Evaluation of Ceftobiprole in a Rabbit Model of Aortic Valve Endocarditis Due to Methicillin-Resistant and Vancomycin-Intermediate Staphylococcus aureus

Ceftobiprole is a novel broad-spectrum cephalosporin that bindswith high affinity to PBP 2a, the methicillin-resistance determinantof staphylococci, and is active against methicillin- and vancomycin-resistantStaphylococcus aureus. Ceftobiprole was compared to vancomycinin a rabbit model of methicillin-resistant S. aureus aorticvalve endocarditis. Ceftobiprole and vancomycin were equallyeffective against endocarditis caused by methicillin-resistantS. aureus strain 76, whereas ceftobiprole was more effectivethan vancomycin against the vancomycin-intermediate S. aureusstrain HIP5836. The activity of ceftobiprole against drug-resistantstrains of S. aureus warrants its further clinical development.

INTRODUCTION

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Introduction

Ceftobiprole (abbreviation, BPR; formerly BAL9141) (Fig. 1)is a novel, broad-spectrum, bactericidal cephalosporin withMICs of $≤$ 4 µg/ml for clinical isolates of methicillin-resistantstaphylococci (MRS), including vancomycin-intermediate Staphylococcusaureus (VISA) and vancomycin-resistant S. aureus (3). The anti-MRSactivity of ceftobiprole stems from its high affinity for PBP2a, the penicillin binding protein chiefly responsible for themethicillin-resistant phenotype of staphylococci. Ceftobiproleis stable to class A penicillinases produced by S. aureus andenteric gram-negative organisms and is relatively stable tosome class C beta-lactamases produced by enteric gram-negativeorganisms (10). In mouse septicemia models, ceftobiprole provedefficacious against infections caused by methicillin-susceptibleS. aureus, methicillin-resistant S. aureus (MRSA), streptococci,and gram-negative bacilli, including Escherichia coli (10).These properties make ceftobiprole an attractive candidate asan empirical monotherapy for a variety of infections while cultureresults are pending, as well as for definitive therapy of infectionscaused by MRS. To improve solubility, ceftobiprole is administeredas its dioxolenylmethyl carbamate prodrug, ceftobiprole medocaril(formerly BAL5788) (Fig. 1). The purpose of the present studywas to compare the efficacy of ceftobiprole to that of vancomycinagainst antibiotic-resistant S. aureus in a rabbit model ofaortic valve endocarditis.

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FIG. 1. Structures of ceftobiprole and its dioxolenylmethyl carbamate prodrug, ceftobiprole medocaril.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains. S. aureus strain 76 is a homogeneous, ß-lactamase-producing,highly methicillin-resistant clinical isolate (nafcillin MIC= 256 µg/ml) (5). S. aureus strain HIP5836 is a methicillin-resistant(nafcillin MIC = 16 µg/ml), ß-lactamase-producingVISA clinical isolate from a patient in New Jersey (originallydesignated strain 992) (2, 7, 21).

Susceptibility testing. MICs were determined by the standard microdilution method withMueller-Hinton broth, using an inoculum of 2 x 10⁶ CFU/ml. MICswere read after incubating microtiter plates for 24 h at 37°C.

Time-kill studies were performed with 50-ml centrifuge tubescontaining 10 ml of Mueller-Hinton broth inoculated with 10⁷CFU of MRSA strain 76 or VISA strain HIP5836 per ml. Sampleswere incubated at 37°C on a reciprocating shaker at 200rpm. Sample volumes (100 µl) were removed at 0, 4, and24 h for quantitative culture to determine viable counts (numbersof CFU per milliliter). The threshold of detection for thismethod is 1 log₁₀ CFU/ml.

Rabbit endocarditis model. To establish endocarditis, a cut-down over the right carotidartery was performed and a polyethylene catheter was positionedacross the aortic valve of a 2.4- to 2.6-kg New Zealand Whiterabbit; the catheter was secured in place for the duration ofthe experiment. Forty-eight hours after the positioning of thecatheter, 1 ml of 0.9% saline containing approximately 10⁶ CFUof either strain 76 or strain HIP5836 was injected intravenouslyinto each rabbit. Antibiotic treatment was commenced 16 to 18h after infection. Rabbits were randomized to one of three groups:(i) an untreated control group, which was euthanized at initiationof therapy to determine baseline bacterial burdens in aorticvalve vegetations, spleens, and kidneys; (ii) a treated controlgroup given vancomycin at a dose of 30 mg/kg of body weightintravenously every 12 h for 4 days; and (iii) a group treatedwith ceftobiprole medocaril at a dose of 25 mg/kg (equivalentto 19 mg of active drug [ceftobiprole] per kg) intramuscularlyevery 8 h for 4 days.

