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PD) parameters of gatifloxacin on its bactericidal activity and resistance selectivity against clinical isolates of Streptococcus pneumoniae
J Infect Chemother (2003) 9:210–214 DOI 10.1007/s10156-003-0243-9
© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2003
ORIGINAL ARTICLE Hiroyuki Ebisu · Ryuta Kishii · Masaya Takei Hideyuki Fukuda
The effect of pharmacokinetic/pharmacodynamic (PK/PD) parameters of gatifloxacin on its bactericidal activity and resistance selectivity against clinical isolates of Streptococcus pneumoniae
Received: October 9, 2002 / Accepted: March 19, 2003
Abstract The impact of the pharmacokinetic/pharmacodynamic (PK/PD) parameters (the 24h area under the concentration-time curve [AUC24h]/minimum inhibitory concentration [MIC] and maximum concentration in serum [Cmax]/MIC ratio) after single oral dosing of gatifloxacin on its bactericidal activity and resistance selectivity against quinolone-susceptible clinical isolates of Streptococcus pneumoniae J-69 was investigated using an in vitro PK model. The MICs of gatifloxacin, levofloxacin, and ciprofloxacin were 0.25, 1, and 1 µg/ml, respectively. When the range of AUC24h/MIC ratios was varied from 9.0 to 36 with a constant Cmax/MIC ratio of 3.4, the bactericidal activity was correlated with the AUC24h/MIC ratios. Eradication was observed at an AUC24h/MIC ratio of 36. On the other hand, the resistance selectivity was associated with the Cmax/ MIC ratio. Mutant strains were selected at a Cmax/MIC ratio of 0.84, but not 1.7 with a constant AUC24h/MIC ratio of 9.0. These results suggested that an AUC24h/MIC ratio of 36 and a Cmax/MIC ratio of 1.7 might be possible benchmarks to show enough bacterial eradication and prevention of emergence of resistant strains to gatifloxacin, respectively. When the serum concentrations after clinical oral dosing of gatifloxacin (200 mg b.i.d.), levofloxacin (100 mg t.i.d.), and ciprofloxacin (200 mg t.i.d.) were simulated, the bactericidal activity of gatifloxacin was higher than those of levofloxacin and ciprofloxacin. Moreover, no resistant strain was obtained by the exposure to gatifloxacin and levofloxacin, whereas ciprofloxacin selected resistant strains. The clinically relevant oral dosage of gatifloxacin was anticipated to result in a high AUC24h/MIC90 ratio of 81 and a Cmax/MIC90 ratio of 4.4, suggesting that this agent is clinically effective in the treatment of pneumococcal infections.
Introduction Streptococcus pneumoniae remains the leading cause of bacterial pneumonia and is the most common pathogen associated with otitis media, sinusitis, and meningitis. Recent increases in the incidence of resistance to β-lactam and macrolide antibiotics among S. pneumoniae1,2 have meant that quinolones are now being considered for use in the treatment of these infections. However, the activities of quinolones against gram-positive organisms have been variable among the agents. Newer quinolones, such as gatifloxacin, have demonstrated increased activity against S. pneumoniae1,3,4 and also offer many advantages over the older quinolones in terms of broad antibacterial spectrum and favorable pharmacokinetics. The contributions of these favorable profiles of newer quinolones to efficacy have been examined in many studies using in-vitro pharmacokinetic (PK) models. The 24h area under the concentration-time curve (AUC)24h/minimum inhibitory concentration (MIC) ratios have been identified as possible predictors of the bactericidal effect of quinolones.5–9 The optimal efficacy of quinolones has been observed at AUC24h/MIC ratios of 30–60.5–9 Some investigators have also suggested that maximum concentration in serum (Cmax)/MIC ratios ranging from 2 to 10 are required to prevent the selection of resistance to quinolones.5,10–12 In this study, we investigated the effect of pharmacokinetic/pharmacodynamic (PK/PD) parameters of gatifloxacin on its bactericidal activity and resistance selectivity against S. pneumoniae, and compared its efficacy with those of levofloxacin and ciprofloxacin by simulating the serum concentration according to the Japanese clinical regimen.
Key words Streptococcus pneumoniae · In vitro pharmacokinetic model · Gatifloxacin · AUC24h/MIC · Cmax/MIC
Materials and methods H. Ebisu (*) · R. Kishii · M. Takei · H. Fukuda Infectious Diseases, Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd., 2399-1, Nogi, Nogi-machi, Shimotsuga, Tochigi 329-0114, Japan Tel. 81-280-562201; Fax 81-280-571293 e-mail: [email protected]
Quinolones Gatifloxacin and levofloxacin were synthesized at Kyorin Pharmaceutical (Tokyo, Japan). Ciprofloxacin
211 Table 1. Pharmacokinetic parameters at clinical doses of gatifloxacin, levofloxacin, and ciprofloxacin simulated in the in vitro pharmacodynamic model Drug
Dose, regimen
Cmax (µg/ml)
t1/2 (h)
AUC24h µg/ml·h
Cmax/MIC
AUC/MIC
Reference no.
Gatifloxacin Levofloxacin Ciprofloxaci
200 mg b.i.d 100 mg t.i.d 200 mg t.i.d
1.71 1.36 1.37
7.1 5.1 2.8
31.7 28.0 19.6
6.8 1.4 1.4
127 28 20
13 14 15
MIC, minimum inhibitory concentration; Cmax, maximum concentration; AUC24h, the 24h area under the concentration-time curve
was purchased from Apin (Abimgdon, Oxfordshire, UK). Bacterial strain The clinical strain of S. pneumoniae J-69 was isolated from a patient with respiratory tract infection in 1996 in Japan. The MICs of gatifloxacin, levofloxacin, and ciprofloxacin for the strain were 0.25, 1, and 1 µg/ml, respectively, which correspond to the MIC90 for clinical isolates.1 The strain was found to lack mutations in the quinolone resistancedetermining region (QRDR) of gyrA and parC.
