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Antimicrobial Agents and Chemotherapy, January 2008, p. 65-76, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.00853-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vitro and In Vivo Antibacterial Activities of DC-159a, a New Fluoroquinolone{triangledown}

Kazuki Hoshino,* Kazue Inoue, Yoichi Murakami, Yuichi Kurosaka, Kenji Namba, Yoshinori Kashimoto, Saori Uoyama, Ryo Okumura, Saito Higuchi, and Tsuyoshi Otani

Biological Research Laboratories IV, Daiichi Sankyo Co., Ltd., 16-13, 1-Chome Kitakasai, Edogawa-ku, Tokyo 134-8630

Received 29 June 2007/ Returned for modification 27 July 2007/ Accepted 21 September 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DC-159a is a new 8-methoxy fluoroquinolone that possesses a broad spectrum of antibacterial activity, with extended activity against gram-positive pathogens, especially streptococci and staphylococci from patients with community-acquired infections. DC-159a showed activity against Streptococcus spp. (MIC90, 0.12 µg/ml) and inhibited the growth of 90% of levofloxacin-intermediate and -resistant strains at 1 µg/ml. The MIC90s of DC-159a against Staphylococcus spp. were 0.5 µg/ml or less. Against quinolone- and methicillin-resistant Staphylococcus aureus strains, however, the MIC90 of DC-159a was 8 µg/ml. DC-159a was the most active against Enterococcus spp. (MIC90, 4 to 8 µg/ml) and was more active than the marketed fluoroquinolones, such as levofloxacin, ciprofloxacin, and moxifloxacin. The MIC90s of DC-159a against Haemophilus influenzae, Moraxella catarrhalis, and Klebsiella pneumoniae were 0.015, 0.06, and 0.25 µg/ml, respectively. The activity of DC-159a against Mycoplasma pneumoniae was eightfold more potent than that of levofloxacin. The MICs of DC-159a against Chlamydophila pneumoniae were comparable to those of moxifloxacin, and DC-159a was more potent than levofloxacin. The MIC90s of DC-159a against Peptostreptococcus spp., Clostridium difficile, and Bacteroides fragilis were 0.5, 4, and 2 µg/ml, respectively; and among the quinolones tested it showed the highest level of activity against anaerobic organisms. DC-159a demonstrated rapid bactericidal activity against quinolone-resistant Streptococcus pneumoniae strains both in vitro and in vivo. In vitro, DC-159a showed faster killing than moxifloxacin and garenoxacin. The bactericidal activity of DC-159a in a murine muscle infection model was revealed to be superior to that of moxifloxacin. These activities carried over to the in vivo efficacy in the murine pneumonia model, in which treatment with DC-159a led to bactericidal activity superior to those of the other agents tested.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Multidrug-resistant Streptococcus pneumoniae and community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are emerging as key pathogens in community-acquired infections (4, 6). Vaccination against pneumococcal disease has proved effective in preventing S. pneumoniae respiratory tract infections; however, resistant variants of nonvaccine serotypes have been reported, and vaccination itself is not completely effective (8, 14). Quinolone antibacterials are beneficial for the empirical treatment of respiratory infections in the community because of their extended antibacterial spectra, including atypical pathogens, with preferable pharmacokinetic (PK) and safety profiles (30); however, several cases of treatment failure because of quinolone resistance-conferring mutations have been reported (5). Several risk factors existed in patients who were infected with quinolone-resistant mutants (11, 13, 29). It is well known that quinolone resistance was acquired in a stepwise fashion, and therefore, not only treatment for highly resistant variants but also prevention of the selection of first-step mutations should be considered in controlling resistance (21). On the other hand, CA-MRSA strains, unlike nosocomial strains, are presently sensitive to several antibiotics, but the development of resistance, including resistance to quinolones, is likely to emerge under increasing selective pressure (3, 15, 22, 26). Thus, the need to develop agents effective for the aggressive treatment of common community-acquired infections, especially those caused by resistant pneumococci and staphylococci, remains. While the newer quinolones, moxifloxacin and gemifloxacin, have improved activities, their MICs against quinolone-resistant S. pneumoniae strains are often above the breakpoint. Consequently, a quinolone with robust activity against quinolone-resistant pathogens would provide substantial benefit in the empirical treatment of these infections. In this study, we assessed the in vitro and in vivo pharmacological activities of DC-159a, (+)-7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid hemihydrate (Fig. 1).


Figure 1
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  		FIG. 1. Chemical structure of DC-159a.

 
(This work was presented in part at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA [8a].)


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antimicrobial agents. The antibacterial agents used in these studies were obtained from the following sources. DC-159a (1/2 H2O form of DC-159; in this study, another salt form, 0.75 ethanol·0.25 H2O was used in the in vitro time-kill study and in vivo bactericidal study), levofloxacin (LVFX), ciprofloxacin (CPFX), moxifloxacin (MFLX), garenoxacin (GRNX), gemifloxacin (GMFX), azithromycin (AZM), clarithromycin (CAM), and telithromycin (TEL) were synthesized at Daiichi Pharmaceuticals, Co., Ltd., Tokyo, Japan. Metronidazole was purchased from Sigma Aldrich Japan (Tokyo, Japan). Each drug was used as the anhydrous free-base equivalent.

