Efficacy of Ceftaroline Fosamil in a Staphylococcal Murine Pneumonia Model

Abstract

Ceftaroline fosamil is a cephalosporin with activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). The objective of this study was to characterize the dose-response relationship of ceftaroline fosamil against S. aureus in an immunocompromised murine pneumonia model, as well as to evaluate the efficacy of the humanized regimen of 600 mg intravenously (i.v.) every 12 h. Seventeen S. aureus (2 methicillin-susceptible Staphylococcus aureus [MSSA], 15 MRSA) isolates with ceftaroline MICs of 0.5 to 4 μg/ml were utilized. The pharmacokinetics of ceftaroline in serum and epithelial lining fluid (ELF) were evaluated to determine bronchopulmonary exposure profiles in infected and uninfected animals, using single and human-simulated doses. Serum fT>MIC (the percentage of time that free drug concentrations remain above the MIC) of 17% to 43% was required to produce a 1-log₁₀ kill in the dose-ranging studies. These targets were readily achieved with the humanized exposure profile, where decreases of 0.64 to 1.95 log₁₀ CFU were observed against 13 MRSA and both MSSA isolates tested. When taken as a composite, the fT>MICs required for stasis and a 1-log₁₀ kill were 16% and 41%, respectively. ELF concentrations were similar to serum concentrations across the dosing interval in infected and uninfected animals. The serum fT>MIC targets required in this lung infection model were similar to those observed with ceftaroline against S. aureus in a murine thigh infection model. Exposures simulating the human dose of 600 mg i.v. every 12 h achieved pharmacodynamic targets against MRSA and MSSA considered susceptible by current U.S. FDA breakpoints.

INTRODUCTION

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) continues to rise and poses a significant health care burden (10, 14). MRSA is one of the many potential pathogens that contribute to community- and hospital-acquired pneumonias (CAP and HAP, respectively). A survey among 59 U.S. hospitals between January 2002 and January 2004 identified MRSA as the pathogen responsible for 8.9% of CAP and 22.9% of HAP cases (10). Frequently, pneumonias caused by MRSA, especially strains that produce the Panton-Valentine leukocidin (PVL) toxin, can result in severe cases of necrotizing pneumonia, which is difficult to treat (14).

Ceftaroline, the active metabolite of the prodrug ceftaroline fosamil, has a broad spectrum of in vitro activity against common Gram-negative and Gram-positive pathogens, including Streptococcus pneumoniae and MRSA (5, 15). Ceftaroline displays high affinity for penicillin binding protein PBP2a, which plays an important role in methicillin resistance in MRSA strains (12, 15). Ceftaroline fosamil is currently approved in the United States for the treatment of acute bacterial skin and skin structure infections and non-MRSA community-acquired bacterial pneumonia at a dose of 600 mg intravenously (i.v) every 12 h (5).

The objective of this study was to evaluate the dose-response relationship of ceftaroline in the neutropenic murine lung infection model using various clinical MRSA isolates. Additionally, we assessed the efficacy of a human-simulated regimen of 600 mg ceftaroline fosamil every 12 h, administered as a 1-h i.v. infusion in both MRSA and methicillin-susceptible S. aureus (MSSA) isolates.

MATERIALS AND METHODS

Antimicrobial test agents.

Analytical-grade ceftaroline (lot FMD-CEF-035; Forest Laboratories, Inc., New York, NY) was used for the in vitro MIC analyses, and commercially available ceftaroline fosamil (lots 001D06 and 002D16) was used for the in vivo experiments. Prior to each in vivo experiment, ceftaroline fosamil was reconstituted with 20 ml of sterile water for injection. The solution was subsequently diluted with 0.9% normal saline to achieve the required concentrations. The ceftaroline fosamil solution was stored under refrigeration and discarded within 24 h of reconstitution.

Bacterial isolates.

Seventeen clinical isolates of S. aureus (fifteen MRSA and two MSSA isolates) with various phenotypic and genotypic profiles were used in this study. Ceftaroline MICs were determined by broth microdilution in triplicate according to CLSI guidelines, and the modal MIC was reported (3). Isolates were stored frozen at −80°C in double-strength skim milk (Remel, Lenexa, KS) and then subcultured twice onto Trypticase soy agar with 5% sheep blood (Becton, Dickinson, and Co., Sparks, MD) and grown for 18 to 24 h at 37°C prior to use in the experiments.

Neutropenic lung infection model.