In vitro pharmacokinetic (PK) model A dilutional in vitro PK model (PASS-400; Dainipponseiki, Kyoto, Japan) was used to simulate serum concentrations of the quinolones based on the PK parameters decided upon. The bacteria was grown in Todd-Hewitt broth (Difco, Sparks, MD, USA) at 37°C. A starting inoculum size of 106–107 CFU/ml was prepared from overnight culture for all the experiments. To ensure that the bacteria were in logarithmic growth phase, the bacteria were left in Todd-Hewitt broth for 2 h before exposure to the drugs in the model. All experiments were performed in duplicate to confirm reproducibility. The effect of PK/PD parameters on the bactericidal activity and the resistance selectivity of gatifloxacin We performed three experiments, as follows. Experiment I Based on the PK parameters of single oral dosing of 200 mg of gatifloxacin,13 serum concentrations of gatifloxacin after oral dosing of 12.5, 25, 50, 100, and 200 mg were proportionally simulated while maintaining a constant half-life (7.1 h).
Experiment III Cmax and half-lives were varied to provide Cmax/MIC ratios of 0.86 and 1.7 with a constant AUC24h/MIC ratio of 9.0, which was only the ratio at which the occurrence of a resistant mutant was observed in experiment I. Comparative pharmacodynamic (PD) profiles of gatifloxacin, levofloxacin, and ciprofloxacin According to the Japanese clinical regimen, the serum concentrations after oral dosing of gatifloxacin (200 mg b.i.d.), levofloxacin (100 mg t.i.d.), and ciprofloxacin (200 mg t.i.d.) were simulated. An appropriate amount of quinolone was added to a culture flask at 0 and 12 h for gatifloxacin and at 0, 8, and 16 h for levofloxacin and ciprofloxacin. The simulated PK parameters13–15 of the test quinolones are shown in Table 1. Pharmacodynamic (PD) analysis At 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h, samples were removed from the culture flask (100 ml) and viable bacteria were counted by plating 100 µl of serial ten fold dilutions onto Heart Infusion Agar (Eiken, Tokyo, Japan) supplemented with 5% (v/v) defibrinated horse blood (Nippon Bio-test Laboratories, Tokyo, Japan). The limit of detection of bacterial numbers was 102 CFU/ml. Quantitative evaluation of bactericidal activity was expressed as the area above the killing-curve (AAKC). To evaluate the resistance selectivity, regrown bacteria were plated onto an agar plate containing the respective quinolones at 2, 4, 8, and 16 MIC. If the strains grew on the agar plate containing the quinolones, their drug susceptibility and gene mutations in the QRDR of gyrA and parC of the representative strain were investigated as described previously.16
Results Experiment II Serum half-lives were varied to provide AUC24h/MIC ratios of 9.0, 18, and 36, with a constant Cmax/MIC ratio of 3.4, which was the minimum ratio needed to eradicate the bacteria in experiment I.
The effect of PK/PD parameters on the bactericidal activity and the resistance selectivity of gatifloxacin In order to study the impact of PK/PD parameters (AUC24h/ MIC and Cmax/MIC ratios) on the bactericidal activities and
212
Fig. 1. Bactericidal activity of gatifloxacin simulating different single oral doses against Streptococcus pneumoniae J-69. Closed circles, 12.5 mg; closed triangles, 25 mg; closed squares, 50 mg; open circles, 100 mg; open triangles, 200 mg; closed diamonds growth control. Dotted line indicates lower limit of detection
Fig. 2. Bactericidal activity of gatifloxacin against S. pneumoniae J-69 at different AUC24h/minimum inhibitory concentration (MIC) ratios, of 9.0 (closed circles); 18 (closed triangles); and 36 (closed squares), with a constant maximum concentration in serum (Cmax)/MIC ratio of 3.4. The numbers in parentheses and the dotted line indicate the area above the killing curve (AAKC; log CFU/ml·h) and the lower limit of detection, respectively
the resistance selectivity of gatifloxacin, experiments were performed under conditions of various PK/PD parameters. Experiment I Time-kill curves for gatifloxacin at the simulated serum concentration after oral dosing of 12.5, 25, 50, 100, and 200 mg are shown in Fig. 1. At doses of 100 mg (corresponding to AUC24h/MIC and Cmax/MIC ratios of 36 and 3.4, respectively), gatifloxacin rapidly reduced bacteria below the limit of detection (102 CFU/ml) within 5 h after exposure. In contrast, at doses of 50 mg, decreases in viable bacterial counts slowed, and then the bacteria increased, compared with the original inoculum size, in 24 h. Resistant strains that possessed gyrA mutation were observed after exposure to gatifloxacin only at the simulated serum concentration after 25 mg oral dosing (corresponding to AUC24h/MIC and Cmax/MIC ratios of 9.0 and 0.86, respectively). Experiment II Serum half-lives were varied to provide AUC24h/MIC ratios of 9.0, 18, and 36, with a constant Cmax/MIC ratio of 3.4, which was the minimum ratio needed to eradicate the bacteria in experiment I. The viable bacteria in this experiment were reduced below the limit of detection by at least 4 h after exposure to gatifloxacin (Fig. 2). However, bacteria increased rapidly, exceeding the counts in the original inoculum size, under the conditions of AUC24h/MIC ratios of 9.0 and 18. When the AUC24h/MIC ratio was adjusted to 36, no bacterial regrowth was observed over 24 h. The AUC24h/MIC ratios were associated with bactericidal activity (AAKC; Fig. 2), and eradication was observed at the AUC24h/MIC ratio of 36. No resistant strains to gatifloxacin were detected by experiment II.
