Therapeutic efficacy of J-111,225, a novel trans-3,5-disubstituted pyrrolidinylthio-1ß-methylcarbapenem, against experimental murine systemic infections

Kaneyoshi Shibata, Rie Nagano, Terutaka Hashizume* and Hajime Morishima

Banyu Tsukuba Research Institute, Okubo 3, Tsukuba 300-2611, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a murine model of systemic infection with methicillin-resistant Staphylococcus aureus (MRSA), J-111,225 showed an ED50 value of 5.83 mg/kg, which was comparable to vancomycin (ED50 4.84 mg/kg), whereas imipenem failed to cure infected mice (ED50 >100 mg/kg). Against a mixed infection caused by MRSA and Pseudomonas aeruginosa, monotherapy with J-111,225 showed an ED50 value of 7.23 mg/kg, whereas combined treatment with vancomycin plus imipenem (1:1) had an ED50 of 20.86 mg/kg. J-111,225 showed good therapeutic efficacy against methicillin-susceptible S. aureus, penicillin-resistant Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae and P. aeruginosa. The unusually broad spectrum suggests that monotherapy with this novel carbapenem may be suitable for polymicrobial infections associated with MRSA.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for many nosocomial infections and remains a serious problem. Vancomycin is available for the management of such staphylococcal infections, despite its limited antimicrobial spectrum, slow clinical response1 and potential for causing adverse effects.2 However, the emergence of vancomycin resistance in organisms such as enterococci3 and MRSA4 may lead to a lack of reliable therapy for these infections. Therefore, the development of new agents for the treatment of such resistant bacterial infections has become important.

Marketed carbapenems completely lack anti-MRSA activity, and no carbapenems have been reported with adequate activity against both methicillin-resistant staphylococci and Pseudomonas aeruginosa. In the course of our study on 1ß-methylcarbapenems, a novel trans-3,5-pyrrolidinylthio-1ß-methylcarbapenem (J-111,225) was identified as an exceptionally broad-spectrum agent covering MRSA and P. aeruginosa in addition to other common Gram-positive and Gram-negative organisms.

In this report, we describe the in vivo therapeutic efficacy of J-111,225 in murine experimental systemic infections.


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

J-111,225 and reference carbapenems were synthesized at the Tsukuba Research Institute (Banyu Pharmaceutical Co., Ltd., Tsukuba, Japan). Imipenem, cilastatin sodium and penicillin G were the products of Banyu Pharmaceutical Co. The following drugs were obtained commercially: vancomycin (Sigma Chemical Co., St Louis, MO, USA) and ceftazidime (Tanabe Pharmaceutical Co., Ltd, Japan). Solutions of the antimicrobial agents were freshly prepared on the day of use.

Bacterial strains

Bacterial strains used in the experiment had been collected over the past several years. MRSA strain BB6226 is ß-lactamase-negative, homogeneously resistant and produces coagulase type II. All bacteria were maintained in glycerol broth at –80°C.

Susceptibility testing

MICs were determined by the broth microdilution method using Mueller–Hinton broth (Difco Laboratories, Detroit, MI, USA). Todd Hewitt Broth (Difco) supplemented with 5% haemolysed horse blood was used for penicillin-resistant Streptococcus pneumoniae (PRSP). A culture grown at 37°C for 6 h in broth was diluted to 107 cfu/mL, and each dilution was inoculated into drug-containing broth with an inoculation apparatus (MIC-2000; Dinatech Lab., Inc., Chantilly, VA, USA). The final inoculum size was 105 cfu/mL. The MIC was defined as the lowest antibiotic concentration that completely prevented visible growth after incubation at 37°C for 20 h.