Bacterial strains. Bacterial strains were collected by the LVFX Surveillance Group from patients in Japan in 2002 (28) or were obtained from BML, Inc. (Saitama, Japan); BCL, Inc. (Tokyo, Japan); or ATCC. LVFX-intermediate and -resistant S. pneumoniae strains were from the GLOBAL Surveillance 2003 program.

Antimicrobial susceptibility testing. MICs were determined according to the standard agar dilution method recommended by the Clinical and Laboratory Standards Institute (formerly NCCLS) (17) for bacterial species other than Haemophilus influenzae and anaerobes, for which the agar dilution method recommended by the Japanese Society of Chemotherapy was used (10). Mueller-Hinton agar (MHA; Difco, Becton, Dickinson and Company [BD], Sparks, MD) supplemented with 5% sheep blood was used for streptococci (other than LVFX-intermediate and -resistant strains) and Moraxella catarrhalis, and GC agar (Difco, BD) with 1% supplement B was used for Neisseria gonorrhoeae. MHA supplemented with 5% Fildes enrichment was used for H. influenzae, and modified Gifu anaerobe medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) agar was used for anaerobic bacteria. Buffered charcoal yeast extract alpha agar (Eiken Chemical Co., Ltd., Tokyo, Japan) was used for preculture, and buffered starch-yeast extract agar without charcoal was used for MIC determination for Legionella pneumophila (20). SP-4 broth (25) was used for preculture and MIC determination for Mycoplasma pneumoniae, and sucrose-phosphate-glutamic acid medium was used for Chlamydophila pneumoniae and Chlamydia trachomatis (9). Drug-containing agar plates were incubated with 1 loopful (5 µl) of an inoculum corresponding to about 104 CFU per spot for aerobic bacteria and 105 CFU per spot for anaerobic bacteria. The inoculated agar plates were incubated at 35°C for 18 h. N. gonorrhoeae was incubated under 10% CO2. L. pneumophila and M. pneumoniae were cultivated for 96 h and 14 days, respectively. The MIC was defined as the lowest drug concentration that prevented the visible growth of the bacteria. MIC testing for anaerobic organisms was performed with an AnaeroPack-Anaero (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan). The quality control strains recommended by the Clinical and Laboratory Standards Institute were included as internal controls throughout the study.

Multistep resistance selection. Three strains each of S. pneumoniae, Streptococcus pyogenes, S. aureus, and H. influenzae randomly selected from stock clinical isolates were used. Fourteen days of serial passage with DC-159a, CPFX, LVFX, or MFLX was performed as follows. A suspension of the strains selected from an overnight agar culture was prepared to produce a 0.5 McFarland standard (1 x 108 to 2 x 108 CFU/ml) in broth and was then diluted further by adding 100 µl to 10 ml of test broth (1:100 dilution). This inoculum was then added to each tube of the antimicrobial dilution series (1 ml) plus an antimicrobial-free control tube to produce a final inoculum of 5 x 105 CFU/ml. The tubes were incubated for 20 to 24 h at 35 ± 1°C in air, and the MIC was recorded as the lowest concentration of antimicrobial that completely inhibited visible macroscopic growth (passage 0). For passage 1, 100 µl of the tube with 0.5x MIC for each antimicrobial dilution series produced from passage 0 was transferred to separate 100-ml volumes of fresh broth medium. To perform the passage, 0.5 ml of each newly prepared inoculum (from the previous day's tube with 0.5x MIC) was transferred to a fresh antimicrobial dilution series. These steps were repeated from day 2 until 14 passages were completed. The MIC was confirmed by using the culture from passage 14 for all isolates that produced a rise in the MIC during passage. Mueller-Hinton broth was used as the basal medium for S. aureus, brain heart infusion broth was used for S. pneumoniae and S. pyogenes, and Haemophilus test medium broth was used for Haemophilus influenzae.

In vitro time-kill study. S. pneumoniae 1026523 (ParC, Ser79Phe) and 104835 (GyrA, Ser81Phe; ParC, Ser79Phe) were cultured overnight at 35°C on MHA with 5% horse blood. Colonies precultured on the agar plates were suspended in 2 ml of cation-adjusted Mueller-Hinton broth (BBL, BD) to adjust the turbidity optically so that it was comparable to a 0.5 McFarland standard (BBL, BD). To obtain a bacterial suspension of approximately 106 CFU/ml, 0.1 ml of the 0.5 McFarland standard suspension was transferred into a glass bottle; and the bottle, which contained 100 ml of cation-adjusted Mueller-Hinton broth with 2% lysed horse blood, was sealed with a silicone rubber plug and incubated for 3 h at 37°C with shaking. In order to examine the effects of the drugs on bacterial growth, the bacterial suspensions were grown at 37°C in an L-type test tube containing 10 ml of the medium without drug (growth control) or with the drugs under shaking conditions. Samples were removed at time intervals of 0, 0.5, 1, 2, 4, 8, and 24 h of exposure. The number of viable cells was determined by the spiral plating technique with an Eddy Jet apparatus (IUL, S.A., Barcelona, Spain). The plates were incubated at 35°C overnight, and the numbers of colonies were counted.

