 | ||
| Ciprofloxacin | 0.12 | 0.12 | 100.23 | 31.02–261.40 | ||
| CiprPseudomonas aeruginosa B-464 | 2.0 × 108 | E-4767 | 1 | 2 | 94.96 | 82.05–110.54 |
| E-5065 | 1 | 2 | 99.27 | 88.57–112.23 | ||
| Ciprofloxacin | 8 | 8 | >200 | |||
| Staphylococcus aureus HS-93 | 8.0 × 109 | E-4767 | 0.008 | 0.008 | 1.57 | 0.08–3.16 |
| E-5065 | 0.06 | 0.12 | 21.31 | 14.71–29.51 | ||
| Ciprofloxacin | 0.12 | 0.12 | 41.71 | 16.82–114.31 | ||
| CiprStaphylococcus aureus 73777 | 6.0 × 109 | E-4767 | 0.25 | 0.25 | 47.48 | 39.08–62.42 |
| E-5065 | 1 | 1 | 184.61 | 126.29–345.12 | ||
| Ciprofloxacin | 16 | 16 | >200 | |||
| CiprStaphylococcus aureus 73940 | 3.0 × 109 | E-4767 | 0.25 | 0.25 | 30.89 | 25.89–36.61 |
| E-5065 | 1 | 1 | 111.27 | 84.55–165.89 | ||
| Ciprofloxacin | 16 | 32 | >200 | |||
| Streptococcus pneumoniae 1625 | 2.8 × 107 | E-4767 | 0.003 | 0.006 | 7.15 | 4.01–9.22 |
| E-5065 | 0.03 | 0.03 | 45.47 | 40.41–50.69 | ||
| Ciprofloxacin | 0.25 | 0.25 | 70.71 | 61.89–80.79 | ||
| Streptococcus pneumoniae 84551 | 1.9 × 103 | E-4767 | 0.03 | 0.03 | 15.23 | 13.40–16.93 |
| E-5065 | 0.25 | 0.25 | 50.93 | 25.77–84.65 | ||
| Ciprofloxacin | 1 | 1 | >200 | |||
| Streptococcus pneumoniae 1752 | 6.0 × 107 | E-4767 | 0.007 | 0.015 | 16.92 | 9.91–20.49 |
| E-5065 | 0.06 | 0.06 | 80.99 | 75.44–86.94 | ||
| Ciprofloxacin | 0.25 | 0.5 | >200 |
Mice were inoculated intraperitoneally with 0.5 ml of bacterial suspension, i.e., approximately five times the minimal lethal dose.
Mice infected with S. aureus HS-93, E. coli HM-42, and P. aeruginosa HS-116 were given a single oral dose immediately after bacterial challenge. Mice infected with S. pneumoniae were given four consecutive oral doses at 0, 6, 12, and 24 h postinfection. Mice infected with Cipr bacterial strains were given two consecutive oral doses at 0 and 6 h postinfection.
ED50s were calculated by probit analysis (15).
95% confidence intervals were calculated by the method of Litchfield and Wilcoxon (14).
All compounds showed a consistently high protective effect against lethal systemic infections produced by E. coli HM-42. However, the capacity of E-4767 and E-5065 to protect mice infected with this strain (ED50s of 3.3 and 2.7 mg/kg, respectively) was approximately twofold higher than that of ciprofloxacin (ED50, 7.5 mg/kg).
The two new fluoroquinolones demonstrated similar protective effects against experimental infections produced by P. aeruginosa. Against P. aeruginosa HS-116 infection, the ED50s of ciprofloxacin, E-4767, and E-5065 were 100.2, 31.49, and 83.8 mg/kg. Against Cipr P. aeruginosa strain B-464 (ED50 of ciprofloxacin, >200 mg/kg), the ED50s of E-4767 and E-5065 were 94.9 and 99.3 mg/kg, respectively.
Regarding gram-positive pathogens, E-4767 was clearly the most effective compound. Against S. aureus HS-93, the ED50 of E-4767 was 1.6 mg/kg, i.e., 25 times lower than the ED50 of ciprofloxacin. E-5065 demonstrated a therapeutic efficacy similar to that of ciprofloxacin in this infection model. Against Cipr S. aureus strains, both new chlorofluoroquinolones showed in vivo protective activity. The efficacy of E-4767 in protecting mice infected with S. aureus 73777 or S. aureus 73940 (ED50s of 47.5 and 30.9 mg/kg, respectively) was between three- and fourfold greater than that of E-5065 (ED50s of 184.6 and 111.3 mg/kg, respectively).
Against infections produced by S. pneumoniae strains, the efficacies of E-4767 and E-5065 were also much greater than that of ciprofloxacin. The ED50s of E-4767 for infections produced by S. pneumoniae strains ranged from 7.2 to 16.9 mg/kg. For E-5065, this range was 45.5 to 81 mg/kg. The lower protective effect of ciprofloxacin against S. pneumoniae infection was reflected by an ED50 range for the strains tested of 70.7 to >200 mg/kg.