Fig. 3. Bactericidal activity of gatifloxacin against S. pneumoniae J-69 at different Cmax/MIC ratios, of 0.86 (closed circles) and 1.7 (closed triangles), with a constant AUC24h/MIC ratio of 9.0. The numbers in parentheses and the dotted line indicate AAKC (log CFU/ml·h)and the lower limit of detection, respectively
Experiment III In order to study the impact of Cmax/MIC ratios on the bactericidal activities and resistance selectivity of gatifloxacin, Cmax and half-lives were varied to provide Cmax/ MIC ratios of 0.86 and 1.7, with a constant AUC24h/MIC ratio of 9.0, which was only the ratio at which the occurrence of resistant mutants was observed in experiment I. Mutants resistant to gatifloxacin were not obtained at a Cmax/MIC ratio of 1.7, but were obtained at a ratio of 0.86, although the bactericidal activity (AAKC) was almost the same (Fig. 3). The MIC of gatifloxacin for the resistant strains obtained here was 0.5 µg/ml. The resistant strains
213 Table 2. Gene mutation in the QRDR and antibacterial activity of various quinolones against the representative resistant mutant obtained after exposure to ciprofloxacin MIC (µg/ml) CPFX Parent
1
Mutant
32
GFLX 0.25 4
Gene mutation LVFX 1 16
gyrA parC gyrA parC
None None Ser81 Æ Phe Ser79 Æ Tyr
CPFX, ciprofloxacin; GFLX, gatifloxacin; LVFX, levofloxacin; QRDR, quinolone resistance-determining region
Fig. 4. Bactericidal activities of fluoroquinolones against S. pneumoniae J-69, simulating serum concentrations after clinical dosing. Closed circles, 200 mg 2 gatifloxacin; closed triangles, 100 mg 3 levofloxacin; closed squares, 200 mg 3 ciprofloxacin; closed diamonds, growth control. Dotted line indicates lower limit of detection
were only twofold less susceptible to gatifloxacin than the parent strains, and the representative resistant strains possessed single-point mutations in the QRDR of gyrA (Ser81 Æ Phe). Comparative pharmacodynamic (PD) profiles of gatifloxacin, levofloxacin, and ciprofloxacin According to the Japanese clinical regimen, serum concentrations after the oral dosing of gatifloxacin (200 mg b.i.d.), levofloxacin (100 mg t.i.d.), and ciprofloxacin (200 mg t.i.d.) were simulated. Time-kill curves of these quinolones are presented in Fig. 4. Gatifloxacin and levofloxacin reduced bacteria below the limit of detection. A more rapid rate of killing was observed with gatifloxacin than with levofloxacin. On the other hand, ciprofloxacin demonstrated lower bactericidal activity over the first 12 h, with the occurrence of subsequent regrowth of bacteria by 24 h. The MIC of ciprofloxacin against regrown bacteria was 32 µg/ml, although the MIC against the parent J-69 was 1 µg/ml. The MICs of gatifloxacin and levofloxacin against these resistant strains increased to 4 µg/ml and 16 µg/ml, respectively. These highly resistant strains possessed single-point mutations in the QRDR of both gyrA (Ser81 Æ Phe) and parC (Ser79 Æ Tyr) (Table 2).
Discussion Recently, the pharmacodynamics of fluoroquinolones have been investigated well, using in vitro PK models. It was suggested that AUC/MIC ratios of 30–60 were sufficient to eradicate S. pneumoniae.5–9 However, because fluoroquinolones kill bacteria in a concentration-dependent manner,4
not only the AUC/MIC but also the Cmax/MIC might affect their pharmacodynamics. Therefore, it was important to consider the effect of the AUC/MIC ratio and the Cmax/MIC ratio on the pharmacodynamics of fluoroquinolones against S. pneumoniae. In order to investigate the impact of AUC/ MIC ratios on the antipneumococcal activities of gatifloxacin, S. pneumoniae were exposed to gatifloxacin at various AUC/MIC ratios with a constant Cmax/MIC ratio of 3.4. The AUC24h/MIC ratios were associated with bactericidal activity (AAKC), and eradication was observed at the AUC24h/MIC ratio of 36 (Fig. 2). Also, no resistant strains to gatifloxacin were detected by experiment II. These results suggest that the bactericidal activity of gatifloxacin is mainly dependent on AUC24h/MIC. Moreover, AUC/MIC ratios of 36 might be sufficient for the bacterial eradication. Our results agreed with the ideas from previous PK/PD studies in which AUC/MIC ratios of approximately 30 to 60 were sufficient for the eradication of S. pneumoniae.5–9 Resistance selectivity is one of the important PD factors in antibacterial agents. Pharmacokinetic parameters, as well as the target preference of quinolones, are thought to be associated with their resistance selectivity. The Cmax/MIC ratio has been suggested as a predictive parameter for resistance selectivity, and Cmax/MIC ratios ranging from 2 to 10 might be necessary for the prevention of resistance selection.5,10–12 We compared the resistance selectivity at Cmax/ MIC ratios of 0.87 and 1.7 with a constant AUC24h/MIC ratio of 9.0. Resistant mutants to gatifloxacin were not obtained at a Cmax/MIC ratio of 1.7 but were obtained at a ratio of 0.86, although the bactericidal activity (AAKC) was almost the same (Fig. 3). These results suggest that the selection of resistance after exposure to gatifloxacin in the PK model appeared to be mainly dependent on the Cmax/MIC ratio, and a Cmax/MIC ratio of 1.7 might be necessary to prevent the occurrence of a resistant strain. We focused on relationships between the AUC24h/MIC or Cmax/MIC and pharmacodynamics, because quinolones kill bacteria in a concentration-dependent manner. Also, the time of concentration above the MIC (T MIC) is known to be affected by the pharmacodynamics of antibacterial agents, especially time-dependent ones such as the β-lactams and macrolides. In this study, eradication of S. pneumoniae J-69 required that the concentrations of gatifloxacin remain above the MIC for 14 h (data not shown). However, because there were limited data for T MIC, further studies about T MIC will be needed.