In vivo efficacy against experimental infections. Five- to 6-week-old female Crj:CD-1 (ICR) mice and 3-week-old male CBA/JNCrlj mice (Charles River Japan, Inc., Kanagawa, Japan) were used for the neutropenic muscle infection model for the in vivo bactericidal study and for the pneumococcal pneumonia model, respectively. They were maintained in animal rooms maintained at 23 ± 2°C with 55% ± 20% relative humidity. All experimental procedures with the animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Daiichi Pharmaceutical Co. Ltd.

(i) In vivo bactericidal study. Macrolide-resistant S. pneumoniae J24 was used as the challenge organism. The organism was suspended in 10% skim milk, seeded in Todd-Hewitt broth (Becton Dickinson, Sparks, MD) at a volume of 5 ml and was cultured anaerobically in a biochamber (BCP-120F; Tietech Co., Ltd., Nagoya, Japan) for 9 h at 35°C. The culture was centrifuged in a centrifugal separator at 10,000 rpm for 10 min at 4°C. The supernatant was removed, and the precipitate was resuspended in Mueller-Hinton broth at a volume of 5 ml; this suspension was used as the inoculum. The mice were rendered neutropenic by injecting cyclophosphamide intraperitoneally 4 days (150 mg/kg of body weight) and 1 day (100 mg/kg) before infection. The mice were used in groups of 8 mice each for the therapeutic groups and 12 mice for the control group and were anesthetized with an intramuscular injection of a mixture of ketamine hydrochloride (Ketalar; 50 mg/ml; Sankyo Co., Ltd., Tokyo, Japan), xylazine hydrochloride (Selactar; 20 mg/ml; Bayer Ltd., Tokyo, Japan), and distilled water for injection at a ratio of 2:1:4 at a volume of 70 µl/mouse. Then, the inoculum was intramuscularly inoculated into the calf at a volume of 40 µl/mouse. Therapy (subcutaneous dosing) with DC-159a or MFLX was initiated 2 h after inoculation. The mice were exsanguinated by cutting the axillary artery and vein while the mice were under anesthesia with diethyl ether (Kishida Chemical Co., Ltd., Osaka, Japan). Each calf muscle was removed aseptically and then homogenized, and the resultant homogenate was used as the original bacterial suspension. The number of colonies per muscle was calculated. The detection limit was ≥1.48 log10 CFU/muscle. For statistical comparisons, culture-negative samples were considered to contain 1.48 log10 CFU/muscle.

For the PK analysis, DC-159a or MFLX at a dose of 32 mg/kg each was subcutaneously injected into the infected mice. Blood and calf tissue samples were obtained from three mice each at various time intervals (0.25, 0.5, 1, 2, 4, and 6 h) after administration of the drugs tested.

(ii) Pneumococcal pneumonia in mice. Experimental pneumonia was induced in CBA/JNCrj mice by a slight modification of the method reported by Tateda et al. (24). S. pneumoniae 104835 suspended in saline was intranasally inoculated into the mice, which were anesthetized with the same mixture of ketamine and xylazine described above, at a volume of 50 µl/mouse. One day after the infection, the animals (in groups of four mice each) were treated with DC-159a, MFLX, or GRNX subcutaneously twice a day at a dose of 50, 100, or 200 mg/kg/day for 3 consecutive days. The number of bacteria in the lungs was examined on day 4 after inoculation, the day following the final administration of drug. The lungs were removed aseptically and weighed, and then the viable bacterial counts were determined. The detection limit was ≥2.30 log10 CFU/g of lung. For statistical comparisons, culture-negative samples were considered to contain 2.30 log10 CFU/g of lung.

For the PK analysis, DC-159a, MFLX, or GRNX at a dose of 15 mg/kg each was subcutaneously injected into the infected mice. Blood and lung tissue samples were obtained from three mice each at various time intervals (0.08, 0.25, 0.5, 1, 2, 4, and 6 h) after administration of the drugs tested.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The antibacterial activities of DC-159a and the comparator drugs against gram-positive and -negative bacteria are shown in Table 1.


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  		TABLE 1. Antibacterial activities of DC-159a

 
The MIC90 of DC-159a against methicillin-susceptible Staphylococcus aureus (MSSA) and quinolone-susceptible MRSA was 0.06 µg/ml. Against MSSA, DC-159a was 32-fold more active than CPFX, 8-fold more active than LVFX, 2-fold more active than MFLX and GMFX, and 2-fold less active than GRNX at the MIC90 level. Against quinolone-resistant MRSA, DC-159a (MIC90, 8 µg/ml) showed the highest activity compared with the activities of the other quinolones tested.

The MIC90 of DC-159a against methicillin-susceptible coagulase-negative staphylococci and methicillin-resistant coagulase-negative staphylococci was 0.5 µg/ml. Against methicillin-resistant coagulase-negative staphylococci, DC-159a showed the highest activity compared with the activities of the other quinolones tested.

