Banyu Tsukuba Research Institute, Okubo 3, Tsukuba 300-2611, Japan
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Abstract |
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Introduction |
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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.
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Materials and methods |
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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 MuellerHinton 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 brainheart infusion broth (Difco) or saline containing 35% 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 WilcoxonLitchfield 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 (UR06). 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
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Results |
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The protective efficacies against single and mixed systemic infections with MRSA BB6226 and/or P. aeruginosa BB6190 were determined (Table I). 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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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 (t) in plasma and greater AUC0
(13.5 min and 401.7 mgmin/L, respectively) than imipenem (8.9 min and 360.3 mgmin/L, respectively). J-111,225 and imipenem had similar urinary recovery rate from 0 to 6 h (UR06) (52.0% and 53.7%, respectively). Vancomycin showed slower elimination from plasma (t
= 26.2 min), greater AUC0
(713.3 mgmin/L) and higher UR06 (95.7%) than the carbapenems tested.
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Discussion |
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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.
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Acknowledgments |
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Notes |
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References |
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Received 14 July 1999; returned 11 October 1999; revised 21 October 1999; accepted 18 November 1999