214
The incidence of S. pneumoniae with multidrug resistance has increased worldwide. Older fluoroquinolones such as ciprofloxacin have demonstrated poor activity against gram-positive pathogens, including S. pneumoniae. The respiratory fluoroquinolones, such as gatifloxacin and moxifloxacin, have demonstrated good activity against these organisms. According to the Japanese clinical regimen, serum concentrations after the oral dosing of gatifloxacin (200 mg b.i.d.), levofloxacin (100 mg t.i.d.), and ciprofloxacin (200 mg t.i.d.) were simulated. In this comparative experiment, we showed that gatifloxacin demonstrated better bactericidal activity than ciprofloxacin and levofloxacin. Also, resistant strains appeared in the regrown bacteria only after exposure to ciprofloxacin. Reduced bactericidal activity of ciprofloxacin was observed at the second and third doses compared with the activity at the first dose. At the first dose, the bactericidal activity of ciprofloxacin was exerted against the wild-type population. On the other hand, the bactericidal activity at the second and third doses might have been exerted against the resistant strains selected at the first dose of ciprofloxacin. Therefore, bactericidal activity of ciprofloxacin might be lower at the second and third doses. The resistant strains selected by ciprofloxacin showed 32-fold less susceptibility to ciprofloxacin and 16-fold less susceptibility to gatifloxacin and levofloxacin as compared with those of the parent strain, and possessed mutations in both gyrA and parC, which encode the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV, respectively. We have already shown that resistance acquisition was observed in a static condition and that ciprofloxacin selected gyrA-parC double-mutant strains from parC mutants,17,18 so that in this PK model, ciprofloxacin might select parC mutants first, and then select gyrA mutants, to be gyrAparC double-mutants. Other investigators reported that ciprofloxacin readily selected resistant strains in comparison with the respiratory fluoroquinolones in PK models.10–12,19 Our results also suggest that ciprofloxacin might be more likely to select a resistant mutant than gatifloxacin or levofloxacin in the clinical setting. In summary, AUC24h/MIC ratios of 36 and Cmax/MIC ratios of 1.7 appear to be needed for bacterial eradication and for the prevention of the possible emergence of S. pneumoniae strains resistant to gatifloxacin, respectively. In the Japanese clinical regimen, gatifloxacin (200 mg b.i.d.) demonstrated better PD profiles against S. pneumoniae than ciprofloxacin and levofloxacin. This clinically relevant oral dosage of gatifloxacin is anticipated to result in a high AUC24h/MIC90 ratio1 of 81 and a Cmax/MIC90 ratio of 4.4, suggesting that this agent is clinically effective in the treatment of pneumococcal infections.
2.
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4. 5.
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12.
13. 14. 15. 16. 17. 18.
19.
References 1. Tsurumaki Y, Manda H, Takei M, Hosaka M. In vitro antimicrobial activity of gatifloxacin against 873 clinical isolates from respi-
ratory tract, urinary tract and surgical infections during 1997–1998 in Japan. J Antimicrob Chemother 2000;45:685–9. Doerm GV, Pfaller MA, Kugler K, Freeman J, Joes RN. Prevalence of antimicrobial resistance among respiratory tract isolates of Streptococcus pneumoniae in North America: 1997 results from the SENTRY antimicrobial surveillance program. Clin Infect Dis 1998;27:764–70. Hosaka M, Yasue T, Fukuda H, Tomizawa H, Aoyama H, Hirai K. In vitro and in vivo antibacterial activity of AM-1155, a new 6-fluoro-8-methoxy quinolone. Antimicrob Agents Chemother 1992;36:2108–17. Wakabayashi E, Mitsuhashi S. In vitro antibacterial activity of AM-1155, a novel 6-fluoro-8-methoxy quinolone. Antimicrob Agents Chemother 1994;38:594–601. Lister PD. Pharmacodynamics of gatifloxacin against Streptococcus pneumoniae in an in vitro pharmacokinetic model: impact of area under the curve/MIC ratios on eradication. Antimicrob Agents Chemother 2002;46:69–74. Lacey MK, Xiaowei WL, Tessier PR, Nicolau DP, Quintilliani R, Nightngale CH. Pharmacodynamic comparisons of levofloxacin, ciprofloxacin and ampicillin against Streptococcus pneumoniae in an in vitro model of infection. Antimicrob Agents Chemother 1999;43:672–7. Lister PD, Sanders CC. Pharmacodynamics of trovafloxacin, ofloxacin and ciprofloxacin against Streptococcus pneumoniae in an in vitro pharmacokinetic model. Antimicrob Agents Chemother 1999;43:1118–23. Hershberger E, Rybak MJ. Activities of trovafloxacin, clinafloxacin, sparfloxacin, levofloxacin, and