Against S. pneumoniae, including penicillin-resistant and/or macrolide-resistant strains, the MIC90 of DC-159a was 0.12 µg/ml. DC-159a was 8- to 16-fold more active than LVFX, was 16- to 32-fold more active than CPFX, was 2-fold more active than MFLX, had activity comparable to that of GRNX, and had activity comparable to or 2- to 4-fold less than that of GMFX. DC-159a was two- to fourfold more active than TEL. The MIC90 values for AZM and CAM against S. pneumoniae were more than 64 and 32 µg/ml, respectively. The MIC90 of DC-159a against quinolone-resistant isolates was 1 µg/ml. DC-159a was as active as GRNX, 4-fold more active than MFLX, 32-fold more active than LVFX, 64-fold more active than CPFX, and 2-fold less active than GMFX.

The MIC90 of DC-159a for Streptococcus pyogenes was 0.12 µg/ml; it was eightfold more active than LVFX, was twofold more active than MFLX, and had activity comparable to the activities of GRNX and GMFX.

The MICs of DC-159a against a small number of quinolone-resistant S. pyogenes strains (four strains) were examined. DC-159a showed MICs that ranged from 0.5 to 1 µg/ml, which was 16-fold more active than LVFX and 4-fold more active than MFLX (data not shown).

The MIC90s of DC-159a against Enterococcus faecalis and Enterococcus faecium were 4 and 8 µg/ml, respectively. DC-159a was 8-fold more active than LVFX and 8- to 16-fold more active than CPFX against Enterococcus spp.

The MIC90s of DC-159a against important causative pathogens in respiratory tract infections, such as H. influenzae, M. catarrhalis, and Klebsiella pneumoniae, were 0.015, 0.06, and 0.25 µg/ml, respectively. The activity of DC-159a against K. pneumoniae was comparable to the activities of LVFX and MFLX. Against M. catarrhalis, DC-159a had activity similar to the activities of the other quinolones tested at the MIC90 level. DC-159a was highly active against ampicillin-resistant H. influenzae strains, such as β-lactamase-positive ampicillin-resistant strains and β-lactamase-negative ampicillin-resistant strains, and inhibited the growth of all Haemophilus isolates at 0.25 µg/ml or less.

Against Escherichia coli, Serratia marcescens, Proteus mirabilis, indole-positive Proteus, Citrobacter spp., and Enterobacter spp., the MIC90s of DC-159a were 4, 4, 4, 8, 8, and 2 µg/ml, respectively. Against E. coli, DC-159a showed activity comparable to the activities of the other quinolones tested. Overall, the activity of DC-159a was almost comparable to the activities of LVFX and MFLX.

DC-159a inhibited the growth of 90% isolates of Acinetobacter spp., Salmonella spp., and Stenotrophomonas maltophilia at 2, 0.12 and 1 µg/ml, respectively. Against quinolone-susceptible Pseudomonas aeruginosa strains that cause respiratory tract and urinary tract infections, the MIC90 of DC-159a was 4 µg/ml. The activity of DC-159a was comparable to the activities of MFLX and GRNX.

Against N. gonorrhoeae, DC-159a (MIC90, 2 µg/ml) inhibited the growth of all isolates, including CPFX-resistant strains, at 2 µg/ml or less. DC-159a showed higher levels of activity than the other quinolones tested.

For anaerobic bacteria, the MIC90s of DC-159a against Peptostreptococcus spp., Clostridium difficile, and Bacteroides fragilis were 0.5, 4, and 2 µg/ml, respectively. In particular, DC-159a showed 32-fold or greater activity than LVFX and 4- to 8-fold greater activity than MFLX against gram-positive anaerobic bacteria, such as Peptostreptococcus spp. and C. difficile.

The MIC90 of DC-159a against L. pneumophila was 0.03 µg/ml and was comparable to the MIC90s of MFLX and GMFX. DC-159a showed an MIC of 0.12 µg/ml against all Mycoplasma pneumoniae strains tested. The antibacterial activity of DC-159a was generally eightfold higher than that of LVFX, twofold higher than that of GMFX, and comparable to that of MFLX.

The activity of DC-159a against C. pneumoniae was comparable to the activities of MFLX, GMFX, and AZM; and DC-159a was four- to eightfold more active than LVFX. The MIC of DC-159a against all strains of C. trachomatis tested was 0.063 µg/ml. The activity of DC-159a was comparable to that of MFLX and was fourfold greater than that of LVFX.

Multistep resistance. A comparison of the initial MICs and the final MICs at passage 14 for the four fluoroquinolones against all 12 isolates is shown in Table 2. All three pneumococcal isolates had a DC-159a MIC of 0.12 µg/ml at the start of the passage experiment. At the final passage this had increased by only 1 dilution for one pneumococcal strain and 2 to 3 dilutions for the other two pneumococcal strains. In comparison, MFLX had the same initial MIC as DC-159a, but 14 passages raised the MFLX MIC by 3 to 5 dilutions. The CPFX and the LVFX MICs also increased during passage.


