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Antimicrobial Agents and Chemotherapy, April 2009, p. 1698-1700, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01549-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Efficacy of Piperacillin Combined with the Penem β-Lactamase Inhibitor BLI-489 in Murine Models of Systemic Infection

Peter J. Petersen,^1* C. Hal Jones,¹ Aranapakam M. Venkatesan,² and Patricia A. Bradford¹

Infectious Disease Research,¹ Chemical Sciences, Wyeth Research, Pearl River, New York²

Received 20 November 2008/ Returned for modification 22 December 2008/ Accepted 22 January 2009

Top ABSTRACT INTRODUCTION REFERENCES

 
The in vivo efficacy of piperacillin in combination with the penem inhibitor BLI-489 was determined using acute lethal systemic infections in mice. On the basis of preliminary results with various ratios, a dosing ratio of 8:1 was found to be optimal for retention of enhanced efficacy. Piperacillin-BLI-489 dosed at an 8:1 ratio was efficacious against murine infections caused by class A (including extended-spectrum β-lactamases), class C (AmpC), and class D β-lactamase-expressing pathogens.

Top ABSTRACT INTRODUCTION REFERENCES

 
The increased resistance to β-lactam antibacterial agents due to β-lactamase-producing organisms in the hospital and community settings has been acknowledged as a global medical problem (4, 9, 21). The extensive use of expanded-spectrum cephalosporins has resulted in the emergence of diverse extended-spectrum β-lactamase (ESBL) variants of class A and class D as well as AmpC (class C) enzymes (9, 21). Further adding to the problem is the prevalence of ESBL-producing isolates that are multiply resistant to other classes of antimicrobial agents (e.g., aminoglycosides and quinolones) (9). The currently available β-lactamase inhibitors (tazobactam, clavulanic acid, and sulbactam) fail to target class C-producing strains and are marginally effective against many ESBL-producing strains (13). In addition, cefepime, the most effective extended-spectrum cephalosporin, has been associated with clinical failures against a variety of pathogens (1, 2, 11, 17, 18).

Previous reports have shown that the penem bicyclic β-lactamase inhibitors conferred activity and efficacy as an inhibitor of class A (including ESBL), class D, and class C β-lactamase enzymes (19, 20, 22). This study, using acute lethal systemic infections in mice, was performed to determine the optimal ratio of piperacillin to BLI-489 (Fig. 1) that would provide in vivo efficacy. Once the ratio was determined, it was employed to evaluate the efficacy of piperacillin-BLI-489 against a broad spectrum of β-lactamase-producing pathogens.

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FIG. 1. Chemical structure of the penem β-lactamase inhibitor BLI-489.

The organisms used in this study were clinical isolates expressing characterized β-lactamases, as determined by standard laboratory techniques (e.g., isoelectric focusing, PCR, and nucleotide sequencing), from the Wyeth Research culture collection. Quality control strains for in vitro testing were from the American Type Culture Collection (Rockville, MD), as recommended by the Clinical and Laboratory Standards Institute (6, 7). Standard powders were obtained from Wyeth Research, Pearl River, NY. The in vitro activities of the antibiotics were determined as previously described (22), by following the Clinical and Laboratory Standards Institute recommended methodology (6).

Female mice, strain CD-1 (Charles River Laboratories), 20 ± 2 g each, were challenged by intraperitoneal injection of bacterial cells suspended in 5% hog gastric mucin. Five animals were infected at each of five twofold dose levels of the test compound. Subcutaneous dosing solutions were prepared as piperacillin alone or piperacillin combined with a β-lactamase inhibitor at various ratios. A second dose was administered for the more-virulent infections. The bacterial inoculum level was sufficient to result in the deaths of untreated controls within 48 h. The 7-day survival ratios for three separate tests were pooled for determination of the median effective dose (ED₅₀) by a computerized probit analysis (8). All procedures were carried out using protocols approved by the Wyeth Research Animal Care and Use Committee.

The in vivo efficacies of various ratios of piperacillin-BLI-489 (2:1, 4:1, 6:1, 8:1, 10:1, 12:1, and 14:1) against two β-lactamase-producing strains (Escherichia coli GC 6265 [TEM-1, class A] and Enterobacter cloacae GC 4142 [AmpC, class C]) were measured to determine the optimal ratios of piperacillin to BLI-489 that would retain maximum efficacy (Table 1). The in vivo protection demonstrated by piperacillin-BLI-489 was statistically equivalent, on the basis of the 95% confidence limits, for the E. coli infection at ratios of 2:1 through 14:1 (ED₅₀s, 8.5 to 16 mg/kg). Against the E. cloacae infection, piperacillin-BLI-489 showed similar efficacies at the 2:1 through 8:1 ratios (ED₅₀s, 30 to 32 mg/kg). Although not statistically significant, there was a slight decrease in efficacy at the 10:1 ratio (ED₅₀, 47 mg/kg). This trend continued at the 12:1 and 14:1 ratios (ED₅₀s, 60 and 69 mg/kg, respectively), with these ratios being statistically less efficacious than the 2:1 through 8:1 ratios. On the basis of this analysis, subsequent in vivo studies were conducted using the 8:1 ratio of piperacillin to BLI-489 because it was the smallest amount of inhibitor that could be used and still demonstrated a significant enhancement in the efficacy of piperacillin.