Systemic infection models in mice

ICR mice, 4 weeks of age, were obtained from Charles River Japan, Inc. (Yokohama, Japan). Seven mice were used for each dose of drug. The late-exponential-phase growth was harvested and suspended in brain–heart infusion broth (Difco) or saline containing 3–5% gastric mucin. A 0.5 mL portion of bacterial suspension was intraperitoneally inoculated into each mouse; this bacterial challenge was at least ten times higher than the 50% lethal dose (LD50). Under these conditions, all untreated mice died within 3 days. In treatment studies, cilastatin was co-administered with carbapenems to eliminate the effects of mouse dehydropeptidase-I (DHP-I).5 Mice surviving at each dose were counted 6 days after infection, and the 50% effective doses (ED50s) were calculated by the Probit6 or Wilcoxon–Litchfield method.7

In order to mimic polymicrobial infections associated with MRSA, a mixed infection model was established with MRSA BB6226 and P. aeruginosa BB6190 as the co-infectants. In this model the infected mice received monotherapy with J-111,225, imipenem or vancomycin, or combination therapy with imipenem and vancomycin (1:1) 1 h after infection.

Pharmacokinetic studies

Mice (n = 3) were injected subcutaneously with 10 mg J-111,225 or 10 mg imipenem with 40 mg cilastatin per kg of body weight to estimate the pharmacokinetics in the mouse efficacy study. Vancomycin was injected alone at a dose of 10 mg/kg. Blood samples were drawn 5, 10, 30 and 60 min after drug administration. Urine samples were collected for 6 h after drug administration (UR0–6). Concentrations of carbapenems in plasma and urine were determined by disc diffusion bioassay using antibiotic medium 1 (Difco) inoculated with Bacillus subtilis ATCC12432 as the indicator organism, and those of vancomycin were determined by the same method using 1% sodium citrate supplemented nutrient agar (Difco). Pharmacokinetic parameters were calculated according to the moment method.8


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Efficacy against murine systemic infections

The protective efficacies against single and mixed systemic infections with MRSA BB6226 and/or P. aeruginosa BB6190 were determined (Table IGo). In single-organism infections, J-111,225 showed good therapeutic efficacy, which was comparable to that of vancomycin and imipenem against MRSA and P. aeruginosa, respectively. In a mixed systemic infection caused by MRSA and P. aeruginosa, monotherapy with J-111,225 showed good efficacy (ED50 7.23 mg/kg) compared with that of combination therapy with imipenem and vancomycin (1:1; ED50 20.9 mg/kg). Neither vancomycin nor imipenem monotherapy was effective against the mixed infection; this was expected, since MRSA and P. aeruginosa are resistant to imipenem and vancomycin, respectively.


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Table I. In vivo efficacy against single and mixed systemic infections with MRSA BB6226 and P. aeruginosa BB6190a
 
J-111,225 exhibited good in vivo therapeutic efficacy against systemic infections with methicillin-susceptible S. aureus (MSSA), PRSP, Escherichia coli and Klebsiella pneumoniae, which are generally susceptible to known carbapenems (Table IIGo). The ED50 of J-111,225 against MSSA strain Smith was 0.040 mg/kg, which was similar to that of imipenem and lower than that of meropenem, biapenem and vancomycin. J-111,225, with an ED50 of 0.29 mg/kg, was about ten times more effective than penicillin against PRSP BB6230, or as effective as the reference carbapenems and vancomycin. With E. coli ML4707 and K. pneumoniae BB5710, meropenem showed the greatest therapeutic efficacy, and J-111,225 was comparable to or better than imipenem, biapenem and ceftazidime in terms of ED50.


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Table II. Protective efficacies of antibiotics against systemic infections in micea
 
Pharmacokinetic parameters

The Cmax values of J-111,225, imipenem and vancomycin given at a single subcutaneous dose of 10 mg/kg were similar (12.0, 11.9 and 13.1 mg/L, respectively). J-111,225 showed longer half-life (t1/2) in plasma and greater AUC0–{infty} (13.5 min and 401.7 mg•min/L, respectively) than imipenem (8.9 min and 360.3 mg•min/L, respectively). J-111,225 and imipenem had similar urinary recovery rate from 0 to 6 h (UR0–6) (52.0% and 53.7%, respectively). Vancomycin showed slower elimination from plasma (t1/2 = 26.2 min), greater AUC0–{infty} (713.3 mg•min/L) and higher UR0–6 (95.7%) than the carbapenems tested.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the course of our attempts to modify 1ß-methylcarbapenem, to increase its anti-MRSA activity, J-111,225 was obtained in crystalline form. The addition of a trans-3,5- disubstituted 5-arylpyrrolidin-3-ylthio moiety to the C2 position of 1ß-methylcarbapenem contributes to the potent antibacterial activity of this compound against both MRSA and P. aeruginosa, although previously reported anti-MRS ß-lactam antibiotics such as L-695,2569 and SM-1746610 have weak activity against Gram-negative organisms and/or completely lack antipseudomonal activity.