ciprofloxacin against penicillin-resistant Streptococcus pneumoniae in an in vitro infection model. Antimicrob Agents Chemother 2000;44:598–601. Zhanel GG, Roberts D, Waltky A, Laing N, Nichol K, Hoban DJ. Pharmacodynamic activity of fluoroquinolones against ciprofloxacin-resistant Streptococcus pneumoniae. J Antimicrob Chemother 2002;49:807–12. Madaras-Kelly KJ, Demasters TA. In vitro characterization of fluoroquinolone concentration/MIC antimicrobial activity and resistance while simulating clinical pharmacokinetics of levofloxacin, ofloxacin, or ciprofloxacin against Streptococcus pneumoniae. Diagn Microbiol Infect Dis 2000;37:253–60. Thorburn CE, Edwards D. The effect of pharmacokinetics on the bactericidal activity of ciprofloxacin and sparfloxacin against Streptococcus pneumoniae and the emergence of resistance. J Antimicrob Chemother 2001;48:15–22. Klepser ME, Ernst EJ, Petzold CR, Rhomberg P, Doern GV. Comparative bactericidal activities of ciprofloxacin, clinafloxacin, grepafloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Streptococcus pneumoniae in a dynamic in vitro model. Antimicrob Agents Chemother 2001;45:673–8. Nakashima M, Uematsu T, Kosuge K, Kusajima H, Ooie T, Uchida H. Phase I study of gatifloxacin, a new quinolone I.Single-dose study. Jpn J Chemotherapy 1999;47(S-2):175–207. Nakajima M, Uematsu T, Kanamaru M, Okazai O, Hakusui H. Phase I study of levofloxacin, (S)-(-)-ofloxacin. Jpn J Clin Pharmacol Ther 1992;23:515–20. Matsumoto F, Kobayashi H, Takamura K, Takeda H. Pharmacokinetics with BAY O 9867 (ciprofloxacin). Jpn J Chemotherapy 1985;33(S-7):140–88. Fukuda H, Hiramatsu K. Primary targets of fluoroquinolones in Streptococcus pneumoniae. Antimicrob Agents Chemother 1999;43:410–12. Fukuda H. Genetic study of the mechanism of fluoroquinolones in Streptococcus pneumoniae. Jpn J Chemother 2000;48:243–50. Fukuda H, Kishii R, Takei M, Hosaka M. Contributions of the 8-methoxy group of gatifloxacin to resistance selectivity, target preference and antibacterial activity against Streptococcus pneumoniae. Antimicrob Agents Chemother 2001;45:1649–53. Madaras-Kelly KJ, Ostergaad BE, Hovde LB, Rotscafer JC. Twenty-four-hour area the concentration-time curve/MIC ratio as a generic predictor of fluoroquinolone antimicrobial effect by using three strains of Pseudomonas aeruginosa and in vitro pharmacodynamic model. Antimicrob Agents Chemother 1996;40:627– 32.
© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2003
ORIGINAL ARTICLE Hiroyuki Ebisu · Ryuta Kishii · Masaya Takei Hideyuki Fukuda
The effect of pharmacokinetic/pharmacodynamic (PK/PD) parameters of gatifloxacin on its bactericidal activity and resistance selectivity against clinical isolates of Streptococcus pneumoniae
Received: October 9, 2002 / Accepted: March 19, 2003
Abstract The impact of the pharmacokinetic/pharmacodynamic (PK/PD) parameters (the 24h area under the concentration-time curve [AUC24h]/minimum inhibitory concentration [MIC] and maximum concentration in serum [Cmax]/MIC ratio) after single oral dosing of gatifloxacin on its bactericidal activity and resistance selectivity against quinolone-susceptible clinical isolates of Streptococcus pneumoniae J-69 was investigated using an in vitro PK model. The MICs of gatifloxacin, levofloxacin, and ciprofloxacin were 0.25, 1, and 1 µg/ml, respectively. When the range of AUC24h/MIC ratios was varied from 9.0 to 36 with a constant Cmax/MIC ratio of 3.4, the bactericidal activity was correlated with the AUC24h/MIC ratios. Eradication was observed at an AUC24h/MIC ratio of 36. On the other hand, the resistance selectivity was associated with the Cmax/ MIC ratio. Mutant strains were selected at a Cmax/MIC ratio of 0.84, but not 1.7 with a constant AUC24h/MIC ratio of 9.0. These results suggested that an AUC24h/MIC ratio of 36 and a Cmax/MIC ratio of 1.7 might be possible benchmarks to show enough bacterial eradication and prevention of emergence of resistant strains to gatifloxacin, respectively. When the serum concentrations after clinical oral dosing of gatifloxacin (200 mg b.i.d.), levofloxacin (100 mg t.i.d.), and ciprofloxacin (200 mg t.i.d.) were simulated, the bactericidal activity of gatifloxacin was higher than those of levofloxacin and ciprofloxacin. Moreover, no resistant strain was obtained by the exposure to gatifloxacin and levofloxacin, whereas ciprofloxacin selected resistant strains. The clinically relevant oral dosage of gatifloxacin was anticipated to result in a high AUC24h/MIC90 ratio of 81 and a Cmax/MIC90 ratio of 4.4, suggesting that this agent is clinically effective in the treatment of pneumococcal infections.