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TABLE 1. In vivo efficacies of piperacillin alone and in combination with BLI-489 at various ratios against β-lactamase-producing E. coli and E. cloacae strains

Administered alone, piperacillin showed poor efficacy (ED₅₀s, 103 to 1,121 mg/kg) against the intraperitoneal murine infection models caused by class A, C, and D β-lactamase-producing strains (Table 2). Piperacillin combined with BLI-489 at an 8:1 ratio significantly reduced piperacillin ED₅₀s for all infections evaluated. Piperacillin-BLI-489 demonstrated efficacy that was equivalent to that of piperacillin-tazobactam against infection with either a class A-producing E. coli (TEM-1) strain (ED₅₀s, 13 and 11 mg/kg, respectively) or a class A-producing Klebsiella pneumoniae (SHV-1) strain (ED₅₀s, 23 and 24 mg/kg, respectively). Similarly, BLI-489 or tazobactam in combination with piperacillin demonstrated comparable efficacies against infection with an E. coli strain expressing a class A ESBL (TEM-10) enzyme (ED₅₀s, 40 and 25 mg/kg, respectively). However, BLI-489 statistically exceeded the efficacy of tazobactam in combination with piperacillin against an infection with a K. pneumoniae strain that produces both class A (SHV-1) and class A ESBL (SHV-5) enzymes (ED₅₀s, 28 and 58 mg/kg, respectively). In addition, piperacillin-BLI-489 with an ED₅₀ of 45 mg/kg was significantly more efficacious than piperacillin-tazobactam (ED₅₀, 152 mg/kg) against an infection with a different class A ESBL enzyme (CTX-M-5), produced by a strain of Salmonella enterica serovar Typhimurium.

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TABLE 2. In vivo efficacies of piperacillin alone and in combination with BLI-489 or tazobactam following acute lethal murine infection caused by β-lactamase-producing pathogens

Against infections with class C (AmpC)-producing organisms (Table 2), BLI-489 significantly enhanced the efficacy of piperacillin tested alone and statistically exceeded the efficacy of piperacillin-tazobactam. The ED₅₀ of piperacillin was reduced from 285 mg/kg to 38 mg/kg when tested in combination with BLI-489 against an E. cloacae infection expressing a class C (AmpC) β-lactamase, whereas piperacillin-tazobactam was statistically less efficacious (ED₅₀, 71 mg/kg). Against an infection with a class C (AmpC)-producing Pseudomonas aeruginosa strain, the ED₅₀ of piperacillin (estimate of 980 mg/kg) was reduced to 103 mg/kg in combination with BLI-489 but only to 246 mg/kg when combined with tazobactam. Similarly, piperacillin-BLI-489 with an ED₅₀ of 13 mg/kg was statistically more efficacious than piperacillin or piperacillin-tazobactam (ED₅₀s, 103 and 45 mg/kg, respectively) against infection with an E. coli strain expressing a class C (ACT-1) enzyme.

BLI-489 combined with piperacillin also demonstrated in vivo efficacy against a class D (OXA-1)-producing E. coli strain. The ED₅₀ of piperacillin was reduced from 980 mg/kg alone to 86 mg/kg when combined with BLI-489, whereas the combination of piperacillin-tazobactam was less efficacious (ED₅₀, 270 mg/kg).

The increased resistance to β-lactam antibacterial agents due to β-lactamase enzymes in the hospital and community settings has long been acknowledged as a global medical problem (4, 9, 21). In response, β-lactam antibacterial agents have been designed to resist hydrolysis or have been combined with β-lactamase inhibitors (3, 14, 15). The current commercially used β-lactamase inhibitors (tazobactam, clavulanic acid, and sulbactam) lack significant activity against the ESBL-producing class A strains as well as class D- and class C (AmpC)-expressing organisms (5, 13, 14). Tigecycline, carbapenems, and the polymyxins remain the only current existing therapeutic alternatives for these problematic pathogens (10, 12, 16). A number of new compounds, including non-β-lactam and β-lactam inhibitors, penems, cephalosporins, and carbapenems, are currently in development to address this unmet medical need (3). The potent activities demonstrated by the penem β-lactamase inhibitors against class A ESBL and class C and class D β-lactamases may provide valuable additions to the inhibitor armament (19, 20, 22).

In this study, the combination of piperacillin with BLI-489 at the 8:1 ratio restored the in vivo efficacy of piperacillin, similar to that shown by the combination in vitro. Piperacillin-BLI-489 showed enhanced efficacy over piperacillin-tazobactam against organisms producing class A ESBLs and demonstrated significant improvement in efficacy against the strains expressing class C and class D enzymes. This new combination (piperacillin-BLI-489) offers an advantage over the current commercial inhibitors and warrants further development.

 
* Corresponding author. Mailing address: Infectious Disease Research, Wyeth Research, Bldg. 200, Rm. 3301, 401 N. Middletown Rd., Pearl River, NY 10965. Phone: (845) 602-3070. Fax: (845) 602-5671. E-mail: petersp{at}wyeth.com

Published ahead of print on 2 February 2009.

Top ABSTRACT INTRODUCTION REFERENCES

 

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Antimicrobial Agents and Chemotherapy, April 2009, p. 1698-1700, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01549-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Petersen, P. J. Articles by Bradford, P. A. Search for Related Content PubMed PubMed Citation Articles by Petersen, P. J. Articles by Bradford, P. A.