Efficacy of a novel tetracycline derivative, glycylcycline, against penicillin-resistant Streptococcus pneumoniae in a mouse model of pneumonia

Kenichiro Murakami, Kazuhiro Tateda*, Tetsuya Matsumoto, Shuichi Miyazaki and Keizo Yamaguchi

Department of Microbiology, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ohta-ku, Tokyo 143-8540, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MIC90 of glycylcycline (<=0.06 mg/L) against 55 strains of Streptococcus pneumoniae was 100-fold lower than that of minocycline or tetracycline. In a mouse model of penicillin-resistant S. pneumoniae (PRSP) pneumonia, glycylcycline (10 mg/kg) decreased bacterial counts in the lungs from 106 cfu to <102 cfu, whereas no apparent reduction of bacterial numbers was observed with minocycline or penicillin G. Pharmacokinetic studies showed that the half-life and area under the curve of glycylcycline were superior to those of minocycline and penicillin G in the lungs. These results show a preferential distribution of glycylcycline in the lungs and potent in vivo bactericidal activity in PRSP pneumonia.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The novel tetracycline derivative, glycylcycline (N,N-dimethylglycylamido 6-demethyl-6-deoxytetracycline), is reported to have potent in vitro antimicrobial activity against S. pneumoniae isolates with acquired resistance to several antimicrobial agents.13 Although potent in vitro activity of glycylcycline against antibiotic-resistant pneumococci has been reported by several investigators, to our knowledge, there are no studies that have examined its pharmacokinetic properties or in vivo efficacy.

In this study, we examined the in vivo antimicrobial activities of glycylcycline against penicillin-resistant S. pneumoniae (PRSP) in a non-compromised mouse model of pneumonia.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains

Clinical isolates of S. pneumoniae from Toho University Hospital (Tokyo, Japan) collected from 1992 to 1994 were used for in vitro antibiotic susceptibility testing. Isolates for which the MIC of ampicillin was <=0.06 mg/L were designated as penicillin susceptible, whereas those with MICs of ampicillin >1 mg/L were designated as penicillin resistant. Strain TUM19, a penicillin-resistant pneumococcus (MIC of penicillin G, 2 mg/L), was used for the preparation of the mouse model of pneumonia.

Antibiotics used

The following drugs were kindly provided by the following companies: glycylcycline, tetracycline and minocycline, Lederle Japan, Tokyo, Japan; penicillin G and ampicillin, Meiji Seika, Tokyo; erythromycin, Shionogi & Co., Osaka, Japan.

Antimicrobial susceptibility testing

MICs of antibiotics were determined by a broth dilution method using Mueller–Hinton broth (Difco, Detroit, MI, USA) supplemented with 5% lysed horse blood. An inoculum of 5.0 x 104 cfu/well was incubated with the antibiotic in microtitre plates at 35°C for 18 h. The MIC was defined as the lowest concentration of the antimicrobial agent that inhibited visible growth of S. pneumoniae.4

Mouse pneumonia model

Non-compromised CBA/J mice (5-week-old females, Charles River Japan) were used for experimentally induced pneumonia, as described previously.5,6 Briefly, the bacterial suspension (40 µL) was inoculated intranasally in anaesthetized mice. Antibiotics (10 mg/kg) were injected sc into mice daily for 3 days commencing 48 h after infection. Twenty-four hours after treatment, the mice were killed and the lungs were removed immediately. The lungs were homogenized in saline, and 0.1 mL of serial 10-fold dilutions of the homogenate was plated on blood agar for determination of viable bacterial counts. Results were expressed as the mean log cfu per lungs ± s.d. (n = 5 for each group).

Pharmacokinetic studies

Serum and lung samples were collected from three mice in each group, at 5, 15, 30, 60, 120, 240 and 360 min following sc injection of 50 mg/kg body weight of each antibiotic. Antibiotic concentrations in the serum and lung homogenates were determined by the paper disc method using Bacillus subtilis ATCC6633 for minocycline and penicillin G and Bacillus cereus ATCC11778 for glycylcycline as bioassay reference strains.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic susceptibility testing

Glycylcycline showed the most potent antibacterial activity (Table IGo). Regardless of susceptibility to penicillin, MICs of glycylcycline against all strains examined were <=0.06 mg/L. The MIC90 of glycylcycline was 100-fold lower than that of minocycline (8 mg/L) or tetracycline (32 mg/L).


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Table I. MICs of glycylcycline compared with three other agents against Streptococcus pneumoniae
 
Comparative antibiotic efficacies in the clearance of PRSP TUM-19

The MICs of glycylcycline, minocycline and penicillin G against S. pneumoniae TUM19 were, respectively, 0.031, 8 and 2 mg/L. Glycylcycline decreased bacterial counts in the lungs from 106 cfu to below 102 cfu after a single administration. In contrast, no apparent reduction in the bacterial count was observed with minocycline or penicillin G, even after three injections.