Introduction Streptococcus pneumoniae remains the leading cause of bacterial pneumonia and is the most common pathogen associated with otitis media, sinusitis, and meningitis. Recent increases in the incidence of resistance to β-lactam and macrolide antibiotics among S. pneumoniae1,2 have meant that quinolones are now being considered for use in the treatment of these infections. However, the activities of quinolones against gram-positive organisms have been variable among the agents. Newer quinolones, such as gatifloxacin, have demonstrated increased activity against S. pneumoniae1,3,4 and also offer many advantages over the older quinolones in terms of broad antibacterial spectrum and favorable pharmacokinetics. The contributions of these favorable profiles of newer quinolones to efficacy have been examined in many studies using in-vitro pharmacokinetic (PK) models. The 24h area under the concentration-time curve (AUC)24h/minimum inhibitory concentration (MIC) ratios have been identified as possible predictors of the bactericidal effect of quinolones.5–9 The optimal efficacy of quinolones has been observed at AUC24h/MIC ratios of 30–60.5–9 Some investigators have also suggested that maximum concentration in serum (Cmax)/MIC ratios ranging from 2 to 10 are required to prevent the selection of resistance to quinolones.5,10–12 In this study, we investigated the effect of pharmacokinetic/pharmacodynamic (PK/PD) parameters of gatifloxacin on its bactericidal activity and resistance selectivity against S. pneumoniae, and compared its efficacy with those of levofloxacin and ciprofloxacin by simulating the serum concentration according to the Japanese clinical regimen.
Key words Streptococcus pneumoniae · In vitro pharmacokinetic model · Gatifloxacin · AUC24h/MIC · Cmax/MIC
Materials and methods H. Ebisu (*) · R. Kishii · M. Takei · H. Fukuda Infectious Diseases, Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd., 2399-1, Nogi, Nogi-machi, Shimotsuga, Tochigi 329-0114, Japan Tel. 81-280-562201; Fax 81-280-571293 e-mail: [email protected]
Quinolones Gatifloxacin and levofloxacin were synthesized at Kyorin Pharmaceutical (Tokyo, Japan). Ciprofloxacin
211 Table 1. Pharmacokinetic parameters at clinical doses of gatifloxacin, levofloxacin, and ciprofloxacin simulated in the in vitro pharmacodynamic model Drug
Dose, regimen
Cmax (µg/ml)
t1/2 (h)
AUC24h µg/ml·h
Cmax/MIC
AUC/MIC
Reference no.
Gatifloxacin Levofloxacin Ciprofloxaci
200 mg b.i.d 100 mg t.i.d 200 mg t.i.d
1.71 1.36 1.37
7.1 5.1 2.8
31.7 28.0 19.6
6.8 1.4 1.4
127 28 20
13 14 15
MIC, minimum inhibitory concentration; Cmax, maximum concentration; AUC24h, the 24h area under the concentration-time curve
was purchased from Apin (Abimgdon, Oxfordshire, UK). Bacterial strain The clinical strain of S. pneumoniae J-69 was isolated from a patient with respiratory tract infection in 1996 in Japan. The MICs of gatifloxacin, levofloxacin, and ciprofloxacin for the strain were 0.25, 1, and 1 µg/ml, respectively, which correspond to the MIC90 for clinical isolates.1 The strain was found to lack mutations in the quinolone resistancedetermining region (QRDR) of gyrA and parC.
In vitro pharmacokinetic (PK) model A dilutional in vitro PK model (PASS-400; Dainipponseiki, Kyoto, Japan) was used to simulate serum concentrations of the quinolones based on the PK parameters decided upon. The bacteria was grown in Todd-Hewitt broth (Difco, Sparks, MD, USA) at 37°C. A starting inoculum size of 106–107 CFU/ml was prepared from overnight culture for all the experiments. To ensure that the bacteria were in logarithmic growth phase, the bacteria were left in Todd-Hewitt broth for 2 h before exposure to the drugs in the model. All experiments were performed in duplicate to confirm reproducibility. The effect of PK/PD parameters on the bactericidal activity and the resistance selectivity of gatifloxacin We performed three experiments, as follows. Experiment I Based on the PK parameters of single oral dosing of 200 mg of gatifloxacin,13 serum concentrations of gatifloxacin after oral dosing of 12.5, 25, 50, 100, and 200 mg were proportionally simulated while maintaining a constant half-life (7.1 h).
Experiment III Cmax and half-lives were varied to provide Cmax/MIC ratios of 0.86 and 1.7 with a constant AUC24h/MIC ratio of 9.0, which was only the ratio at which the occurrence of a resistant mutant was observed in experiment I. Comparative pharmacodynamic (PD) profiles of gatifloxacin, levofloxacin, and ciprofloxacin According to the Japanese clinical regimen, the serum concentrations after oral dosing of gatifloxacin (200 mg b.i.d.), levofloxacin (100 mg t.i.d.), and ciprofloxacin (200 mg t.i.d.) were simulated. An appropriate amount of quinolone was added to a culture flask at 0 and 12 h for gatifloxacin and at 0, 8, and 16 h for levofloxacin and ciprofloxacin. The simulated PK parameters13–15 of the test quinolones are shown in Table 1. Pharmacodynamic (PD) analysis At 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h, samples were removed from the culture flask (100 ml) and viable bacteria were counted by plating 100 µl of serial ten fold dilutions onto Heart Infusion Agar (Eiken, Tokyo, Japan) supplemented with 5% (v/v) defibrinated horse blood (Nippon Bio-test Laboratories, Tokyo, Japan). The limit of detection of bacterial numbers was 102 CFU/ml. Quantitative evaluation of bactericidal activity was expressed as the area above the killing-curve (AAKC). To evaluate the resistance selectivity, regrown bacteria were plated onto an agar plate containing the respective quinolones at 2, 4, 8, and 16 MIC. If the strains grew on the agar plate containing the quinolones, their drug susceptibility and gene mutations in the QRDR of gyrA and parC of the representative strain were investigated as described previously.16
Results Experiment II Serum half-lives were varied to provide AUC24h/MIC ratios of 9.0, 18, and 36, with a constant Cmax/MIC ratio of 3.4, which was the minimum ratio needed to eradicate the bacteria in experiment I.
