Comparison of In Vivo and In Vitro Pharmacodynamics of a Humanized Regimen of 600 Milligrams of Ceftaroline Fosamil Every 12 Hours against Staphylococcus aureus at Initial Inocula of 10⁶ and 10⁸ CFU per Milliliter

Abstract

In light of the in vivo/in vitro discordance among beta-lactams against Gram-negative pathogens, we compared the in vivo pharmacodynamics of humanized ceftaroline against 9 Staphylococcus aureus strains (MICs of 0.13 to 1 mg/liter) from published in vitro studies using standard and high inocula in the murine thigh infection model. Consistent with the in vitro findings, mean reductions of ≥1 log₁₀ CFU were observed for ceftaroline against all strains using both standard and high inocula. These results suggest in vivo/in vitro concordance with no observed inoculum effect.

TEXT

Discordant in vivo and in vitro pharmacodynamics (PD) have been reported among beta-lactams against Gram-negative pathogens (1–4). The discrepancy has been speculated to be due to the unnatural accumulation of enzymes within in vitro pharmacodynamic models and the resultant, rapid hydrolysis of antimicrobials against Enterobacteriaceae with enzyme-mediated resistance (4, 5). However, the in vivo/in vitro paradox has also been observed in other Gram-negative pathogens, such as Pseudomonas aeruginosa, whose mechanisms of resistance are mainly non-enzyme mediated (e.g., efflux pump or outer membrane proteins) (4, 6).

As for Gram-positive pathogens, such as Staphylococcus aureus, while many have been explored in in vivo and in vitro pharmacodynamic models, usage of varied isolates, initial inoculum concentrations, and antimicrobial regimens makes comparisons between in vivo and in vitro models difficult (7–10). Ceftaroline, a cephalosporin with anti-methicillin-resistant S. aureus (MRSA) activity via its high affinity to penicillin-binding protein 2a, is considered a viable option for resistant S. aureus infections. In lieu of the knowledge that increased ceftaroline MICs against S. aureus are mostly associated with mutations in penicillin-binding protein 2a (11, 12), this study aimed to compare the in vivo and in vitro pharmacodynamics of ceftaroline against Gram-positive pathogens with non-enzyme-mediated resistance. To mimic the published in vitro pharmacodynamic studies that tested ceftaroline (active drug) against S. aureus, we evaluated the in vivo pharmacodynamics of a previously determined humanized regimen (simulating ceftaroline pharmacokinetics in humans versus mice) of 600 mg ceftaroline fosamil (a ceftaroline prodrug) every 12 h against nine phenotypically diverse S. aureus isolates in the setting of two different initial inocula in the neutropenic murine thigh infection model.

Commercially available ceftaroline fosamil (lot 0008D36; Forest Pharmaceuticals, Inc., St. Louis, MO) was used for the in vivo studies. Vials were reconstituted and diluted to the desired concentrations according to the manufacturer's specifications.

Nine S. aureus isolates (ceftaroline MICs of 0.13 to 1 mg/liter) that were previously studied in published in vitro pharmacodynamic models (13, 14) were used for the standard-inoculum in vivo studies. Among these, three MRSA isolates were tested for the high-inoculum in vivo studies (Table 1) (14).

TABLE 1.

Phenotypic profiles and corresponding fT_>MIC of tested S. aureus isolates in standard- and high-inoculum studies^a

S. aureus isolate	Isolate no. from referenced in vitro data (reference)	Phenotypeb	Ceftaroline MIC (mg/liter)	fT>MIC (%)
477	89608 (13)	MSSA	0.13	100
478	89637 (13)	MSSA	0.13	100
479	83771 (13)	CA-MRSA	0.5	81.7
480	84495 (13)	HA-MRSA	0.5	81.7
456	VRS3a (13)	VRSA	0.5	81.7
435	NRS4 (13)	VISA	1	60.8
412c	412 (14)	MRSA	1	60.8
449c	449 (14)	hVISA	1	60.8
454c	454 (14)	VISA	1	60.8

^afT_>MIC data from reference 10.

^bCA, community acquired; HA, health care acquired; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus; VISA, vancomycin-intermediate Staphylococcus aureus; VRSA, vancomycin-resistant Staphylococcus aureus; hVISA, heteroresistant vancomycin-intermediate Staphylococcus aureus.

^cIsolates that were used in both standard- and high-inoculum studies.