Effect of various doses of glycylcycline on pneumonia caused by S. pneumoniae TUM-19

We also examined the effect of three different doses of glycylcycline, 0.4, 2 and 10 mg/kg, each injected sc daily for 3 days from 2 days after infection. A clear dose-dependent effect on lung bacterial counts was noted after administration of glycylcycline. Thus, 10 mg/kg of glycylcycline induced a >4 log decrease in bacterial count in the lungs after a single administration, which was followed by complete clearance of bacteria 6 days after infection. On the other hand, a significant, but less marked reduction of bacterial numbers, approximately 2 log decrease, was also induced by treatment with glycylcycline at 2 mg/kg body weight. However, only a minimal effect was observed in mice treated with 0.4 mg/kg of glycylcycline.

Pharmacokinetic studies

The half-life of glycylcycline in the lungs was 5.56 h, which was about 1.6 and 31 times longer than that for minocycline and penicillin G, respectively (Table IIGo). The AUC of glycylcycline in the lungs was about 7 and 13 times that for minocycline and penicillin G, respectively. Importantly, the ratio of AUC in the lung/serum was higher for glycylcycline (2.2), than for minocycline (1.0) and penicillin G (0.6). Another feature of glycylcycline was its preferential distribution in the lungs compared with the other antibiotics examined.


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Table II. Pharmacokinetic parameters
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An increase in the frequency of tetracycline resistance is becoming a serious problem in several Gram-positive bacteria. These antibiotic-resistant bacteria represent a real therapeutic challenge, emphasizing the need for new effective compounds. Particularly, in the past few years, an alarming increase in multiply drug-resistant strains of S. pneumoniae has been reported all over the world. Glycylcycline, N,N-dimethylglycylamido 6-demethyl-6-deoxytetracycline, is a tetracycline derivative with enhanced activity against tetracycline-resistant bacteria. Eliopoulos et al.1 reported that the MIC90 of glycylcycline against penicillin-resistant pneumococci was 0.5 mg/L while those of tetracycline and minocycline were >128 and 32 mg/L, respectively. Weiss et al.2 reported similar results, in which the MIC90s of glycylcycline and minocycline were <=0.12 and 8 mg/L, respectively. Our MIC90 values are similar to those in these reports.

We have recently developed a lethal mouse model of PRSP pneumonia, in which non-compromised CBA/J mice are infected with the bacteria following intranasal instillation.5,6 This model provided us with the opportunity to investigate the in vivo efficacy of antibiotics against PRSP pneumonia in healthy individuals. Interestingly, a single injection of glycylcycline induced a marked reduction in bacterial count in the lungs from 106 cfu to <102 cfu. In contrast, no apparent reduction in bacterial count was observed when the same dose of minocycline or penicillin G was used, even after three injections.

The MIC of glycylcycline against S. pneumoniae TUM19 was 0.031 mg/L, which was 285- and 64-fold lower than that of minocycline and penicillin G, respectively. The potent in vitro antibacterial effects of glycylcycline were also confirmed against several clinical isolates of pneumococci. Moreover, clear differences in several pharmacokinetic factors were observed between the antibiotics examined in the present study. In particular, the half-life of glycylcycline in the lungs was superior to that of minocycline or penicillin G.

There are substantial differences in pharmacokinetic parameters between mice and humans. In addition, any immediate or late adverse reactions due to glycylcycline may limit the clinical usefulness of this antibiotic. Further animal and clinical studies using PRSP infection are warranted to test the effectiveness of this new compound and also its derivatives.


    Notes
 
*Corresponding author. Tel: +81-3-3762-4151; Fax: +81-3-5493-5415; E-mail: address:kazu{at}med.toho-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Eliopoulos, G. M., Wennersten, C. B., Cole, G. & Moellering, R. C. (1994). In vitro activities of two glycylcyclines against gram-positive bacteria. Antimicrobial Agents and Chemotherapy 38, 534–41.[Abstract]

2 . Weiss, W. J., Jacobus, N. V., Petersen, P. J. & Testa, R. T. (1995). Susceptibility of enterococci, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae to the glycylcyclines. Journal of Antimicrobial Chemotherapy 36, 225–30.[Abstract]

3 . Wise, R. & Andrews, J. M. (1994). In vitro activities of two glycylcyclines. Antimicrobial Agents and Chemotherapy 38, 1096–102.[Abstract]

4 . National Committee for Clinical Laboratory Standards. (1991). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Second Edition: Approved Standard M7-A2. NCCLS, Villanova, PA.

5 . Takashima, K., Tateda, K., Matsumoto, T., Ito, T., Iizawa, Y., Nakao, M. et al. (1996). Establishment of a model of penicillinresistant Streptococcus pneumoniae pneumonia in healthy CBA/J mice. Journal of Medical Microbiology 45, 319–22.[Abstract]

6 . Tateda, K., Takashima, K., Miyazaki, H., Matsumoto, T., Hatori, T. & Yamaguchi, K. (1996). Noncompromised penicillin-resistant pneumococcal pneumonia CBA/J mouse model and comparative efficacies of antibiotics in this model. Antimicrobial Agents and Chemotherapy 40, 1520–5.[Abstract]

Received 14 September 1999; returned 22 November 1999; revised 11 April 2000; accepted 30 May 2000





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