The effect of PK/PD parameters on the bactericidal activity and the resistance selectivity of gatifloxacin In order to study the impact of PK/PD parameters (AUC24h/ MIC and Cmax/MIC ratios) on the bactericidal activities and
212
Fig. 1. Bactericidal activity of gatifloxacin simulating different single oral doses against Streptococcus pneumoniae J-69. Closed circles, 12.5 mg; closed triangles, 25 mg; closed squares, 50 mg; open circles, 100 mg; open triangles, 200 mg; closed diamonds growth control. Dotted line indicates lower limit of detection
Fig. 2. Bactericidal activity of gatifloxacin against S. pneumoniae J-69 at different AUC24h/minimum inhibitory concentration (MIC) ratios, of 9.0 (closed circles); 18 (closed triangles); and 36 (closed squares), with a constant maximum concentration in serum (Cmax)/MIC ratio of 3.4. The numbers in parentheses and the dotted line indicate the area above the killing curve (AAKC; log CFU/ml·h) and the lower limit of detection, respectively
the resistance selectivity of gatifloxacin, experiments were performed under conditions of various PK/PD parameters. Experiment I Time-kill curves for gatifloxacin at the simulated serum concentration after oral dosing of 12.5, 25, 50, 100, and 200 mg are shown in Fig. 1. At doses of 100 mg (corresponding to AUC24h/MIC and Cmax/MIC ratios of 36 and 3.4, respectively), gatifloxacin rapidly reduced bacteria below the limit of detection (102 CFU/ml) within 5 h after exposure. In contrast, at doses of 50 mg, decreases in viable bacterial counts slowed, and then the bacteria increased, compared with the original inoculum size, in 24 h. Resistant strains that possessed gyrA mutation were observed after exposure to gatifloxacin only at the simulated serum concentration after 25 mg oral dosing (corresponding to AUC24h/MIC and Cmax/MIC ratios of 9.0 and 0.86, respectively). Experiment II Serum half-lives were varied to provide AUC24h/MIC ratios of 9.0, 18, and 36, with a constant Cmax/MIC ratio of 3.4, which was the minimum ratio needed to eradicate the bacteria in experiment I. The viable bacteria in this experiment were reduced below the limit of detection by at least 4 h after exposure to gatifloxacin (Fig. 2). However, bacteria increased rapidly, exceeding the counts in the original inoculum size, under the conditions of AUC24h/MIC ratios of 9.0 and 18. When the AUC24h/MIC ratio was adjusted to 36, no bacterial regrowth was observed over 24 h. The AUC24h/MIC ratios were associated with bactericidal activity (AAKC; Fig. 2), and eradication was observed at the AUC24h/MIC ratio of 36. No resistant strains to gatifloxacin were detected by experiment II.
Fig. 3. Bactericidal activity of gatifloxacin against S. pneumoniae J-69 at different Cmax/MIC ratios, of 0.86 (closed circles) and 1.7 (closed triangles), with a constant AUC24h/MIC ratio of 9.0. The numbers in parentheses and the dotted line indicate AAKC (log CFU/ml·h)and the lower limit of detection, respectively
Experiment III In order to study the impact of Cmax/MIC ratios on the bactericidal activities and resistance selectivity of gatifloxacin, Cmax and half-lives were varied to provide Cmax/ MIC ratios of 0.86 and 1.7, with a constant AUC24h/MIC ratio of 9.0, which was only the ratio at which the occurrence of resistant mutants was observed in experiment I. Mutants resistant to gatifloxacin were not obtained at a Cmax/MIC ratio of 1.7, but were obtained at a ratio of 0.86, although the bactericidal activity (AAKC) was almost the same (Fig. 3). The MIC of gatifloxacin for the resistant strains obtained here was 0.5 µg/ml. The resistant strains
213 Table 2. Gene mutation in the QRDR and antibacterial activity of various quinolones against the representative resistant mutant obtained after exposure to ciprofloxacin MIC (µg/ml) CPFX Parent
1
Mutant
32
GFLX 0.25 4
Gene mutation LVFX 1 16
gyrA parC gyrA parC
None None Ser81 Æ Phe Ser79 Æ Tyr
CPFX, ciprofloxacin; GFLX, gatifloxacin; LVFX, levofloxacin; QRDR, quinolone resistance-determining region
Fig. 4. Bactericidal activities of fluoroquinolones against S. pneumoniae J-69, simulating serum concentrations after clinical dosing. Closed circles, 200 mg 2 gatifloxacin; closed triangles, 100 mg 3 levofloxacin; closed squares, 200 mg 3 ciprofloxacin; closed diamonds, growth control. Dotted line indicates lower limit of detection
were only twofold less susceptible to gatifloxacin than the parent strains, and the representative resistant strains possessed single-point mutations in the QRDR of gyrA (Ser81 Æ Phe). Comparative pharmacodynamic (PD) profiles of gatifloxacin, levofloxacin, and ciprofloxacin According to the Japanese clinical regimen, serum concentrations after the oral dosing of gatifloxacin (200 mg b.i.d.), levofloxacin (100 mg t.i.d.), and ciprofloxacin (200 mg t.i.d.) were simulated. Time-kill curves of these quinolones are presented in Fig. 4. Gatifloxacin and levofloxacin reduced bacteria below the limit of detection. A more rapid rate of killing was observed with gatifloxacin than with levofloxacin. On the other hand, ciprofloxacin demonstrated lower bactericidal activity over the first 12 h, with the occurrence of subsequent regrowth of bacteria by 24 h. The MIC of ciprofloxacin against regrown bacteria was 32 µg/ml, although the MIC against the parent J-69 was 1 µg/ml. The MICs of gatifloxacin and levofloxacin against these resistant strains increased to 4 µg/ml and 16 µg/ml, respectively. These highly resistant strains possessed single-point mutations in the QRDR of both gyrA (Ser81 Æ Phe) and parC (Ser79 Æ Tyr) (Table 2).