Concentrations of vancomycin in sera were determined from bloodsamples obtained 1 h after dosing. Vancomycin was assayed byagar diffusion with antibiotic medium 8 and Bacillus subtilisATCC 6633 as the indicator strain. Serum ceftobiprole concentrationswere determined from blood samples obtained 1 h after dosing.The assays were performed by the manufacturer by high-pressureliquid chromatography and mass spectrometry as described previously(1). The half-life of ceftobiprole was determined for one uninfectedrabbit from blood samples obtained at 1, 2, and 4 h after dosing.

Rabbits that died any time after initiation of therapy werescored for mortality, but only those surviving beyond the first24 h of treatment were included in an analysis of tissue bacterialtiters. Surviving rabbits were euthanized 16 h after the lastdose of drug. Aortic valve vegetations, spleens, and kidneyswere removed. Tissues were homogenized in 0.5 ml of 0.9% saline,and 100-µl volumes were quantitatively cultured on bloodagar to determine the number of bacteria present. The limitof detection of this method is 5 CFU per vegetation, or approximately1.70 log₁₀CFU/g. Emergence of resistance to ceftobiprole wasassayed by inoculation of 100 µl of homogenized vegetationsfrom control and ceftobiprole-treated rabbits onto Trypticasesoy agar plates containing 5 µg of ceftobiprole per ml.

Data analysis. The bacterial titers in the aortic valve vegetation, kidney,and spleen of each rabbit was expressed as log₁₀ numbers ofCFU per gram of tissue. Cultures yielding no growth were scoredas sterile and were assigned a value of 1.70 log₁₀CFU/g whenmeans were calculated. Differences between mean titers of thetreatment groups were tested for statistical significance (definedas a P of <0.05) by analysis of variance. Significant differencesin rates of mortality were determined by Fisher's exact test.

RESULTS

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Results

MICs of ceftobiprole were 2 µg/ml for MRSA strain 76 and0.5 µg/ml for VISA strain HIP5836. MICs of vancomycinwere 0.5 and 4 µg/ml for strains 76 and HIP5836, respectively.The MIC of 4 µg/ml for HIP5836 instead of 8 µg/ml,the breakpoint for VISA, probably reflects instability of thephenotype (2). In time-kill studies, ceftobiprole at a concentrationof 1 or 5 µg/ml was bactericidal towards strain 76, producingan $~$ 3-log₁₀-CFU/ml decrease in viable counts by 24 h (Fig. 2).Vancomycin at a concentration of 10 µg/ml was also bactericidaltowards strain 76, whereas this strain was not inhibited bynafcillin at a concentration of 32 µg/ml. Ceftobiproleat a concentration of 1 or 5 µg/ml and vancomycin at 10µg/ml were each bactericidal towards strain HIP5836, achieving $≥$ 6-log₁₀ decreases in viable counts by 24 h (Fig. 3). Nafcillinat 32 µg/ml, which is just above the MIC of 16 µg/mlfor this strain, also was bactericidal towards strain HIP5836,with a decrease in viable counts of $~$ 3 log₁₀ by 24 h.

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FIG. 2. Time-kill studies of MRSA strain 76 comparing ceftobiprole at 1 and 5 µg/ml (BPR 1 and BPR 5, respectively) with vancomycin at 10 µg/ml (Van 10) and nafcillin at 32 µg/ml (Naf 32).

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FIG. 3. Time-kill studies of VISA strain HIP5836 comparing ceftobiprole at 1 and 5 µg/ml (BPR 1 and BPR 5, respectively) with vancomycin at 10 µg/ml (Van 10) and nafcillin at 32 µg/ml (Naf 32).

Mean serum vancomycin concentrations (± standard deviations)achieved 1 h after dosing were 29 ± 9 µg/ml (ninerabbits), similar to those observed in humans. The half-lifeof vancomycin in this model is approximately 80 min (6). Meanserum ceftobiprole concentrations (± standard deviations)1 h after dosing were 29 ± 7 µg/ml (16 rabbits),similar to those achieved in humans 1 h after an intravenousinfusion of 500 to 750 mg (17). The half-life of ceftobiprolewas estimated to be approximately 1 h, compared to approximately3 h in humans (17).

Ceftobiprole and vancomycin produced similar reductions in tissueburdens for infection caused by strain 76 (Table 1). Mortalityfor ceftobiprole- and vancomycin-treated rabbits was high; 10of 21 rabbits (48%) and 9 of 19 rabbits (47%) died, respectively,with most deaths occurring during the first 2 days of treatment(Fig. 4). No resistance to ceftobiprole by strain 76 was detectedin aortic valve vegetation, spleen, or kidney samples of untreatedcontrols or ceftobiprole-treated rabbits.

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TABLE 1. Organism titers in vegetations, spleens, and kidneys of untreated and antibiotic-treated rabbits infected with MRSA strain 76

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FIG. 4. Survival curves for rabbits with endocarditis caused by MRSA strain 76, comparing treatment with ceftobiprole to treatment with vancomycin (Vanco).