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  		TABLE 2. Comparison of initial MICs (passage 0) and MICs after 14 consecutive passages at 0.5x MIC with four quinolones against 12 bacterial isolates

 
For Streptococcus pyogenes, no change in the DC-159a MIC was observed for one strain, and the other two strains had merely 1- to 2-dilution increases in the DC-159a MIC over 14 passages. Similar results were observed for MFLX, and greater MIC increases occurred with CPFX and LVFX. For two strains of Staphylococcus aureus, the DC-159a MIC increased by only 1 or 2 dilutions over the 14 passages. For a third strain, the DC-159a MIC increased by 4 dilutions. After passage, the DC-159a final MIC was no greater than 0.5 µg/ml against any S. aureus strain. Greater increases in the MICs developed during passage with the comparator fluoroquinolones. Over 14 passages, the DC-159a MIC increased by only 1 to 2 dilutions against all three strains of H. influenzae. As with the other bacterial species tested, the increases in the MICs of the other fluoroquinolones were higher with H. influenzae.

Bactericidal activity. The bactericidal activity of DC-159a against S. pneumoniae strains harboring various mutations in the quinolone resistance-determining region was compared with the activities of MFLX and GRNX. DC-159a (0.75 ethanol·0.25 H2O was used) showed rapid, concentration-dependent killing of the strains tested. The concentration dependency of DC-159a compared with the dependencies of the other quinolones tested was noteworthy, especially at the earlier time points. Both ParC Ser79Phe mutants (Fig. 2) and GyrA Ser81Phe and ParC Ser79Phe mutants (Fig. 3) were killed earlier by DC-159a than by MFLX or GRNX.


Figure 2
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  		FIG. 2. Bactericidal activities of DC-159a and reference compounds against S. pneumoniae 1026523 (ParC, Ser79Phe).

 

Figure 3
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  		FIG. 3. Bactericidal activities of DC-159a and reference compounds against Streptococcus pneumoniae 104835 (GyrA, Ser81Phe; ParC Ser79Phe).

 
Bactericidal activity in neutropenic murine calf muscle. The bactericidal activity of DC-159a in a neutropenic murine muscle infection model with a macrolide-resistant S. pneumoniae strain (MRSP; CAM MIC, >100 µg/ml) was compared with that of MFLX. The MICs of DC-159a and MFLX against this strain were both 0.1 µg/ml. The serum area under the concentration-time curve (AUC) values and the muscle AUC/serum AUC ratios in mice given DC-159a and MFLX subcutaneously at doses of 5 and 10 mg/kg were comparable (Table 3).


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  		TABLE 3. MICs and AUCs for DC-159a and MFLX in neutropenic murine muscle infection model with macrolide-resistant S. pneumoniae

 
There were significant differences between the untreated and the drug-treated groups with respect to the number of viable MRSP isolates in the calf tissues of the mice throughout the experiment. Similarly, DC-159a significantly reduced the numbers of viable MRSP cells in the calf tissues in a dose-dependent manner compared with the reduction achieved with the corresponding dose in the MFLX-treated group. Whereas a 10-mg/kg dose of MFLX resulted in an approximate 1-log10 reduction in the numbers of CFU at 4 and 6 h postadministration, the same dose of DC-159a effected a 3-log10 reduction in the numbers of CFU at the same time points. These findings indicate that the in vivo bactericidal activity of DC-159a against MRSP is superior to that of MFLX. (Fig. 4)


Figure 4
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  		FIG. 4. In vivo bactericidal activity of DC-159a against MRSP J24. Symbols: x, control; {square}, MFLX at 5 mg/kg; {blacksquare}, MFLX at 10 mg/kg; {circ}, DC-159a at 5 mg/kg; •, DC-159a at 10 mg/kg. Values are the mean bacterial number after common logarithmic conversion and their standard errors. The horizontal broken line represents the mean bacterial number at the onset of therapy. The bacterial numbers in the calf tissues of mice were measured at 2, 4, 6, and 24 h after treatment. Statistical analysis was performed by using the bacterial numbers in the calf tissues after common logarithmic conversion. The up- and down-pointing arrows represent the times of administration and inoculation, respectively. *, P < 0.05 for DC-159a compared to the results for MFLX at a dose of 5 mg/kg; ***, P < 0.001 for DC-159a compared to the results for MFLX at a dose of 5 mg/kg; ##, P < 0.01 for DC-159a compared to the results for MFLX at a dose of 10 mg/kg; ###, P < 0.001 DC-159a compared to the results for MFLX at a dose of 10 mg/kg (Dunnett's multiple comparison test).

 
Murine pneumonia. The therapeutic efficacy of DC-159a in a murine pneumonia model due to S. pneumoniae resistant to penicillin, macrolide, and quinolone (MDRSP) was compared with the efficacies of MFLX, GRNX, and GMFX (Fig. 5). The MICs of DC-159a, MFLX, GRNX, and GMFX for this strain were 1, 4, 1, and 0.5 µg/ml, respectively. Subcutaneous treatment with all test drugs at 25, 50, and 100 mg/kg was started 1 day after infection. Thereafter, treatment was administered twice a day for 3 consecutive days. DC-159a exhibited apparent bactericidal activity, with reductions of 2 log10 CFU/g at 50 mg/kg twice a day and 3 log10 CFU/g at 100 mg/kg twice a day compared with the counts for the nontreated mice at the onset of treatment. In contrast, the comparator drugs effected slight decreases of less than 1 log10 CFU/g, even at a dose of 100 mg/kg twice a day. Of the quinolones tested, DC-159a was the only drug that significantly eliminated MDRSP from the lungs.


