Research Laboratories of Toyama Chemical Co., Ltd, 2-4-1 Shimookui, Toyama 930-0817, Japan
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Abstract |
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Introduction |
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While the spectra and antibacterial activities of fluoroquinolones such as ciprofloxacin (developed in the 1980s) have been improved to include most Gram-negative bacteria, their activities against Gram-positive bacteria remained limited. The later 1980s and 1990s, however, have seen the synthesis and development of several agents with strong antibacterial activity against S. pneumoniae, including both penicillin-susceptible and -resistant strains. These new agents include tosufloxacin, 2,34 temafloxacin 5,6 and sparfloxacin. 7,8
Quinolones exhibit bactericidal activity against S. pneumoniae in a manner similar to that seen with other Gram-positive and Gram-negative bacteria. In addition, all quinolones have a paradoxical lethal effect on bacteria, 9 i.e. there is an increase in bactericidal activity with an increase in drug concentration up to an optimum bactericidal concentration (OBC), above which higher concentrations are somewhat less bactericidal.
Although several investigators have recently demonstrated the in-vivo efficacy of fluoroquinolones, 10,11,1213 there has not been any detailed evaluation of the in-vitro bactericidal profiles of quinolones against S. pneumoniae and the relationship between bactericidal activity and chemical structure. This report describes the characteristics of in-vitro bactericidal profiles of quinolones against S. pneumoniae.
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Materials and methods |
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For timekill studies, the penicillin-susceptible S. pneumoniae strains IID553 and TUH-39 and the penicillin-resistant strain TMS-3 were used. 14,15 The latter two strains were clinical isolates kindly provided by Professor Keizo Yamaguchi (Toho University School of Medicine, Tokyo, Japan). The strains were frozen at -135°C in skimmed milk until use. The MICs for each strain were as follows: tosufloxacin; 0.25 mg/L for strain IID553 and 0.5 mg/L for strains TUH-39 and TMS-3; sparfloxacin: 0.25 mg/L for strain IID553, 0.5 mg/L for strain TUH-39 and 1 mg/L for strain TMS-3; ciprofloxacin and levofloxacin: 1 mg/L for strain IID553 and 2 mg/L for strains TUH-39 and TMS-3.
Media and chemicals
MuellerHinton agar (MHA) and MuellerHinton broth (MHB) were purchased from Difco Laboratories (Detroit, MI, USA). Tosufloxacin, sparfloxacin, ciprofloxacin, levofloxacin, enoxacin, norfloxacin and compounds IVIII (see Table II) were synthesized at the Research Laboratories of Toyama Chemical Co., Ltd. (Toyama, Japan). Other chemicals (guaranteed grade) were purchased from Nakalai Tesque, Inc. (Kyoto, Japan).
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MICs were determined by the broth microdilution method using 0.1 mL of cation-adjusted MHB (CAMHB), i.e. MHB cation-adjusted with calcium and magnesium, supplemented with 5% lysed horse blood. Each well contained a two-fold dilution of antibiotics and a final bacterial concentration of 5 x 10 4 cfu/well. The plates were incubated at 37°C overnight and the MIC was defined as the lowest concentration of antibiotic that did not allow visible growth.
The bactericidal activity was determined by a timekill study using the same broth dilution method. Antibiotics were used at 4 x MIC. After inoculation of bacterial cells, the microtitre plate was incubated at 37°C and an aliquot was removed at 0, 0.25, 0.5, 1, 2, 4 and 8 h. Each sample was serially diluted 10-fold with physiological saline, and 0.05 mL of each sample was spread on 20 mL of 5% sheep blood agar. Plates were incubated for up to 48 h at 37°C. Drug carryover did not affect colony formation in this condition. To determine the optimum bactericidal:MIC ratio (OBMR) of each quinolone, the cells of S. pneumoniae TMS-3 were incubated with a range of drug concentrations from the MIC to 48 x MIC, with an inoculum size of about 10 6 cfu/mL, for 2 h at 37°C. After incubation, viable counts were made on solid blood agar. Experiments were performed in triplicate, and log reduction at OBMRs are given as mean ± S.D.