Pathogen-free, female BALB/c mice weighing approximately 20 g were acquired from Harlan Laboratories (Indianapolis, IN). This study was reviewed and approved by the Hartford Hospital Institutional Animal Care and Use Committee. Animals were maintained and used in accordance with National Research Council recommendations and were provided food and water ad libitum. Mice were rendered neutropenic by administering intraperitoneal injections of cyclophosphamide (Baxter, Deerfield, IL) at doses of 250 mg/kg of body weight and 100 mg/kg, 4 days and 1 day, respectively, prior to inoculation. Uranyl nitrate at a dose of 5 mg/kg was also administered 3 days prior to inoculation to induce a predictable degree of renal impairment. Mice were anesthetized with isoflurane and inoculated with 0.05 ml of 10⁷ CFU suspension of the infecting S. aureus isolate in 3% mucin (Sigma-Aldrich, St. Louis, MO). The inoculum was administered into the mouths of the mice, and immediately thereafter their nares were blocked to induce aspiration.

Protein binding.

Protein binding studies were conducted in triplicate using Amicon Centrifree Micropartition devices (Millipore, Bedford, MA) with 3,000-molecular-weight-cutoff filters according to the manufacturer's package insert. An aqueous stock solution of 1,000 μg/ml ceftaroline was prepared in normal saline. Freshly collected mouse serum was used, and dilutions were made to yield final concentrations of 5 μg/ml and 50 μg/ml. The concentrations were selected based on serum concentration profiles of the doses that were utilized in the pharmacodynamic studies. Each solution of serum and drug was placed in a shaking water bath at 37°C for 10 min. Then, 0.9 ml of each serum solution was transferred into three ultrafiltration devices and centrifuged for 45 min at 10°C at 2,000 × g to generate an ultrafiltrate volume of ∼250 μl. Nonspecific binding of the drug to the filter device was also assessed by following the same procedure, with the exception that normal saline was used instead of mouse serum.

Percent protein binding was calculated using the following equation: %PB = [(S − SUF)/S] × 100, where S is the total drug concentration and SUF is the concentration in the ultrafiltrate (i.e., free or unbound drug).

Pharmacokinetic studies and determination of dosing regimen.

For single-dose pharmacokinetic studies, mice were prepared and infected as described for the neutropenic lung infection model. Single doses of ceftaroline at 6.25, 12.5, and 50 mg/kg were administered in 0.2-ml volumes subcutaneously 3 h after inoculation. Groups of six mice were euthanized by carbon dioxide exposure. Blood samples were collected via intracardiac puncture at various time points over an 8-h period. Additionally, epithelial lining fluid (ELF) was collected via bronchoalveolar lavage (BAL) at 4 time points for the 12.5- and 50-mg/kg dosing groups. BAL was performed by inserting a catheter in the trachea and instilling four aliquots of 0.4 ml normal saline followed by immediate removal of fluid. Serum and BAL samples were separated by centrifugation and stored at −80°C until analysis. Ceftaroline concentrations were analyzed by Eurofins Medinet, Inc. (Chantilly, VA) using a validated liquid chromatography-tandem mass spectrometry (LC/MS-MS) assay. The lower limit of detection for the assay was 7.5 ng/ml. The interday coefficient of variation of this assay was <12%.

Serum and BAL samples were also utilized for urea determination (urea nitrogen reagent set; Teco Diagnostics, Anaheim, CA). The interday and intraday coefficients of variation of this assay were <6%. The ceftaroline concentrations in ELF were calculated using the following equation: CPT_BAL × (urea_serum/urea_BAL). The terms urea_serum and urea_BAL represent the concentrations of urea in the serum and BAL fluid, respectively. CPT_BAL is the concentration of ceftaroline in the BAL fluid. The resultant concentration of ceftaroline in ELF was assumed to be free drug (9).

Pharmacokinetic parameter values for single doses of ceftaroline in mice were calculated using an appropriate compartmental model with first-order input and elimination (WinNonlin version 5.2; Pharsight, Mountain View, CA). In addition to visual inspection of the fit, compartment model selection was based on Akaike's information criterion and correlation coefficient values.

(i) Single-dose studies.

Individual parameter estimates from single-dose pharmacokinetic studies were used to calculate mean pharmacokinetic parameters. These mean parameter estimates were utilized to predict the percentage of time that free drug concentrations remain above the MIC (fT>MIC) for the ceftaroline regimens used in the dose-ranging studies.

(ii) Human-simulated regimen.