DISCUSSION
Fluoroquinolones have proven to be a very useful therapeutic class of agents for the treatment of infectious diseases, including those of the respiratory tract, urinary tract, skin and soft tissues, and even those that are transmitted sexually. Marketed compounds, such as ciprofloxacin, ofloxacin, levofloxacin, and others, have been extensively used because of their potent activity against gram-negative organisms. However, their activities are not optimal against certain gram-positive pathogens, such as staphylococci and streptococci, or against anaerobes. This situation has pointed out the need to develop new compounds capable of overcoming these drawbacks.
In general, fluoroquinolones with C-7 azetidine-1-yl substituents seem to improve potency against gram-positive and anaerobic organisms (6, 8, 9). On the other hand, C-8 fluoro- or chloro- derivatives are more effective in vivo than their quinolone counterparts with no substitution at the C-8 position (4).
E-4767 and E-5065 are two new 8-chlorofluoroquinolones with a 7-azetidin ring substituent, which show increased in vitro activity and in vivo efficacy not only against gram-positive but also against gram-negative organisms.
Against staphylococci, streptococci, and enterococci, including coagulase-negative and Metr Staphylococcus spp. strains, E-4767 was 2- to 8-fold more potent in vitro than E-5065 but 4- to 32-fold and 8- to 64-fold more potent than tosufloxacin and ciprofloxacin, respectively. E-4767 and E-5065 were active against Cipr S. aureus strains, with MIC90s of 0.25 and 2 μg/ml, respectively. The MIC50s of ciprofloxacin, fleroxacin, ofloxacin, and tosufloxacin for resistant strains were all >4 μg/ml. In addition, the two new 8-chlorofluoroquinolones demonstrated excellent activity against anaerobes. E-4767 showed twofold-greater potency than E-5065 and ciprofloxacin against isolates of Clostridium spp. Against Bacteroides spp., E-5065 was 32-fold more potent than ciprofloxacin but 8-fold less potent than E-4767. Both E-4767 and E-5065 have demonstrated activity against all members of the Enterobacteriaceae family tested, including S. marcescens and P. rettgeri, with MIC90s of 0.25 and ≤0.5 μg/ml. Against enteric bacteria, E-4767 was about twice as potent as E-5065. Furthermore, against this group of organisms, E-4767 showed 2- to 16-fold-greater in vitro activity than ciprofloxacin and 4- to 32-fold-greater activity than tosufloxacin. Against P. aeruginosa, the activities of E-4767 and E-5065 were comparable to that of ciprofloxacin, fourfold higher than that of tosufloxacin, and eightfold higher than those of ofloxacin and fleroxacin.
As has been reported for most quinolones, E-4767 and E-5065 exhibited substantial reductions in activity when the medium pH decreased to under 4.8. The documented antagonistic effect of magnesium ions on quinolone antimicrobial activity was also observed with E-4767 and E-5065 in the presence of 5 or 10 mM Mg2+. The in vitro activities of E-4767 and E-5065 were lowered by the presence of horse serum or human urine at pH 7.2, although the activities were only reduced two- to fourfold compared to those observed in broth. Bacteriostatic and bactericidal activities of both drugs were usually achieved at similar compound concentrations.
In general, E-4767 and E-5065 have demonstrated excellent in vitro properties against a broad range of pathogenic bacteria. However, it is well known that the final outcome of any anti-infective treatment is a consequence of the in vitro activity and pharmacokinetic properties. This is the reason why recent efforts have focused on the development of new compounds not only with improved in vitro activities against gram-positive and anaerobic organisms but also with improved pharmacokinetic performance. Preliminary pharmacokinetic studies with E-4767 and E-5065 showed that after a single oral administration of 50 mg/kg, both compounds reached significantly higher concentrations in mouse serum than did ciprofloxacin. The latter was selected as the reference compound for in vivo studies since it is the most widely used of the marketed quinolones. The pharmacokinetic results showed E-4767 and E-5065 to be rapidly absorbed, reaching concentrations in serum of 5.7 and 6.2 μg/ml, respectively, within 30 min, compared to 2.3 μg/ml of ciprofloxacin in this same amount of time. The AUC was also greater than that obtained with ciprofloxacin. It is well known that compounds having a C-7 azetidine-1-yl substituent achieve significantly higher concentrations in serum and consequently show increased therapeutic efficacy in protection tests (6, 14).
E-4767 and E-5065 were effective in the treatment of a variety of experimental infections in mice, including those produced by gram-positive and -negative pathogens. The two new chlorofluoroquinolones were similar to or more effective than ciprofloxacin against all experimental infections studied. Against infections produced by gram-positive pathogens such as S. aureus and S. pneumoniae, E-4767 was between 3 and 14 times more effective than E-5065 and up to 25-fold more effective than ciprofloxacin. E-4767 and E-5065 were also effective in protecting mice infected with E. coli or P. aeruginosa strains. The protective effects shown by E-4767 and E-5065 against gram-positive cocci may be related to their higher in vitro activities and perhaps better pharmacokinetic properties.
In conclusion, E-4767 and E-5065 are new broad-spectrum compounds with potent in vitro activities against gram-negative organisms, gram-positive cocci, and anaerobes. Both drugs exhibit greater therapeutic efficacies in murine experimental infection models involving gram-positive and -negative organisms than ciprofloxacin, possibly because of their better in vitro activities against gram-positive cocci linked to their improved pharmacokinetic properties, these being key factors for enhancing in vivo drug efficacy.
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