Determination of PAE
The PAE experiment was performed according to the method of Bustamante et al. 16 with slight modifications. Using a sterile cotton swab, cells of S. pneumoniae TMS-3 grown on 5% sheep blood agar plates were suspended in physiological saline at approximately 1 x 10 9 cfu/mL. The cell suspensions were inoculated into CAMHB supplemented with 5% lysed horse blood to give a final bacterial concentration of 10 7 cfu/mL. A final concentration at the OBMR of each drug was added and, after a 2 h exposure at 37°C, cultures were diluted 1000-fold with fresh broth to avoid the influence of drug carryover. At 1 h intervals, aliquots were withdrawn and were serially diluted in 10-fold increments, with a 0.04 mL aliquot of each being plated on to 5% sheep blood agar plates. For each plate, the viable cell count was determined after incubation for up to 48 h at 37°C. As described by Craig & Gudmundsson, 17 the PAE was the difference in time required by test and control cultures to increase 1 log after removal of the antibiotic. The suppressive effects on growth were not seen with this condition. Experiments were performed in triplicate, and PAEs are given as averages and ranges.
Statistical analysis
The data of log10 reduction of viable cell counts and PAEs were compared by Student's t-test and by the Tukey procedure with a cutoff of P = 0.05 for significance (SAS version 4.0, SAS Institute Japan, Tokyo, Japan).
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Results |
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The killing curves of each quinolone against the three strains of S. pneumoniae are shown in Figure 1. In drug-free medium, the bacterial count increased logarithmically from approximately 10 510 6 cfu/mL at 8 h after incubation. When each quinolone was tested at 4 x MIC, tosufloxacin showed the most rapid bactericidal activity, although there was some variation in the reduction of bacterial count for each strain. Although it had a lower MIC than ciprofloxacin and levofloxacin, sparfloxacin showed the least reduction in bacterial count after 2 h of all quinolones tested. Studies were repeated three times with similar results. To investigate the bactericidal activity of quinolones in detail, we determined the OBMRs, ranging from the MIC to 48 x MIC and the reduction of bacterial counts at each OBMR against S. pneumoniae TMS-3 (Figure 2, Table I). The OBMR of each quinolone was 4 x MIC for ciprofloxacin, 8 x MIC for tosufloxacin and levofloxacin and 12 x MIC for sparfloxacin. Tosufloxacin showed the greatest log reduction in bacterial count at 2 h at the OBMR among the quinolones tested. Similar results were obtained for the other two strains (data not shown).
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Relationship between bactericidal activity, PAE and chemical structure
The relationships between bactericidal activity, PAE and chemical structure were investigated using tosufloxacin, ciprofloxacin, norfloxacin, enoxacin and compounds IVIII (Table II). With naphthyridine derivatives, the MIC of compounds I, II and IV were four, two and eight times higher, respectively, than that of tosufloxacin. The MIC of compound III and enoxacin was eight and 32 times higher, respectively, than that of tosufloxacin. Similarly, with quinoline derivatives, the MICs of compounds VI, VII and VIII were two, two and eight times higher, respectively, than that of compound V. The MICs of ciprofloxacin and norfloxacin were, respectively, four and 16 times that of compound V. When the OBMRs were compared following 2 h incubation after the addition of each compound, compounds having the same group at the N-1 position of the quinolone nucleus showed similar OBMRs. The OBMRs of compounds having ethyl, cyclopropyl and 2,4-difluorophenyl group at the N-1 position of the quinolone nucleus were 12, 4 and 8 x MIC, respectively. However, differences were seen in the log reduction in bacterial counts at the OBMR. First, where the type of nucleus was varied without changing the groups in positions N-1 and C-7, compounds with a quinoline nucleus tended to have more bactericidal activity than those with a naphthyridine nucleus. When the group at the N-1 position was varied without changing the quinolone nucleus or the group at the C-7 position, compounds having a 2,4-difluorophenyl group at the N-1 position had significantly more bactericidal activity than those with a cyclopropyl or ethyl group. Finally, when the group at the C-7 position was varied without altering the quinolone nucleus or the group at the N-1 position, compounds with an aminopyrrolidinyl group in the C-7 position tended to show more bactericidal activity than those with a piperazinyl group at the C-7 position, except for compounds with a quinolone nucleus having an ethyl or cyclopropyl group in the N-1 position. The degree of bactericidal activity was affected more by changing the group in the N-1 position than by changing the nucleus or the group in the C-7 position.
Compounds having the same group at the N-1 position of the quinolone nucleus showed similar PAEs. The PAEs of compounds having an ethyl, cyclopropyl and 2,4-difluorophenyl group in the N-1 position of the quinolone nucleus were 0.040.16, 0.410.67 and 0.771.45 h, respectively. The PAEs of compounds having a 2,4-difluorophenyl group in the N-1 position in the quinolone nucleus were about 5.536 times longer than those having an ethyl group and about 1.23.5 times longer than those having a cyclopropyl group. In addition, a good relationship was observed between the bactericidal activity and PAE of each compound (Figure 3). Similar results were obtained from the other two strains (data not shown).