A human-simulated dosing regimen based on serum concentrations was developed using the same mean pharmacokinetic parameters as noted above. This regimen simulated the fT>MIC, peak concentration (C_max), and area under the curve (AUC) as observed in humans following treatment with 600 mg ceftaroline fosamil i.v. every 12 h as a 1-h infusion. Exposure for ceftaroline in humans was derived from pharmacokinetic data in healthy volunteers. The protein binding for ceftaroline in humans was 20% (7, 19). To ensure appropriate target exposures were reached with the simulated dosing regimen, confirmatory pharmacokinetic studies were conducted in neutropenic-infected and uninfected mice. The purpose of this was to determine whether there were notable differences in ELF penetration between infected and uninfected models. For both infected and uninfected groups, blood and ELF samples were collected from groups of six mice at 3 time points over the 12-h dosing interval.

In vivo efficacy.

Seventeen clinical S. aureus isolates (2 MSSA, 15 MRSA) were studied using the neutropenic lung infection model. Beginning 3 h after inoculation, groups of six mice received treatment with ceftaroline over a 24-h period. Ceftaroline fosamil doses were administered as 0.2-ml subcutaneous injections. Control animals were administered normal saline at the same volume, route, and frequency as the treatment regimens.

Groups of six untreated control mice were sacrificed just prior to the initiation of therapy (0 h) to serve as a baseline measurement of lung bacterial density. All other control and treatment mice were sacrificed 24 h after initiation of therapy. Mice that did not survive to 24 h had their lungs removed at the time of expiration. Following sacrifice, lungs were aseptically harvested and then homogenized in 1.0 ml of normal saline. Serial dilutions of lung homogenate were plated onto 5% sheep blood agar and Columbia nutrient agar (Remel Inc., Lenexa, KS) and incubated at 37°C for 24 h. The change in bacterial density was calculated as the difference in log₁₀ CFU from ceftaroline-treated mice after 24 h from the 0-h control animals.

(i) Dose-ranging studies.

Ten S. aureus (MRSA) isolates were utilized in the dose-ranging studies against ceftaroline fosamil. Dosing regimens from 0.5 to 75 mg/kg/day administered in 1 to 3 doses were given to provide a range of exposures with regard to fT>MIC.

(ii) Human-simulated studies.

Fifteen S. aureus isolates (13 MRSA and 2 MSSA) were tested against the human-simulated regimen of 600 mg ceftaroline fosamil every 12 h, administered as a 1-h infusion. This dosing regimen was repeated every 12 h for 24 h.

Pharmacodynamic target determination.

The sigmoidal maximum effect (E_max) model was used to describe the relationship between the pharmacodynamic target (fT>MIC) and efficacy. The stasis exposure values and 1-log₁₀ reduction in bacterial density were calculated from the composite curve of all 17 S. aureus isolates evaluated in dose-ranging and human-simulated studies.

RESULTS

Bacterial isolates.

The phenotypic and genotypic profiles for the S. aureus isolates used in this study are listed in Table 1. The ceftaroline MICs for these isolates ranged from 0.5 to 4 μg/ml.

Table 1.

Genotypic and phenotypic profiles of the Staphylococcus aureus test isolates for ceftaroline^a

S. aureus isolate	USA type	PVL	SCCmec	CPT MIC (μg/ml)	Dosing approach used
					Dose ranging	Human-simulated regimen
487	USA300	Pos	4	0.5	+	+
489	USA300	Pos	4	0.5	+	+
152	USA100	Neg	ND	0.5	+
56	USA100	ND	ND	1	+
*465		Neg	ND	1		+
*466		Neg	ND	1		+
491	USA300	Pos	4	1	+	+
492	USA100	Neg	2	1		+
493	USA100	Neg	2	1		+
494		Neg	3	1	+	+
495		Neg	3	1		+
497	USA100	Neg	2	2	+	+
498	USA100	Neg	2	2		+
499	USA100	Neg	2	2	+	+
506		Neg	2	2		+
473		Neg	ND	4	+	+
476		Neg	ND	4	+	+

^aPhenotypic profiles (MICs) were determined by broth microdilution. The dosing approaches used against each isolate are also listed in the two far right columns (+ indicates that the dosing approach was used). ND, not determined; Neg, negative; Pos, positive;

^*, MSSA (all other isolates are MRSA).

Protein binding.

Ceftaroline protein binding in mouse serum was found to be 31.4% ± 6.84 and 34.5% ± 9.96 for the 50 and 5 μg/ml concentrations, respectively. These values are similar to the previous report of 35% protein binding in mouse serum; therefore, this value was used throughout the study (7).

