Journal of Antimicrobial Chemotherapy (2000) 45, 489-492
© 2000 The British Society for Antimicrobial Chemotherapy
Comparative therapeutic efficacy of clinafloxacin in a pneumococcal meningitis mouse model
Department of Infectious Diseases, Parke-Davis Pharmaceutical Research, Division of Warner–Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
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Abstract |
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The therapeutic efficacy of clinafloxacin, a fluoroquinolone in clinical trials, was compared with that of ciprofloxacin and ceftriaxone in a novel pneumococcal meningitis mouse model. Mice were challenged by the intracerebral ventricular route with 50 ÌL of a lethal bacterial suspension and treated subcutaneously 2 h later. Both penicillin-susceptible and multidrug-resistant pneumococcal strains were used for evaluation. Survival percentages were calculated as the median curative dose (CD50) using log-probit statistical methods. Ceftriaxone was the most active agent against the penicillin-susceptible strain (CD50 = 2 mg/kg), but showed a 30-fold decrease in potency against the resistant strain. Clinafloxacin was equally effective against both strains, and proved to be the most active agent against the penicillin-resistant pneumococcus.
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Introduction |
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Streptococcus pneumoniae and Neisseria meningitidis are the most common causes of community-acquired bacterial meningitis throughout the world.1 Pneumococcal meningitis is associated with high mortality and frequent development of neurological sequelae, particularly ototoxicity. In recent years, the treatment of pneumococcal meningitis has become problematical owing to the emergence of penicillin- and cephalosporin-resistant strains.2 Currently, the standard empirical treatment of this disease involves the use of a broad-spectrum, third-generation cephalosporin such as ceftriaxone.3 Several reports over the past few years have cited cephalosporin treatment failures in the management of patients with pneumococcal meningitis.4 The newer fluoroquinolones currently under development have shown excellent pharmacokinetic profiles and show great improvement over their predecessors with regard to absorption, distribution volume and tissue penetration.5 In addition, these newer agents exhibit longer serum half-lives which potentially allow for once-daily dosing. Clinafloxacin, a novel aminopyrrolidinyl fluoroquinolone currently in clinical trials, has shown outstanding in vitro activity against multi-resistant S. pneumoniae, including recent clinical isolates with mutations in the genes for DNA gyrase (gyrA) and topoisomerase IV (parC and parE).6 In this study, a novel mouse pneumococcal meningitis model was used to evaluate the potential therapeutic efficacy of clinafloxacin compared with that of ciprofloxacin and ceftriaxone.
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Materials and methods |
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Bacterial strains
S. pneumoniae SVI (ATCC 10015), a penicillin-susceptible strain, was obtained from the American Type Culture Collection (Rockville, MD, USA). S. pneumoniae 2521, a multiple drug-resistant strain, was obtained from Dr Anna Marton of the National Institute of Public Health in Budapest, Hungary. MIC values of penicillin G and ceftriaxone for this organism were 8 mg/L and 4 mg/L, respectively.
Antimicrobial agents
Clinafloxacin was synthesized at Parke-Davis Pharmaceutical Research (Ann Arbor, MI, USA). Ciprofloxacin and ceftriaxone were obtained from Sigma Chemical Co. (St Louis, MO, USA).
Animals
All tests were done using 18–22 g female CD-1 mice (Charles River Laboratories, Portage, MI, USA). These studies were approved by the Institutional Animal Care and Use Committee (Parke-Davis Pharmaceutical Research).
Antimicrobial susceptibility tests
MICs were determined according to the guidelines of the National Committee for Clinical Laboratory Standards.7
Preparation of inoculum
The culture was grown overnight on Tryptic Soy Agar (TSA) containing 5% sheep blood at 35°C and suspended in Todd Hewitt Broth (THB) to an optical density of 0.8 at 600 nm. Dilutions necessary to give the desired number of cells for challenge were made in THB. The challenge level was 100 times the median lethal dose (LD50), as determined by virulence titration studies. Specifically, the inoculum contained 6.0 x 106 and 5.0 x 104 cfu/mL of S. pneumoniae SVI and S. pneumoniae 2521, respectively. For the resistant strain, 5% horse serum and 2% Debittered Brewer's Yeast (Champlain Industries, Clifton, NJ, USA) were used as adjuvant.
Mouse meningitis model
To establish this model, the mice were infected by the intracerebral ventricular (icv) route. This was accomplished by injection of the inoculum through a soft spot in the cranium located 1–2 mm in front of the coronal suture, and 1 mm on either side of the sagittal suture, which runs vertically down the midline of the cranium.8 A 27-gauge, 1/8 inch stainless steel needle was used to administer 50 µL of inoculum. To verify the establishment of a localized infection in the brain, various organs were examined for bacterial growth over time. Samples were harvested from the blood, liver, kidneys and brain at 0, 2, 6 and 24 h post-inoculation. The organs were processed in physiological saline using a Stomacher Lab-Blender 80 (Seward Medical, London, UK). Colony counts were performed and the geometric mean cfu/g of tissue was then calculated.
Mouse protection test procedure
Groups of five mice were infected with a predetermined number (100 LD50) of pneumococcal cells by the icv route, and treated with a single subcutaneous dose of drug 2 h after challenge. In an initial probe test, four-fold serial decremental doses were used to establish the therapeutic range of the drug. If active, they were titrated in two-fold decremental doses using at least two levels in common with the initial test. Two untreated challenge control groups were included as virulence controls for each test. Final survival percentages at each dose level were used to calculate the median curative dose (CD50) and its 95% confidence limits using the log-probit method of statistical analysis.9 For the resistant pneumococcal strain, four-fold decremental dosing was used for both tests, owing to the shallow dose–response relationship observed with two-fold dosing.