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Discussion |
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The MICs of tosufloxacin against S. pneumoniae were lowest and the bactericidal activity at OBMR was superior to those of sparfloxacin, ciprofloxacin and levofloxacin. The PAE of tosufloxacin was about 2.32.6 times longer than those of the other quinolones. These results indicate that tosufloxacin possesses potent in-vitro bactericidal activity and PAE against S. pneumoniae.
Recently, the pharmacokinetic parameters of tosufloxacin in healthy volunteers were reported. The maximum concentration in serum (Cmax) of tosufloxacin was 0.88 ± 0.28 mg/L after administration of 300 mg of tosufloxacin tosilate which is equivalent to 204 mg of the free base. 18 This concentration corresponded to approximately three to four times the MIC for S. pneumoniae, since the MIC90 of tosufloxacin was 0.25 mg/L against recent isolates of S. pneumoniae including penicillin-resistant strains. 19 However, in general, the concentrations of quinolone in pulmonary tissue are often several times higher than those in serum. For example, we have found that the Cmax in lungs (1.33 ± 0.088 µg/g) after administration of 10 mg/kg of tosufloxacin was 3.6-fold higher than that in serum (0.37 ± 0.035 mg/L) using CBA/j mice infected with S. pneumoniae TMS-3 (data not shown).
We considered that the concentration at OBMR of tosufloxacin (8 x MIC) would be attainable in vivo, since the concentration/MIC ratio in infected pulmonary tissue would be larger than that in serum. The investigation using newly synthesized compounds demonstrated that the MICs of quinolones depended on groups at both the N-1 and C-7 positions and that compounds with a 2,4-difluorophenyl group at the N-1 position in the quinolone nucleus exhibited potent bactericidal activity against S. pneumoniae. Similar results were obtained with temafloxacin 5,6 and trovafloxacin (CP-99,219), 20,21 which had a 2,4-difluorophenyl group as a common structure with tosufloxacin in the N-1 position of the quinoline and naphthyridine nucleus (data not shown). In addition, compounds with a 2,4-difluorophenyl group showed a longer PAE than those with an ethyl or cyclopropyl group at the N-1 position in the quinolone nucleus. Interestingly, although the bactericidal activities of the quinolones were well correlated with the PAE, they did not always correlate with the MIC, suggesting that a 2,4-difluorophenyl group at the N-1 position in quinolones plays an important role in the expression of bactericidal activity and PAE rather than in expression of antibacterial activity. It might be expected that the bactericidal activity and PAE are governed by factors other than those that provided the MIC in S. pneumoniae. The mechanisms of bacterial killing and PAE by quinolones are still not fully understood. Walters et al. 22 reported that bacterial killing by quinolones was influenced by the degree of induction of the SOS response using a variety of SOS mutants of Escherichia coli. However, it is not obvious whether the quinolone-induced SOS response influences the survival or the damaging effect to quinolone-treated cells. On the other hand, it has been shown that active protein and RNA synthesis are required, in part, for the bactericidal action of quinolones, although their direct inhibition by quinolones is not the primary mechanism of action. 23,24,2526
It has been reported that PAEs induced by quinolones may represent the time required for the quinolones to dissociate from the receptor binding sites and to diffuse out of the bacterium. 27 However, other investigators observed no predictable pattern of DNA synthesis within drug classes in Staphylococcus aureus and E. coli exposed to various antimicrobial agents including ciprofloxacin. 28 The variety of bactericidal activity and PAE in the quinolones observed in this study may be due to the different degree of induction of SOS response or different effects of quinolones on DNA, RNA and protein synthesis. Additional detailed studies are required to determine the precise role of the 2,4-difluorophenyl group in bactericidal activity and PAE.
In conclusion, the bactericidal activity and PAE of quinolones are governed by factors other than those that determine antibacterial activity, and a 2,4-difluorophenyl group in the N-1 position of the quinolone nucleus played an important role in the expression of bactericidal activity and PAE against S. pneumoniae. Thus, our findings suggest a novel approach for development of future quinolones having potent bactericidal activity against Gram-positive bacteria.
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Acknowledgments |
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Notes |
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References |
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Received 8 September 1998; returned 27 January 1999; revised 24 February 1999; accepted 30 March 1999
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