Ceftobiprole was superior to vancomycin for endocarditis causedby VISA strain HIP5836 (Table 2). The burdens of organisms invegetations, spleens, and kidneys were significantly lower inceftobiprole-treated rabbits than in vancomycin-treated animals.Compared to what occurred in the untreated control group, vancomycinhad no effect on the number of bacteria present in vegetations,whereas it did effect some reduction in bacterial counts inspleen and kidney. Mortality for the ceftobiprole-treated group(1 of 12 rabbits [8.3%]) was significantly lower (P = 0.022)than for the vancomycin-treated group (4 of 6 rabbits [67%])(Fig. 5). No ceftobiprole-resistant populations of VISA strainHIP5836 were detected in tissue samples of controls or ceftobiprole-treatedrabbits.

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TABLE 2. Organism titers in vegetations, spleens, and kidneys of untreated and antibiotic-treated rabbits infected with VISA strain HIP5836

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FIG. 5. Survival curves for rabbits with endocarditis caused by VISA strain HIP5836, comparing treatment with ceftobiprole to treatment with vancomycin (Vanco).

DISCUSSION

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Discussion

Ceftobiprole was bactericidal in vitro, and administered asprodrug ceftobiprole medocaril, it was bactericidal in vivotowards both MRSA strain 76 and VISA strain HIP5836. The activityof ceftobiprole was superior to that of vancomycin for rabbitsinfected with the VISA strain and equivalent to that of vancomycinin rabbits infected with MRSA strain 76. No emergence of ceftobiprole-resistantpopulations was observed for either of these strains. Theseresults are concordant with those obtained for a rat model ofMRSA aortic valve endocarditis, in which ceftobiprole provedto be superior to vancomycin following 3 days of treatment forthe two MRSA strains surveyed (8).

Mortality in rabbits infected with strain 76 was high, particularlyduring the first 48 h of therapy, irrespective of treatmentwith either ceftobiprole or vancomycin. This outcome is mostlikely a reflection of the overwhelming infection caused bystrain 76 in this model and is not attributable to treatmentfailure, since surviving rabbits showed significant reductionsin vegetation titers compared to those of untreated controls.Mortality was also high in vancomycin-treated rabbits infectedwith VISA strain HIP5836, which probably is attributable totreatment failure with vancomycin given that no decrease inbacterial counts in vegetations occurred during therapy. Onlyone ceftobiprole-treated rabbit died during the 4-day treatmentperiod, whereas deaths occurred at a more or less steady rateamong vancomycin-treated rabbits. These results suggest thatceftobiprole might be effective in patients in whom vancomycinfails, as it did in the patient from whom HIP5836 was isolated.

The feasibility of synthesizing beta-lactams that bind PBP 2awith high affinity is well established (4, 9, 12, 13, 16, 20).Solution of the PBP 2a crystal structure has shown that criticalinteractions with cephalosporin side chains occur in a pocket,closed in the free PBP 2a enzyme, that binds the 7-acyl aminoside chain and in an extended groove that interacts with the3'-cephem side chain through numerous noncovalent interactions(15). The cephalosporins that are active against MRSA have sidechains that fill both side chain-binding sites. However, sincethis is also true of molecules that are less active againstMRSA, the precise nature of the interactions that favor theformation of the reversible Michaelis complex and thus speedthe acylation of the active-site serine needs further investigation.Predictably, the antimicrobial effect of those compounds thatare effective inhibitors of both PBP 2a and the normal complementof sensitive penicillin binding proteins is inhibition of growthand eventual cell death at therapeutically relevant concentrations.Since resistance to compounds that bind with high affinity toPBP 2a does not occur as a single step (14), the emergence ofresistance during therapy seems unlikely, which is in fact whatwas observed.

Ceftobiprole appears to have sufficient activity to be efficaciousfor human infections caused by MRSA (19), and the present invivo data confirm this. Ceftobiprole has been safe and welltolerated in phase 1 and phase 2 clinical trials, with no severeor serious adverse effects reported (17-19). The activity ofceftobiprole is not expected to be affected by reduced susceptibilityto vancomycin based on the known mechanisms of action of thesecompounds, in vitro data, and the results of endocarditis modelsof MRSA and VISA infection. The findings in this study, consideredtogether with reports (8, 11; T. Bogdanovich, B. Bozdogan, andP. C. Appelbaum, Abstr. 44th Intersci. Conf. Antimicrob. AgentsChemother., abstr. A-928, 2004) on the refractoriness of staphylococcito develop endogenous resistance to ceftobiprole, suggest thatceftobiprole could be a valuable addition to the antibacterialarmamentarium.

ACKNOWLEDGMENTS

Li Basuino and Andres Madrigal provided technical support.

This work was supported by a research grant from Basilea PharmaceuticaAG, Basel, Switzerland.

FOOTNOTES

* Mailing address: Division of Infectious Diseases, San Francisco General Hospital, and University of California—San Francisco, Room 3400, Building 30, 1001 Potrero Ave., San Francisco, CA 94110. Phone: (415) 206-5437. Fax: (415) 648-8425. E-mail: chipc{at}itsa.ucsf.edu.

REFERENCES

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