Pharmacokinetic determination. (i) Single-dose studies.

The pharmacokinetics of ceftaroline were best characterized using a one-compartment model and were linear over the range of doses tested.

(ii) Human-simulated dosing.

The free drug concentration-time profile for 600 mg ceftaroline administered as a 1-h infusion in humans, as well as the human-simulated murine regimen in infected and uninfected mice, is shown in Fig. 1. The resulting regimen involved two doses of ceftaroline fosamil: 11 mg/kg at 0 h, followed by 0.25 mg/kg at 6.5 h. The pharmacodynamic driver of efficacy, fT>MIC, for this class of compounds was similar between humans and our humanized profile in mice (Fig. 1). As noted in Fig. 1, the serum profiles were similar in infected and uninfected mice.

Fig 1.

Simulated free serum drug concentration-time profile for ceftaroline following administration of 600 mg ceftaroline fosamil in human (dashed line) and mice (solid line) with actual obtained concentrations (mean ± SD) in infected mice (black circles) and uninfected mice (black triangles), with corresponding targets in mice and humans listed.

(iii) Pulmonary concentrations.

Ceftaroline concentrations in ELF were similar to their corresponding serum concentrations after humanized exposures (Fig. 2). Similarities between serum and ELF concentrations were also noted in single-dose pharmacokinetic evaluations (data not shown). Figure 2 illustrates the ELF penetration for the human-simulated regimen for the infected and uninfected neutropenic mice.

Fig 2.

Free serum and ELF concentration profile following the human-simulated regimen in infected mice (A) and uninfected mice (B).

In vivo efficacy. (i) Dose ranging.

The mean (±standard deviation) bacterial density for the 0-h control mice was 5.65 ± 0.14 log₁₀ CFU. The mean bacterial density of the 24-h control mice increased to 7.58 ± 0.25 log₁₀ CFU. The results of the 10 isolates used in the dose-ranging studies are shown in Fig. 3. The fT>MIC required to produce an approximately 1-log kill ranged from 17% to 43%.

Fig 3.

Efficacy of ceftaroline fosamil at 24 h compared with 0-h controls for 10 S. aureus isolates with MIC values of 0.5 μg/ml (A and B), 1 μg/ml (C and D), 2 μg/ml (E), and 4 μg/ml (F) in the neutropenic lung infection model.

(ii) Human simulated.

The mean (±standard deviation) bacterial density for the 0-h neutropenic control mice at the start of dosing was 5.79 ± 0.11 log₁₀ CFU. The mean bacterial density of the 24-h control mice increased to 7.60 ± 0.22 log₁₀ CFU. A number of control mice did not survive the entire 24 h. In these cases, lungs were harvested at the time of expiration. Bacterial densities from these animals were found to be similar to those in mice that did survive 24 h and were included in the data analysis. All mice that received treatment with ceftaroline survived 24 h. The human-simulated regimen of ceftaroline produced a 0.64- to 1.95-log₁₀ CFU reduction after 24 h against the various S. aureus isolates in the lung infection model (Fig. 4).

Fig 4.

Change in log₁₀ CFU (mean ± SD) for a human-simulated regimen of ceftaroline fosamil at 24 h relative to 0-h controls for 15 S. aureus isolates with a MIC range of 0.5 to 4 μg/ml in the neutropenic lung infection model.

(iii) Pharmacodynamic target.

The target exposure values for stasis and a 1-log₁₀ reduction in bacterial densities calculated from the composite E_max model were 16% and 41%, respectively (Fig. 5). The correlation coefficient (r²) for this model was 0.79.

Fig 5.

Pharmacodynamic profile of ceftaroline fosamil against 17 S. aureus isolates in the neutropenic lung infection model.

DISCUSSION

The incidence of S. aureus continues to increase, and it is often the pathogen responsible for HAP and some cases of CAP (18). Unfortunately, treatment options remain limited, with vancomycin and linezolid being the preferred Gram-positive agents for treatment of HAP (1). Additionally, poor outcomes and clinical failures have been reported with vancomycin, notably in isolates with vancomycin MICs of 1 to 2 μg/ml (11, 16). Ceftaroline fosamil, the most recent addition to the antimicrobial class of cephalosporins, has bactericidal activity in vitro against S. aureus isolates, including those with increased vancomycin MICs (5, 7). In this study, we determined the in vivo efficacy of ceftaroline fosamil against a variety of S. aureus isolates in a neutropenic murine lung infection model utilizing two different approaches to dosing.