Figure 5
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  		FIG. 5. Therapeutic efficacy of DC-159a against experimental murine pneumonia due to multidrug-resistant Streptococcus pneumoniae 104835. DC, DC-159a; MF, MFLX; GR, GRNX; GM, GMFX. Values are the mean bacterial numbers (log CFU/g in the lungs) and their standard errors. The bacterial numbers in the lungs were determined following infection and just before drug administration started (precontrol) and on the day after final administration (postcontrol; treated groups). Statistical analyses were performed by using the bacterial numbers in the lungs after common logarithmic conversion. **, P < 0.01 compared to the results for the postcontrol (Dunnett multiple comparison test); ***, P < 0.001 compared to the results for the postcontrol (Dunnett multiple comparison test); $$, P < 0.01 compared to the results for the precontrol (Dunnett's multiple comparison test); ##, P < 0.01 DC-159a compared to the results for MFLX at 200 mg/kg (Tukey's multiple comparing test).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The continuous increase in the emergence of resistant pathogens as causes of community-acquired infections is one of the significant concerns in first-line therapy, especially respiratory tract infections (6). Penicillin and macrolide resistance in S. pneumoniae has spread widely throughout the world. Because of the broad spectrum of activity of quinolone antibacterials, their use has been extended to the treatment of pneumonia caused by atypical pathogens, and the use of this class of agents for the treatment of respiratory tract infections has recently become more common (30). On the other hand, resistance resulting from mutations in both target enzymes of the quinolones, DNA gyrase and topoisomerase IV, in clinical isolates, including those with efflux types of mutations, has been reported (2, 27). Several reports have warned of the risk for an increase in highly resistant variants as a result of a single mutation in ParC in S. pneumoniae (12, 16, 21, 23).

Moreover, staphylococci in the community have also been developing resistance to various first-line agents, including quinolone antibacterials, suggesting another unmet need for agents for empirical treatment.

DC-159a was developed to introduce a quinolone for respiratory infections that fills these unmet needs because of a preferable antibacterial spectrum and a low propensity to cause the emergence of resistance in key community-acquired respiratory pathogens. The advantages of DC-159a, especially against quinolone-resistant S. pneumoniae strains, is clearly demonstrated by its in vitro MICs and in vivo efficacies in this study. An advantageous feature of DC-159a is its rapid bactericidal activity against S. pneumoniae both in vitro and in vivo. The superiority of DC-159a, as shown by its therapeutic efficacy against murine pneumonia, might be attributable to these excellent bactericidal activities and PK profiles (Table 4). The rapid killing of quinolone-intermediate and quinolone-resistant mutants of S. pneumoniae may be important in lowering the incidence of the emergence of resistance and in killing highly quinolone-resistant mutants at clinically achievable concentrations. We observed similar properties in evaluations of a structurally related molecule, DK-507k (18). The fluoroquinolone agents marketed to date are not potent enough for the treatment of infections caused by highly resistant mutants (LVFX MIC, more than 4 µg/ml) that possess mutations in both DNA gyrase and topoisomerase IV. GMFX and GRNX showed strong in vitro activity against S. pneumoniae; however, pharmacodynamic indices are not achievable at the usual dosage because of the high levels of protein binding and/or the low levels of exposure at the target organ. DC-159a showed activity (MIC90, 1 µg/ml) against a quinolone-resistant population of S. pneumoniae, including strains with various types of mutations in both target enzymes. The level of serum protein binding of DC-159a is less than 50% in various species, and it has been shown to have a good PK profile, such as a high level of distribution in the lung, which was observed in preclinical examinations (data not shown). In the murine pneumonia study, among the quinolones tested, DC-159a was the only drug that significantly reduced the numbers of viable MDRSP cells in the lungs.


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  		TABLE 4. Serum AUCs of test drugs in mice at study dosea

 
Levofloxacin-nonsusceptible isolates have been reported not only among streptococci and staphylococci but also among anaerobes, such as B. fragilis and C. difficile (1, 7, 19). DC-159a exhibited excellent activity against these species and may play an important role in the treatment of community-acquired infections to reduce the risk of the emergence of resistance in nontargeted species.

Additional studies of both streptococci and staphylococci will help to confirm the superiority and mechanism of action of this compound.

In conclusion, the improved activity of DC-159a against gram-positive organisms compared with the activities of the other quinolones tested and its activity against gram-negative organisms, which is comparable to that of LVFX, as well as it better activity against atypical organisms, such as M. pneumoniae and C. pneumoniae, warrant ascertainment of the role of this agent in the empirical treatment of community-acquired infections. It is suggested that DC-159a will be a beneficial therapeutic option for respiratory tract infections, including those due to multidrug-resistant S. pneumoniae strains.


   ACKNOWLEDGMENTS
 
We thank Subaru Ogiso, Naoe Haga, Hitomi Awaya, and Megumi Chiba for excellent technical assistance. We are grateful to Kenichi Sato and Mayumi Tanaka for helpful discussions. The multistep resistance selection experiments were performed at GR Micro Ltd. London, United Kingdom (research directed by Ian Morrissey).