Discussion Recently, the pharmacodynamics of fluoroquinolones have been investigated well, using in vitro PK models. It was suggested that AUC/MIC ratios of 30–60 were sufficient to eradicate S. pneumoniae.5–9 However, because fluoroquinolones kill bacteria in a concentration-dependent manner,4
not only the AUC/MIC but also the Cmax/MIC might affect their pharmacodynamics. Therefore, it was important to consider the effect of the AUC/MIC ratio and the Cmax/MIC ratio on the pharmacodynamics of fluoroquinolones against S. pneumoniae. In order to investigate the impact of AUC/ MIC ratios on the antipneumococcal activities of gatifloxacin, S. pneumoniae were exposed to gatifloxacin at various AUC/MIC ratios with a constant Cmax/MIC ratio of 3.4. The AUC24h/MIC ratios were associated with bactericidal activity (AAKC), and eradication was observed at the AUC24h/MIC ratio of 36 (Fig. 2). Also, no resistant strains to gatifloxacin were detected by experiment II. These results suggest that the bactericidal activity of gatifloxacin is mainly dependent on AUC24h/MIC. Moreover, AUC/MIC ratios of 36 might be sufficient for the bacterial eradication. Our results agreed with the ideas from previous PK/PD studies in which AUC/MIC ratios of approximately 30 to 60 were sufficient for the eradication of S. pneumoniae.5–9 Resistance selectivity is one of the important PD factors in antibacterial agents. Pharmacokinetic parameters, as well as the target preference of quinolones, are thought to be associated with their resistance selectivity. The Cmax/MIC ratio has been suggested as a predictive parameter for resistance selectivity, and Cmax/MIC ratios ranging from 2 to 10 might be necessary for the prevention of resistance selection.5,10–12 We compared the resistance selectivity at Cmax/ MIC ratios of 0.87 and 1.7 with a constant AUC24h/MIC ratio of 9.0. Resistant mutants to gatifloxacin were not obtained at a Cmax/MIC ratio of 1.7 but were obtained at a ratio of 0.86, although the bactericidal activity (AAKC) was almost the same (Fig. 3). These results suggest that the selection of resistance after exposure to gatifloxacin in the PK model appeared to be mainly dependent on the Cmax/MIC ratio, and a Cmax/MIC ratio of 1.7 might be necessary to prevent the occurrence of a resistant strain. We focused on relationships between the AUC24h/MIC or Cmax/MIC and pharmacodynamics, because quinolones kill bacteria in a concentration-dependent manner. Also, the time of concentration above the MIC (T MIC) is known to be affected by the pharmacodynamics of antibacterial agents, especially time-dependent ones such as the β-lactams and macrolides. In this study, eradication of S. pneumoniae J-69 required that the concentrations of gatifloxacin remain above the MIC for 14 h (data not shown). However, because there were limited data for T MIC, further studies about T MIC will be needed.
214
The incidence of S. pneumoniae with multidrug resistance has increased worldwide. Older fluoroquinolones such as ciprofloxacin have demonstrated poor activity against gram-positive pathogens, including S. pneumoniae. The respiratory fluoroquinolones, such as gatifloxacin and moxifloxacin, have demonstrated good activity against these organisms. According to the Japanese clinical regimen, serum concentrations after the oral dosing of gatifloxacin (200 mg b.i.d.), levofloxacin (100 mg t.i.d.), and ciprofloxacin (200 mg t.i.d.) were simulated. In this comparative experiment, we showed that gatifloxacin demonstrated better bactericidal activity than ciprofloxacin and levofloxacin. Also, resistant strains appeared in the regrown bacteria only after exposure to ciprofloxacin. Reduced bactericidal activity of ciprofloxacin was observed at the second and third doses compared with the activity at the first dose. At the first dose, the bactericidal activity of ciprofloxacin was exerted against the wild-type population. On the other hand, the bactericidal activity at the second and third doses might have been exerted against the resistant strains selected at the first dose of ciprofloxacin. Therefore, bactericidal activity of ciprofloxacin might be lower at the second and third doses. The resistant strains selected by ciprofloxacin showed 32-fold less susceptibility to ciprofloxacin and 16-fold less susceptibility to gatifloxacin and levofloxacin as compared with those of the parent strain, and possessed mutations in both gyrA and parC, which encode the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV, respectively. We have already shown that resistance acquisition was observed in a static condition and that ciprofloxacin selected gyrA-parC double-mutant strains from parC mutants,17,18 so that in this PK model, ciprofloxacin might select parC mutants first, and then select gyrA mutants, to be gyrAparC double-mutants. Other investigators reported that ciprofloxacin readily selected resistant strains in comparison with the respiratory fluoroquinolones in PK models.10–12,19 Our results also suggest that ciprofloxacin might be more likely to select a resistant mutant than gatifloxacin or levofloxacin in the clinical setting. In summary, AUC24h/MIC ratios of 36 and Cmax/MIC ratios of 1.7 appear to be needed for bacterial eradication and for the prevention of the possible emergence of S. pneumoniae strains resistant to gatifloxacin, respectively. In the Japanese clinical regimen, gatifloxacin (200 mg b.i.d.) demonstrated better PD profiles against S. pneumoniae than ciprofloxacin and levofloxacin. This clinically relevant oral dosage of gatifloxacin is anticipated to result in a high AUC24h/MIC90 ratio1 of 81 and a Cmax/MIC90 ratio of 4.4, suggesting that this agent is clinically effective in the treatment of pneumococcal infections.
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4. 5.
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