It should be noted that monotherapy with J-111,225 was effective against mixed infection caused by MRSA and P. aeruginosa. In clinical cases with such infections, combined therapy with vancomycin and imipenem (or other anti-Gram-negative agents) might be considered as a therapeutic regimen. However, not only might the intensive use of vancomycin stimulate the emergence of vancomycin-resistant MRSA,4 but vancomycin therapy, either alone or in combination with conventional broad-spectrum agents, has a relatively high cost and a fairly high potential for causing unexpected adverse reactions. In this context, monotherapy against polymicrobial infections might be advantageous.

Although J-111,225 was co-administered with cilastatin in this study to eliminate the effects of mouse DHP-1, which is distributed in several organs,5 J-111,225 could be developed for clinical use without co-administration of a DHP-I inhibitor, since the stability of J-111,225 to hydrolysis by human DHP-I is comparable to that of meropenem.

J-111,225 has therapeutic advantages over currently available carbapenems because of its exceptionally broad spectrum of activity against clinically important resistant pathogens including MRSA. J-111,225 offers the potential of monotherapy for polymicrobial infections associated with MRSA.


    Acknowledgments
 
This paper was presented in part at the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, USA, 24–27 September 1998.


    Notes
 
* Corresponding author. Tel: +81-298-77-2000; Fax: +81-298-77-2029; E-mail: haszmett{at}banyu.co.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Levine, D. P., Fromm, B. S. & Reddy, B. R. (1991). Slow response to vancomycin plus rifampin in methicillin-resistant Staphylococcus aureus endocarditis. Annals of Internal Medicine 115, 674–80.[ISI][Medline]

2 . Farber, B. F. & Moellering, Jr, R. C. (1983). Retrospective study of the toxicity of preparations of vancomycin from 1974 to 1981. Antimicrobial Agents and Chemotherapy 23, 138–41.[ISI][Medline]

3 . Woodford, N., Johnson, A. P., Morrison, D. & Speller, D. C. (1995). Current perspectives on glycopeptide resistance. Clinical Microbiology Reviews 8, 585–615.[Abstract]

4 . Hiramatsu, K., Hanaki, H., Ino, T., Yabuta, K., Oguri, T. & Tenover, F. C. (1997). Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. Journal of Antimicrobial Chemotherapy 40, 135–6.[Free Full Text]

5 . Hajdu, R., Hayase, K., Sundelof, J., Hara, K., Kropp, H. & Kahan, F. (1985). Cilastatin-sensitive lactamase active on carbapenem and penem antibiotics in the lung of rodents. In Recent Advances in Chemotherapy (Ishigami, J., Ed.), pp. 1211–2. University of Tokyo Press, Tokyo.

6 . Miller, L. C. & Tainter, M. L. (1944). Estimation of ED50 and its error by means of logarithmic-probit graph paper. Proceedings of the Society for Experimental Biology and Medicine 57, 261–4.

7 . Litchfield, J. T. & Wilcoxon, F. (1949). A simplified method of evaluating dose–effect experiments. Journal of Pharmacology and Experimental Therapeutics 96, 99–113.[ISI]

8 . Yamaoka, K., Nakagawa, T. & Uto, T. (1978). Statistical moment in pharmacokinetics. Journal of Pharmacokinetics and Biopharmaceutics 30, 476–8.

9 . Chambers, H. F. (1995). In vitro and in vivo antistaphylococcal activities of L-695,256, a carbapenem with high affinity for penicillin-binding protein PBP2a. Antimicrobial Agents and Chemotherapy 39, 462–6.[Abstract]

10 . Sumita, Y., Nouda, H., Kanazawa, K. & Fukasawa, M. (1995). Antimicrobial activity of SM-17466, a novel carbapenem antibiotic with potent activity against methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 39, 910–6.[Abstract]

Received 14 July 1999; returned 11 October 1999; revised 21 October 1999; accepted 18 November 1999





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