   FOOTNOTES
 
* Corresponding author. Mailing address: Biological Research Laboratories IV, Daiichi Sankyo Co., Ltd., 16-13, 1-Chome Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan. Phone: 81-3-5696-8245. Fax: 81-3-5696-4264. E-mail: hoshino.kazuki.d6{at}daiichisankyo.co.jp Back

{triangledown} Published ahead of print on 15 October 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ackermann, G., Y. J. Tang-Feldman, R. Schaumann, J. P. Henderson, A. C. Rodloff, J. Silva, and S. H. Cohen. 2003. Antecedent use of fluoroquinolones is associated with resistance to moxifloxacin in Clostridium difficile. Clin. Microbiol. Infect 9:526-530.[CrossRef][Medline]
  2. Bast, D. J., D. E. Low, C. L. Duncan, L. Kilburn, L. A. Mandell, R. J. Davidson, and J. C. de Azavedo. 2000. Fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae: contributions of type II topoisomerase mutations and efflux to levels of resistance. Antimicrob. Agents Chemother. 44:3049-3054.[Abstract/Free Full Text]
  3. Blumberg, H. M., D. Rimland, D. J. Carroll, P. Terry, and I. K. Wachsmuth. 1991. Rapid development of ciprofloxacin resistance in methicillin-susceptible and -resistant Staphylococcus aureus. J. Infect. Dis. 163:1279-1285.[Medline]
  4. Fridkin, S. K., J. C. Hageman, M. Morrison, L. T. Sanza, K. Como-Sabetti, J. A. Jernigan, K. Harriman, L. H. Harrison, R. Lynfield, and M. M. Farley. 2005. Methicillin-resistant Staphylococcus aureus disease in three communities. N. Engl. J. Med. 352:1436-1444.[Abstract/Free Full Text]
  5. Fuller, J. D., and D. E. Low. 2005. A review of Streptococcus pneumoniae infection treatment failures associated with fluoroquinolone resistance. Clin. Infect. Dis. 41:118-121.[CrossRef][Medline]
  6. Fuller, J. D., A. McGeer, and D. E. Low. 2005. Drug-resistant pneumococcal pneumonia: clinical relevance and approach to management. Eur. J. Clin. Microbiol. Infect. Dis. 24:780-788.[CrossRef][Medline]
  7. Golan, Y., L. A. McDermott, N. V. Jacobus, E. J. Goldstein, S. Finegold, L. J. Harrell, D. W. Hecht, S. G. Jenkins, C. Pierson, R. Venezia, J. Rihs, P. Iannini, S. L. Gorbach, and D. R. Snydman. 2003. Emergence of fluoroquinolone resistance among Bacteroides species. J. Antimicrob. Chemother. 52:208-213.[Abstract/Free Full Text]
  8. Greene, C. M., M. H. Kyaw, S. M. Ray, W. Schaffner, R. Lynfield, N. L. Barrett, C. Long, K. Gershman, T. Pilishvili, A. Roberson, E. R. Zell, C. G. Whitney, and N. M. Bennett. 2006. Preventability of invasive pneumococcal disease and assessment of current polysaccharide vaccine recommendations for adults: United States, 2001-2003. Clin. Infect. Dis. 43:141-150.[CrossRef][Medline]
  9. Hoshino, K., R. Okumura, K. Inoue, Y Murakami, H. Awaya, T. Hirata, S. Yamachika, S. Higuchi, Y. Kashimoto, H. Haga, T. Otani, and K. Sato. 2006. Abstr. 46th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F1-0478.
  10. Japan Society of Chemotherapy. 1992. Method for in vitro determination of chlamydial susceptibility—standard method of Japanese Society of Chemotherapy. Chemotherapy 40:308-314.
  11. Japan Society of Chemotherapy. 1981. Method for the determination of minimum inhibitory concentration (MIC) of aerobic bacteria by agar dilution method. Chemotherapy 29:76-79.
  12. Jimenez, M. R., J. L. Bellido, and J. A. Garcia Rodriguez. 2005. Risk factors associated with colonization by pneumococci with reduced susceptibility to fluoroquinolones in adult outpatients. J. Clin. Microbiol. 43:1193-1197.[Abstract/Free Full Text]
  13. Kays, M. B., G. G. Zhanel, M. A. Reimann, J. Jacobi, G. A. Denys, D. W. Smith, and M. F. Wack. 2007. Selection of a gyrA mutation and treatment failure with gatifloxacin in a patient with Streptococcus pneumoniae with a preexisting parC mutation. Pharmacotherapy 27:221-226.[CrossRef][Medline]
  14. Kupronis, B. A., C. L. Richards, and C. G. Whitney. 2003. Invasive pneumococcal disease in older adults residing in long-term care facilities and in the community. J. Am. Geriatr. Soc. 51:1520-1525.[CrossRef][Medline]
  15. Kyaw, M. H., R. Lynfield, W. Schaffner, A. S. Craig, J. Hadler, A. Reingold, A. R. Thomas, L. H. Harrison, N. M. Bennett, M. M. Farley, R. R. Facklam, J. H. Jorgensen, J. Besser, E. R. Zell, A. Schuchat, and C. G. Whitney. 2006. Effect of introduction of the pneumococcal conjugate vaccine on drug-resistant Streptococcus pneumoniae. N. Engl. J. Med. 354:1455-1463.[Abstract/Free Full Text]