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Results |
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Model characterization
The course of infection for the two pneumococcal strains over a 24 h period revealed a pattern similar to that shown in the Figure
. While bacterial counts were noted in the blood upon initial challenge, the infection showed progressive concentration in the brain over time. At 24 h, this advancing meningitis led to seeding of peripheral organs, including the liver and kidneys. The level of septicaemia was fairly constant over the 24 h period with bacterial counts remaining at <10,000 cfu/mL in the blood. For the resistant strain, a higher initial inoculum was necessary to achieve the desired virulence (100 LD50).
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), blood (•), kidneys (
), liver (
).Mouse protection tests
Comparative median curative activities of clinafloxacin, ceftriaxone and ciprofloxacin, with their corresponding MICs, are presented in the Table. Survival data at each dose level are provided to demonstrate the dose–response relationship for individual tests. Ceftriaxone was the most active agent against the penicillin-susceptible strain (SVI), with a CD50 of 2 mg/kg. Clinafloxacin also showed good curative activity (CD50 = 23 mg/kg), while ciprofloxacin was ineffective (CD50 > 200 mg/kg). Clinafloxacin was the most effective agent against the multiple drug-resistant pneumococcal strain (2521), with a median curative dose of 21 mg/kg. This was virtually identical to its activity against the susceptible strain. Ceftriaxone was less active against the resistant strain (CD50 = 67 mg/kg), while ciprofloxacin showed no activity (CD50 > 200 mg/kg).
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Discussion |
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This mouse meningitis model was developed as a useful tool in assessing the chemotherapeutic potential of new agents in the treatment of meningitis. It is unique in that the animals used are smaller than in the conventional rabbit meningitis model. It allows more rapid throughput for compound evaluation in early development by requiring smaller amounts of material.
Data analysis for these studies showed that both ceftriaxone and clinafloxacin were effective agents against the penicillin-susceptible strain, with the former exhibiting 11- to 12-fold greater potency. These results are consistent with the current standard therapy, as ceftriaxone and other third-generation cephalosporins remain the drugs of choice for drug-susceptible pneumococcal meningitis.3
Against the multidrug-resistant strain, clinafloxacin was clearly the most active agent tested, with a CD50 value more than three times lower than that of ceftriaxone. Using probit statistical analysis techniques,9 the three-fold increase in the relative potency of clinafloxacin compared with ceftriaxone is statistically significant (P = 0.011). It should be noted that the protective efficacy of clinafloxacin against the sensitive and multidrug-resistant pneumococci was virtually identical, with CD50 values of 23 and 21 mg/kg, respectively. The results from this study are consistent with those found by Friedland et al.10 using an experimental rabbit pneumococcal meningitis model. In those studies, clinafloxacin was compared with four other drugs, including ceftriaxone, cefpirome, meropenem and vancomycin, for its therapeutic efficacy against two multidrug-resistant pneumococcal strains. Results showed that clinafloxacin was the most active single agent against both strains tested. In a more recent study by Ostergaard et al.11 a newer fluoroquinolone, moxifloxacin, was evaluated in a rabbit meningitis model against a penicillin-resistant pneumococcus. Results showed that moxifloxacin was as effective as vancomycin and ceftriaxone in reducing CSF bacterial concentrations at all time points tested (3, 5, 10 and 24 h). Further studies comparing the therapeutic efficacy of clinafloxacin with that of newer fluoroquinolones, such as levofloxacin, sparfloxacin and moxifloxacin, are warranted.
In summary, owing to the rapid emergence of multidrug-resistant pneumococci in recent years, new strategies must be considered in the management of patients with meningitis. The newer fluoroquinolones, with their enhanced pharmacokinetic profile5 and their increased potency against recent S. pneumoniae clinical isolates containing specific resistance mutations,6 may offer a preferable alternative to the standard treatment of pneumococcal meningitis. Based on these studies performed using an experimental meningitis mouse model, clinafloxacin may be a very useful therapeutic option in the treatment of meningitis caused by both susceptible and multidrug-resistant S. pneumoniae.
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Acknowledgments |
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These results were previously presented at the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA.
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Notes |
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* Corresponding author. Tel: +1-734-622-7145; Fax: +1-734-622-7158.
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References |
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1 . Centers for Disease Control and Prevention. (1998). Bacterial Meningitis. [Online]. Division of Bacterial and Mycotic Diseases. http://www.cdc.gov/ncidod/dbmd/diseaseinfo/meningitis_g.htm. [Updated 21 September 1999].
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4 . Sloas, M. M., Barrett, F. F., Chesney, P. J., English, B. K., Hill, B. C., Tenover, F. C. et al. (1992). Cephalosporin treatment failure in penicillin- and cephalosporin-resistant Streptococcus pneumoniae meningitis. Pediatric Infectious Disease Journal 11, 662–6.[Web of Science][Medline]
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Friedland, I. R., Paris, M., Ehrett, S., Hickey, S., Olsen, K. & McCracken, G. H. (1993). Evaluation of antimicrobial regimens for the treatment of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 37, 1630–6.
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Ostergaard, C., Sorensen, T. K., Knudsen, J. D. & Frimodt-Moller, N. (1998). Evaluation of moxifloxacin, a new 8-methoxyquinolone, for treatment of meningitis caused by a penicillin-resistant pneumococcus in rabbits. Antimicrobial Agents and Chemotherapy 42, 1706–12.
Received 13 April 1999; returned 26 October 1999; revised 17 November 1999; accepted 25 November 1999
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