In Vitro and In Vivo Activities of Sitafloxacin against Chlamydia spp.

Abstract

The in vitro and in vivo antichlamydial activity of sitafloxacin was investigated. The MICs and minimal chlamydiacidal concentrations of sitafloxacin for various species of chlamydia ranged from 0.031 to 0.125 μg/ml. Sitafloxacin had an excellent therapeutic effect on experimental Chlamydia psittaci pneumonia and was more potent than tosufloxacin, ofloxacin, and ciproflxacin, although slightly less potent than sparfloxacin.

Sitafloxacin is a new fluoroquinolone antimicrobial agent, which has been reported to have a broader spectrum and more potent activity against gram-positive and gram-negative bacteria than currently available quinolones such as ofloxacin, ciprofloxacin, tosufloxacin, and sparfloxacin (11, 15, 17). In a preliminary study (12), sitafloxacin showed good clinical efficacy against pneumonia and chronic respiratory tract infection. In the present study, the in vitro and in vivo activity of sitafloxacin against chlamydiae was investigated and compared with the activities of other quinolones.

The antimicrobial agents tested were sitafloxacin and ofloxacin (Daiichi Pharmaceutical Co.), sparfloxacin (Dainippon Pharmaceutical Co.), tosufloxacin (Toyama Chemical Co.), and ciprofloxacin (Bayer Yakuhin Co.). These agents were dissolved in solutions according to the instructions of the respective manufacturers.

The standard strains of chlamydia tested were Chlamydia pneumoniae TW-183, AR-39, and AR-388 (purchased from the Washington Research Foundation, Seattle); IOL-207 and Kajaani-6 (obtained from P. Saikku, University of Helsinki, Helsinki, Finland); C. psittaci Budgerigar-1 (obtained from the National Institute of Health, Tokyo, Japan) and California 10 (obtained from A. Matsumoto, Department of Microbiology, Kawasaki Medical School, Kurashiki, Japan); and C. trachomatis D/UW-3/Cx, E/UW-5/Cx, and L2/434/Bu (obtained from the National Institute of Health). The MICs for 15 strains of C. pneumoniae, 7 strains of C. trachomatis, and 5 strains of C. psittaci isolated from patients with chlamydial respiratory and urinary infections were also examined.

The MIC and the minimal chlamydiacidal concentration (MCC) were determined by the standard methods of the Japan Society of Chemotherapy (4, 5). Briefly, confluent monolayers of HEp-2 cells were grown on coverslips and placed into 24-well cell culture plastic plates. The cells were then inoculated with 10⁴ inclusion-forming units (IFU) per well of one of the test organisms (multiplicity of infection of 0.05) by centrifugation (900 × g for 60 min). Next, 1 ml of a culture medium consisting of Eagle minimal essential medium (Nissui Pharmaceuticals Co., Tokyo, Japan), 10% heat-inactivated fetal calf serum (Gibco-BRL/Life Technologies, Inc., Grand Island, N.Y.), and cycloheximide (Nakarai Tesque, Inc., Tokyo, Japan) at a final concentration of 1 μg/ml was applied. The medium also contained one concentration of one of the test agents. The plates were then incubated in 5% CO₂ at 35°C for 72 h for C. pneumoniae, at 37°C for 48 h for C. psittaci, or at 37°C for 72 h for C. trachomatis. After incubation, a genus-specific fluorescein isothiocyanate-conjugated monoclonal antibody (Chlamydia FA Seiken; Denka Seiken, Tokyo, Japan) was used to stain inclusions. Inclusion bodies formed in the cells were observed with a Nikon epifluorescence microscope at ×200 or ×400 magnification, and the MIC was defined as the lowest concentration at which there was complete inhibition of inclusions. The MCC was determined by aspirating the antibiotic-containing medium, washing the wells twice with phosphate-buffered saline, harvesting the cells, infecting the new cells, and adding antibiotic-free medium. Cultures were reincubated under the conditions described above and then fixed and stained as described above. The MCC was the lowest antibiotic concentration that resulted in no inclusions after reincubation with antibiotic-free medium. All tests were performed in triplicate.

The MIC and MCC ranges of sitafloxacin and the other four fluoroquinolones for the three species of chlamydiae are shown in Table 1. Sitafloxacin and sparfloxacin had the most potent in vitro activity among the fluoroquinolones tested, with an MIC range of 0.031 to 0.125 μg/ml. There were no significant differences in the MICs of any of the drugs for the different chlamydial species.

TABLE 1.

In vitro activities of sitafloxacin and other fluoroquinolones against Chlamydia spp.

Chlamydia sp. and agent (n)	MIC (μg/ml)			MCC (μg/ml)
	Range	MIC50	MIC90	Range	MCC90
Chlamydia pneumoniae (20)
Sitafloxacin	0.031–0.125	0.063	0.063	0.031–0.125	0.063
Sparfloxacin	0.031–0.125	0.063	0.063	0.031–0.125	0.063
Tosufloxacin	0.063–0.25	0.125	0.125	0.063–0.25	0.125
Ofloxacin	0.5–2.0	0.5	1.0	0.5–2.0	1.0
Ciprofloxacin	1.0–4.0	1.0	2.0	1.0–4.0	4.0
Chlamydia trachomatis (10)
Sitafloxacin	0.031–0.063	0.063	0.063	0.031–0.063	0.063
Sparfloxacin	0.031–0.063	0.063	0.063	0.031–0.063	0.063
Tosufloxacin	0.063–0.125	0.125	0.125	0.063–0.125	0.125
Ofloxacin	0.5–1.0	0.5	1.0	0.5–2.0	1.0
Ciprofloxacin	1.0–2.0	1.0	2.0	1.0–4.0	4.0
Chlamydia psittaci (7)
Sitafloxacin	0.031–0.063	0.063	0.063	0.031–0.063	0.063
Sparfloxacin	0.031–0.063	0.063	0.063	0.031–0.063	0.063
Tosufloxacin	0.125–0.125	0.125	0.125	0.125–0.125	0.125
Ofloxacin	0.5–1.0	0.5	1.0	0.5–2.0	2.0
Ciprofloxacin	1.0–2.0	1.0	2.0	1.0–4.0	4.0