Ceftaroline fosamil was effective against all 10 MRSA isolates used in the dose-ranging studies and required approximately 17 to 43% fT>MIC to produce a 1-log kill. After 24 h of treatment with the human-simulated regimen, there was considerable activity against all isolates tested, and 14 of the 15 isolates tested had a minimum of a 1-log reduction in bacterial density. The fT>MIC exposure with this regimen ranged from 30% to 73%, depending on the MIC of the isolate. Bacterial reductions in the MSSA isolates were similar to those of the MRSA isolates tested with the same MIC of 1 μg/ml.

The target (fT>MIC) values calculated in the composite E_max model for stasis and 1 log₁₀ CFU reduction were 16% and 41%, respectively (Fig. 5). This was similar to a previous study which administered a human-simulated regimen against various S. aureus isolates in a thigh infection model in which a 27.5% fT>MIC exposure was required to produce efficacy (7). In another study that utilized four S. aureus isolates, a 33% fT>MIC exposure was required to produce a 1-log kill (2). Ceftobiprole, another cephalosporin with activity against MRSA, was previously tested against S. aureus isolates in a murine lung infection model and required a 20 to 30% fT>MIC for maximal efficacy (13). The average reduction in bacterial density after treatment with ceftobiprole was approximately 2 log₁₀ CFU, with maximal kill at 2.5 log₁₀ CFU. The differences among these data are likely the result of the collective sources of variability (i.e., differing growth of isolates, variation in pharmacokinetics within animals, and variation in phenotypic assessments) that are common to these in vivo systems.

Eight MRSA isolates were tested with both dosing approaches (human simulated and dose ranging). For many of these isolates, there was a substantial correlation in bacterial reductions between the dose-ranging and human-simulated dosing approaches. For example, the human-simulated regimen provided fT>MIC exposure of 60% for isolate STA 491. This regimen produced a 1.28 log₁₀ CFU reduction in bacterial density. Likewise, when this isolate was tested in the dose-ranging studies against a regimen of 6.25 mg/kg every 8 h, which provided a 65% fT>MIC, a 1.34 log₁₀ CFU reduction in bacterial densities was observed. These finding are in concordance with a previously published study with ertapenem that utilized two different dosing approaches that provided similar fT>MIC exposures and ultimately displayed parallel results in terms of bacterial killing (4). While dose-ranging studies are beneficial for characterizing pharmacodynamic targets, human-simulated regimens are valuable for assessing the robustness of activity against a range of isolate MICs expected in the clinical setting.

Although all these isolates were tested only in a neutropenic model, clinically this agent will be used in both immunocompetent and immunocompromised patients. In the presence of an intact immune system, it is possible that bacterial reductions may be even further enhanced (17). In this murine lung infection model, ceftaroline showed consistent activity against a range of MRSA and MSSA isolates. Although the FDA-defined susceptibility breakpoint against S. aureus is 1 μg/ml, our study encompassed the highest-reported MIC of 4 μg/ml and displayed substantial activity among all of these isolates (6).

Adequate drug exposure at the site of infection is an important determinant of potential drug efficacy for treating pneumonia (9). The pharmacokinetic results from this study demonstrate that the fT>MIC of ceftaroline in the ELF of mice was essentially the same as that seen in serum (Fig. 2). These results mirror the findings seen with another cephalosporin with anti-MRSA activity, ceftobiprole, which also showed sufficient high penetration into target tissues of mice (13). The bacterial reductions seen in all isolates tested provide reliable evidence that ceftaroline reached the site of infection adequately in this animal model. Previous studies have reported an increase in penetration in infected mice as opposed to uninfected mice (8). Given those findings, we sought to determine if the presence of infection affects the bronchopulmonary penetration of ceftaroline. As noted in Fig. 2, a notable difference in the extent of penetration between the two models was not present. These ELF data are from a murine model and cannot translate to penetration in humans.

In this study, human-simulated and dose-ranging studies with ceftaroline fosamil were effective against a variety of clinical S. aureus isolates, including isolates with MICs up to 4 μg/ml. The target fT>MIC required in this study was similar to previously published data and was readily achieved with the human-simulated regimen of 600 mg every 12 h, administered as a 1-h i.v. infusion (2, 7, 13). While these data are supportive, clinical efficacy data are required to fully assess the viability of ceftaroline fosamil for the treatment of pneumonia due to MRSA in humans.