  16. LeBlanc, L., J. Pepin, K. Toulouse, M. F. Ouellette, M. A. Coulombe, M. P. Corriveau, and M. E. Alary. 2006. Fluoroquinolones and risk for methicillin-resistant Staphylococcus aureus, Canada. Emerg. Infect. Dis. 12:1398-1405.[Medline]
  17. Lim, S., D. Bast, A. McGeer, J. de Azavedo, and D. E. Low. 2003. Antimicrobial susceptibility breakpoints and first-step parC mutations in Streptococcus pneumoniae: redefining fluoroquinolone resistance. Emerg. Infect. Dis. 9:833-837.[Medline]
  18. NCCLS. 2005. Performance standards for antimicrobial susceptibility testing; Fifteenth informational supplement. NCCLS, Wayne, PA.
  19. Otani, T., M. Tanaka, E. Ito, Y. Kurosaka, Y. Murakami, K. Onodera, T. Akasaka, and K. Sato. 2003. In vitro and in vivo antibacterial activities of DK-507k, a novel fluoroquinolone. Antimicrob. Agents Chemother. 47:3750-3759.[Abstract/Free Full Text]
  20. Pepin, J., N. Saheb, M. A. Coulombe, M. E. Alary, M. P. Corriveau, S. Authier, M. Leblanc, G. Rivard, M. Bettez, V. Primeau, M. Nguyen, C. E. Jacob, and L. Lanthier. 2005. Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin. Infect. Dis. 41:1254-1260.[CrossRef][Medline]
  21. Sawatari, K., N. Ito, M. Nagasawa, H. Nakasato, H. Koga, K. Watanabe, et al. 1984. New susceptibility testing medium (B-SYE agar) for Legionella and Legionella-like organisms. Chemotherapy 32:718-723.
  22. Schurek, K. N., H. J. Adam, C. G. Siemens, C. J. Hoban, D. J. Hoban, and G. G. Zhanel. 2005. Are fluoroquinolone-susceptible isolates of Streptococcus pneumoniae really susceptible? A comparison of resistance mechanisms in Canadian isolates from 1997 and 2003. J. Antimicrob. Chemother. 56:769-772.[Abstract/Free Full Text]
  23. Shopsin, B., X. Zhao, B. N. Kreiswirth, G. S. Tillotson, and K. Drlica. 2004. Are the new quinolones appropriate treatment for community-acquired methicillin-resistant Staphylococcus aureus? Int. J. Antimicrob. Agents 24:32-34.[Medline]
  24. Smith, H. J., A. M. Noreddin, C. G. Siemens, K. N. Schurek, J. Greisman, C. J. Hoban, D. J. Hoban, and G. G. Zhanel. 2004. Designing fluoroquinolone breakpoints for Streptococcus pneumoniae by using genetics instead of pharmacokinetics-pharmacodynamics. Antimicrob. Agents Chemother. 48:3630-3635.[Abstract/Free Full Text]
  25. Tateda, K., K. Takashima, H. Miyazaki, T. Matsumoto, T. Hatori, and K. Yamaguchi. 1996. Noncompromised penicillin-resistant pneumococcal pneumonia CBA/J mouse model and comparative efficacies of antibiotics in this model. Antimicrob. Agents Chemother. 40:1520-1525.[Abstract]
  26. Waites, K. B., Y. Rikihisa, and D. T. Robinson. 2003. Mycoplasma and Ureaplasma, p. 972-990. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, vol. 1, 8th ed. ASM Press, Washington, DC.
  27. Weber, S. G., H. S. Gold, D. C. Hooper, A. W. Karchmer, and Y. Carmeli. 2003. Fluoroquinolones and the risk for methicillin-resistant Staphylococcus aureus in hospitalized patients. Emerg. Infect. Dis. 9:1415-1422.[Medline]
  28. Weigel, L. M., G. J. Anderson, R. R. Facklam, and F. C. Tenover. 2001. Genetic analyses of mutations contributing to fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 45:3517-3523.[Abstract/Free Full Text]
  29. Yamaguchi, K., and A. Ohno. 2005. Investigation of the susceptibility trends in Japan to fluoroquinolones and other antimicrobial agents in a nationwide collection of clinical isolates: a longitudinal analysis from 1994 to 2002. Diagn. Microbiol. Infect. Dis. 52:135-143.[CrossRef][Medline]
  30. Yokota, S., K. Sato, O. Kuwahara, S. Habadera, N. Tsukamoto, H. Ohuchi, H. Akizawa, T. Himi, and N. Fujii. 2002. Fluoroquinolone-resistant Streptococcus pneumoniae strains occur frequently in elderly patients in Japan. Antimicrob. Agents Chemother. 46:3311-3315.[Abstract/Free Full Text]
  31. Zhanel, G. G., S. Fontaine, H. Adam, K. Schurek, M. Mayer, A. M. Noreddin, A. S. Gin, E. Rubinstein, and D. J. Hoban. 2006. A review of new fluoroquinolones: focus on their use in respiratory tract infections. Treat. Respir. Med. 5:437-465.[CrossRef][Medline]

Antimicrobial Agents and Chemotherapy, January 2008, p. 65-76, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.00853-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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