In vitro activity of R-95867, the active metabolite of a new oral carbapenem, CS-834, against anaerobic bacteria

Naoki Kato*, Kaori Tanaka, Haru Kato{dagger} and Kunitomo Watanabe

Institute of Anaerobic Bacteriology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The in vitro activity of R-95867, the active metabolite of a new oral carbapenem, CS-834, was compared with those of DU-6859a, cefditoren, ampicillin/sulbactam and clindamycin against a variety of anaerobic bacteria. R-95867 inhibited 90% of anaerobic strains at <=2 mg/L. In general, R-95867 was 2- to 4-fold less active than DU-6859a but more active than other agents tested against strains of peptostreptococci, clostridia, the Bacteroides fragilis group, Porphyromonas spp. and fusobacteria. R-95867 was stable to hydrolysis by ß-lactamase type 2e derived from B. fragilis, Prevotella bivia and Prevotella intermedia, but unstable to hydrolysis by carbapenemase from B. fragilis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antimicrobials active against the majority of clinically significant anaerobes include metronidazole, clindamycin, carbapenems, the ß-lactam/ß-lactamase inhibitor combinations and fluoroquinolones such as DU-6859a1 and trovafloxacin. However, metronidazole is not approved for use in the treatment of anaerobic infections in Japan and is poorly active against non-spore-forming anaerobic Gram-positive bacilli. The incidence of clindamycin-resistant anaerobic bacteria has been increasing.2 Moreover, with the recent pressure to reduce health expenditure, drugs that make outpatient therapy feasible and reduce the need for costly hospital stay are favoured. It is worth developing oral antimicrobials that have potent activity against aerobes and anaerobes including the Bacteroides fragilis group because most anaerobic infections are polymicrobial.

CS-834 is an oral carbapenem newly synthesized by Sankyo Co., Ltd (Tokyo, Japan). R-95867 is the active metabolite of this compound. Here we compared the in vitro activity of R-95867 with other oral agents against a variety of anaerobic bacteria. The stability of R-95867 to hydrolysis by ß-lactamases produced by anaerobic strains was also examined.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antimicrobial agents of known potency were provided by the following pharmaceutical manufacturers: R-95867 (Sankyo); DU-6859a (Daiichi Pharmaceutical Co., Ltd, Tokyo, Japan); cefditoren (Meiji Seika Kaisha, Tokyo, Japan); ampicillin/sulbactam (Pfizer Pharmaceuticals, Inc., Tokyo, Japan); and clindamycin (Japan Upjohn, Tokyo, Japan).

A total of 513 clinical isolates of strictly anaerobic bacteria were tested for antimicrobial susceptibility. The clinical isolates were randomly collected from numerous clinical laboratories in Japan between 1992 and 1996. All strains were identified by a combination of standard methods and the RapID ANA II (Innovative Diagnostic Systems, Atlanta, GA, USA) or Rapid ID 32A (bioMérieux, Marcy-l'Etoile, France).

NCCLS guidelines for agar dilution susceptibility testing of anaerobes3 were used throughout the studies, with a few exceptions. Brucella HK agar (Kyokuto Pharmaceutical Co., Tokyo, Japan) supplemented with 5% laked sheep blood was used as test medium. B. fragilis ATCC 25285 and GAI 5562 were used as quality control strains. Plates were incubated for 2 days at 37°C in an anaerobic chamber (Hirasawa, Tokyo, Japan) with an atmosphere consisting of 82% N2, 10% CO2 and 8% H2.

Effect of medium pH on in vitro activity

A broth microdilution method recommended by NCCLS3 was used to study the effect of medium pH on in vitro activity. Anaerobe broth MIC medium (Difco Laboratories, Detroit, MI, USA) was adjusted with 0.2 M 3-[morpholino]-propanesulphonic acid to pH 7.5, 7.0, 6.5, 6.0 or 5.5. Strains used were ATCC 25285 and seven clinical isolates of B. fragilis. Inoculated microtitre plates were incubated for 2 days at 37°C in the anaerobic conditions described above. The concentration where the most significant reduction of growth was observed was read as the end point.

Crude ß-lactamases were prepared from four strains of B. fragilis, two strains of Prevotella bivia and one strain of Prevotella intermedia. These strains produce ß-lactamase group 2e, except for a single B. fragilis strain that produces ß-lactamase group 3 (carbapenemase); ß-lactamases were categorized using the scheme of Bush.4 The antimicrobial agents used were cephaloridine (Shionogi & Co., Osaka, Japan), R-95867 (Sankyo), imipenem (Banyu Pharmaceutical Co., Tokyo, Japan), cefditoren (Meiji Seika Kaisha) and cefdinir (Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan). ß-Lactamase hydrolysis assay was performed by a spectrophotometric technique as described previously.5 The relative hydrolysis rate of each compound was expressed as a percentage of the hydrolysis rate of cephaloridine.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The MIC ranges and MICs of R-95867, DU-6859a, cefditoren, ampicillin/sulbactam and clindamycin are summarized in Table IGo. The MIC90 of R-95867 for clinical isolates of the anaerobic species tested was <=2 mg/L; 90% of the isolates of most Peptostreptococcus spp., Propionibacterium acnes, Clostridium perfringens, Veillonella spp., B. fragilis, Bacteroides thetaiotaomicron, Bacteroides uniformis, P. bivia, P. intermedia, other Prevotella spp., Porphyromonas spp. and Fusobacterium spp. were inhibited by R-95867 at <=1 mg/L, and R-95867 inhibited 90% of the isolates of Peptostreptococcus anaerobius, Clostridium difficile, Bacteroides caccae and Bacteroides distasonis at 2 mg/L. These activities of R-95867 were 2- to 4-fold lower than those of DU-6859a against all anaerobes tested except for the following: DU-6859a was >=32-fold more active than R-95867 against P. anaerobius and B. caccae and, conversely, R-95867 was twice as active as DU-6859a against P. asaccharolyticus, P. magnus and C. perfringens. Compared with clindamycin at the MIC90, R-95867 was more active against all but P. anaerobius, P. bivia, P. intermedia and other Prevotella spp., against which R-95867 was 4- to 8-fold less active than clindamycin. R-95867 had much greater activity than cefditoren and ampicillin/sulbactam except against P. acnes, for which the cefditoren MIC90 was two-fold lower than that of R-95867. Strains highly resistant to R-95867 were not found, although there were strains that were not inhibited by 128 mg/L of cefditoren, ampicillin/sulbactam or clindamycin. Another study6 demonstrated similar R-95867 activity for P. asaccharolyticus, P. magnus, P. acnes, C. perfringens, B. fragilis and Fusobacterium nucleatum. The susceptibility study of highly ß-lactamase-producing B. fragilis strains revealed that R-95867 was active against ß-lactamase type 2e-producing strains with MICs of 0.25–4 mg/L but had no activity against carbapenemase-producing strains (MICs > 256 mg/L).