The therapeutic effect of sitafloxacin in a mouse model of chlamydial pneumonia was also evaluated. HeLa 229 cells infected with C. psittaci California 10 were sonicated and then diluted with sucrose-phoshate-glutamic acid medium to an appropriate titer. Five-week-old male MCH-ICR mice (CLEA Japan, Inc., Osaka, Japan) were infected with the resulting solution (10⁵ IFU per animal) by nasal instillation, as reported previously (9). The mice were given sitafloxacin (5 to 0.63 mg/kg), sparfloxacin (5 to 0.63 mg/kg), tosufloxacin (5 to 1.25 mg/kg), ofloxacin (10 to 2.5 mg/kg), or ciprofloxacin (10 to 2.5 mg/kg) by gavage twice a day for seven days, starting from the day after infection. Each dosing group consisted of eight mice. One group of mice served as an infected, untreated control. The number of surviving mice was recorded daily for 21 days. The 50% effective dose (ED₅₀) was calculated by probit analysis (8), and the 95% confidence limit was calculated by the method of Litchfield and Wilcoxon (7).

The in vivo antichlamydial activities (ED₅₀) of sitafloxacin and the other four fluoroquinolones against C. psittaci were as follows in milligrams/kilograms/dose (95% confidence limit): sitafloxacin, 1.13 (0.81–1.56); sparfloxacin, 0.95 (0.59–1.34); tosufloxacin, 1.56 (0.64–2.23); ofloxacin, 7.16 (5.07–11.83); and ciprofloxacin, 9.20 (6.99–14.16). The oral ED₅₀ value for sitafloxacin (1.13 mg/kg/dose) was similar to those for sparfloxacin (0.95 mg/kg/dose) and tosufloxacin (1.56 mg/kg/dose) and statistically less than the values for ofloxacin (7.16 mg/kg/dose) and ciprofloxacin (9.20 mg/kg/dose) (P < 0.001). All of the untreated control mice died from 4 to 7 days after inoculation.

Chlamydial species are well-known respiratory pathogens that cause upper and lower respiratory tract infections and pneumonia. C. trachomatis causes pneumonia in neonates, C. psittaci is a causative organism of psittacosis, and C. pneumoniae is recognized as an important respiratory tract pathogen throughout the world (6, 10).

Some of the recently developed quinolones have shown excellent antichlamydial activity in vitro and in vivo, as well as in clinical studies (1–3, 9, 13, 14, 16). According to epidemiological studies of acute respiratory tract infections, good activity against chlamydiae is an important criterion to consider when choosing an oral antibiotic for the treatment of respiratory infections, since such infections are commonly caued by chlamydiae. Recently, Hammerschlag and coworkers reported the microbiologic efficacy of levofloxacin and moxifloxacin against community-acquired pneumonia due to C. pneumoniae by a method that utilized culture (2, 3). In these studies, these quinolones showed high eradication rates.

We also previously reported on the experimental and clinical effectiveness of sparfloxacin against acute chlamydial respiratory infections (9, 16). An animal experiment with the same mouse model of C. psittaci pneumonia as that used in the present study indicated that sparfloxacin was a promising agent for the treatment of chlamydial respiratory infections. Sparfloxacin also showed an clinical efficacy rate of 88.9% in the treatment of 18 proven chlamydial infections in a clinical study. However, these data were obtained by using antigenic and serologic evidence of acute chlamydial infection but were not based on culture.

In the present study, sitafloxacin showed good in vitro antichlamydial activity that was equal to or more potent than that of sparfloxacin, tosufloxacin, ofloxacin, and ciprofloxacin. Our experiments with mice also showed that the therapeutic effect of sitafloxacin against C. psittaci pneumonia was excellent and was almost equal to that of sparfloxacin. The difference in potency might be the result of different pharmacokinetics among the compounds. Therefore, pharmacokinetic information for the five fluoroquinolones was obtained from the respective pharmaceutical companies. Area under the concentration-time curve (AUC) values corrected at a single oral dose of 10 mg/kg in mice were as follows (in micrograms per milliliter per hour): sitafloxacin, 0.34; sparfloxacin, 1.48; tosufloxacin, 2.68; ciprofloxacin, 0.47; and ofloxacin, 3.26 (unpublished results). Sitafloxacin showed the lowest AUC among the test compounds, a finding which indicated that the in vivo potency seen in the study was not due to a better pharmacokinetic profile. On the contrary, sitafloxacin exerted equivalent therapeutic potency compared to tosufloxacin and sparfloxacin in spite of its lower bioavailability in mice. Phase III clinical studies of sitafloxacin are currently under way in Japan. Based on its strong in vitro and in vivo antichlamydial activity, sitafloxacin is expected to have clinical potential for use against chlamydial infections. Prospective studies of sitafloxacin for the treatment of community-acquired pneumonia that detect chlamydiae by culture are needed to determine the role of this drug in the treatment of such infections.