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Table I. In vitro activity of R-95867 and other agents against clinical isolates of anaerobic bacteria
 
The in vitro activity of metronidazole showed that at the MIC90 of metronidazole was comparable to that of R-95867 against the B. fragilis group but 4- to 64-fold, >64-fold and 4- to 8-fold less active than R-95867 against peptostreptococci, P. acnes and C. perfringens, and Prevotella spp. and Porphyromonas spp., respectively (unpublished data). These results indicate that R-95867 is a potent agent against anaerobic bacteria.

The decrease in the pH of the medium was accompanied by an increase in the activity of R-95867 (2- to 8-fold decrease in MICs) but also a gradual decrease in the activity of clindamycin (4- to 8-fold increase in MICs) against B. fragilis strains. R-95867 was 2- to 4-fold more active than clindamycin against seven of the eight strains of B. fragilis tested at pH 6.5. Intra-abdominal abscesses were found to have a wide pH range of 5.5–6.8.7 This may be an advantage for the in vivo activity of R-95867.

R-95867 and imipenem were stable to hydrolysis by ß-lactamase type 2e but not carbapenemase (Table IIGo). ß-Lactamase-producing Prevotella and Porphyromonas strains showed poor susceptibility to many ß-lactam antibiotics. Our results demonstrated that R-95867 was not hydrolysed at all by ß-lactamases produced by P. bivia and P. intermedia strains.


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Table II. Stability of R-95867 and other agents against hydrolysis by ß-lactamases produced by B. fragilis, P. bivia and P. intermedia
 
A previous study showed that R-95867 was active against most aerobes.6 A pharmacokinetic study of CS-834 in healthy volunteers revealed that the maximum concentration of R-95867 in serum after multiple oral doses of CS-834 (150 mg tds for 7 days) was 1.59 mg/L at day 1 and the half-life was 0.81 h at day 1.8

CS-834 warrants clinical trials to evaluate its efficacy against infections with which anaerobic and aerobic bacteria are associated.


    Notes
 
* Corresponding author. Fax: +81-58-265-9001; E-mail: nk19{at}cc.gifu-u.ac.jp Back

{dagger} Present address. Department of Bacteriology, School of Medicine, Kanazawa University, 13 Takara-machi, Kanazawa 920-8640, Japan. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Kato, N., Kato, H., Tanaka-Bando, K., Watanabe, K. & Ueno, K. (1996). Comparison of in vitro activities of DU-6859a and other fluoroquinolones against Japanese isolates of anaerobic bacteria. Clinical Infectious Diseases 23, Suppl. 1, 31–5.[ISI]

2 . Appelbaum, P. C., Spangler, S. K. & Jacobs, M. R. (1993). Susceptibility of 539 Gram-positive and Gram-negative anaerobes to new agents, including RP59500, biapenem, trospectomycin and piperacillin/tazobactam. Journal of Antimicrobial Chemotherapy 32, 223–31.[Abstract]

3 . National Committee for Clinical Laboratory Standards. (1993). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Third Edition: Approved Standard M11-A3. NCCLS, Villanova, PA.

4 . Bush, K. (1989). Classification of ß-lactamases: groups 2c, 2d, 2e, 3, and 4. Antimicrobial Agents and Chemotherapy 33, 271–6.[ISI][Medline]

5 . Bandoh, K., Muto, Y., Watanabe, K., Katoh, N. & Ueno, K. (1991). Biochemical properties and purification of metallo-ß-lactamase from Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 35, 371–2.[ISI][Medline]

6 . Fukuoka, T., Ohya, S., Utsui, Y., Domon, H., Takenouchi, T. & Koga, T. (1997). In vitro and in vivo antibacterial activities of CS-834, a novel oral carbapenem. Antimicrobial Agents and Chemotherapy 41, 2652–63.[Abstract]

7 . Bryant, R. E. (1984). Effect of the suppurative environment on antibiotic activity. In New Dimensions of Antimicrobial Therapy (Root, R. K. & Sande, M. A., Eds), pp. 313–57. Churchill Livingstone, New York.

8 . Umemura, K., Ikeda, Y., Kondo, K., Nakashima, M., Naganuma, H. & Hisaoka, M. (1997). Safety and pharmacokinetics of CS-834, a new oral carbapenem antibiotic, in healthy volunteers. Antimicrobial Agents and Chemotherapy 41, 2664–9.[Abstract]

Received 28 June 1999; returned 23 September 1999; revised 20 October 1999; accepted 16 November 1999