The study protocol was reviewed and approved by the Hartford Hospital Institutional Animal Care and Use Committee. Animals were maintained and used in accordance with the National Research Council recommendations. The previously established neutropenic murine thigh infection model was employed to evaluate efficacy (10). Briefly, pathogen-free, female ICR mice weighing approximately 25 g were acquired from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Mice were rendered neutropenic with intraperitoneal injections of cyclophosphamide (100 and 150 mg/kg of body weight; Baxter Healthcare Corporation, Deerfield, IL) given 1 and 4 days prior to inoculation, respectively. Three days prior to inoculation, mice were also given a single 5 mg/kg intraperitoneal injection of uranyl nitrate to produce a predictable degree of renal impairment to aid in humanizing the drug regimens (10). Two hours prior to the initiation of the antimicrobial therapy, each thigh was inoculated intramuscularly with 0.1 ml solution containing approximately 1 × 10⁷ CFU/ml of the test isolate for the standard-inoculum and 1 × 10⁹ CFU/ml for the high-inoculum studies.

Two hours postinoculation, subcutaneous doses of ceftaroline fosamil were administered as 0.2-ml subcutaneous injections to groups of three mice as two 12-h regimens over a 24-h period as follows: 20 mg/kg at 0 h, 2 mg/kg at 2.5 h and 4 h, and 1 mg/kg at 6 h and 9 h. This regimen was previously described to simulate the human concentration-time profile of 600 mg intravenous ceftaroline fosamil every 12 h, which achieved a percentage of the dosing interval where a free-drug concentration exceeds the MIC (fT_>MIC) of ≥60.8% at an MIC of ≤1 mg/liter (Table 1) (10). The 24-h control groups received normal saline with the same volume, route, and frequency as the treatment regimen. Groups of three untreated mice were assigned to 0-h control groups; they were euthanized by CO₂ exposure, followed by cervical dislocation, and harvested at the initiation of therapy. All other animals were harvested 24 h after the initiation of therapy. Mice that failed to survive to 24 h were harvested at the time of expiration. Following sacrifice, each thigh was harvested and homogenized in 5 ml of normal saline. Serially diluted thigh homogenates were plated onto Trypticase soy agar with 5% sheep blood to determine the bacterial density. The efficacy was calculated as the change in bacterial density in log₁₀ CFU at 24 h compared with that at 0 h.

The efficacy of ceftaroline against the nine isolates in the standard-inoculum study is displayed in Fig. 1A. The respective log₁₀ CFU values at 0 h and in the 24-h controls were 5.5 to 5.8 and 8.3 to 9.6. The ceftaroline treatment resulted in mean reductions of 1.1 to 2.4 log₁₀ CFU, with no relationship to the ceftaroline MIC. These observations were consistent with the findings of Zhanel et al., who evaluated the in vitro pharmacodynamics of the humanized regimen of ceftaroline against isolates 477, 478, 479, 480, 456, and 435 at initial inocula of 1 × 10⁶ CFU/ml (Table 1) (13). In their study, human-simulated ceftaroline treatment against all six strains achieved >3 log₁₀ CFU decreases over 24 h.

FIG 1.

Changes in log₁₀ CFU values for a humanized regimen of ceftaroline at 24 h compared with the 0-h controls for a collection of S. aureus isolates tested against standard inocula (A) and high inocula (B). Bars represent means ± standard deviations. STA, Staphylococcus aureus.

For the high-inoculum in vivo study, the corresponding bacterial densities for controls at 0 h and 24 h were 7.4 to 7.6 and 8.0 to 8.1, respectively. The use of high inocula did not affect the ceftaroline efficacy against the three strains tested (Fig. 1B), with mean decreases of 2.0 to 3.0 log₁₀ CFU. These findings recapitulated the results from an in vitro pharmacodynamic study by Bhalodi et al., who tested the same three S. aureus strains (i.e., 412, 449, and 454 [MICs of 1 mg/liter]) at high inocula (14) and showed decreases of >5 log₁₀ CFU after 24 h. While the study of Bhalodi et al. did not evaluate the efficacy using a standard inoculum against these three strains, our data suggest that ceftaroline does not exhibit an inoculum effect. Although previous in vivo and in vitro studies of penicillins and cephalosporins noted an inoculum effect secondary to a reduction in penicillin-binding proteins during the stationary growth phase (15), the absence of an inoculum effect is consistent with the findings of Lee et al. (16), where another anti-MRSA cephalosporin, ceftobiprole, exhibited the smallest inoculum effect against S. aureus compared with those of daptomycin, linezolid, and vancomycin. Of note, although the 24-h treatment groups in the high-inoculum studies showed greater reductions (2.0 to 3.0) in log₁₀ CFU values than those in standard-inoculum studies (1.1 to 2.4), this was an artifact of the elevated initial bacterial load (i.e., 0 h) against which the efficacy was compared.

While previous data have suggested discordance between in vivo/in vitro efficacy for beta-lactams and Gram-negative pathogens, the findings herein suggest in vivo/in vitro concordance for ceftaroline against S. aureus. This concordance extends to both standard and high inocula, in which ceftaroline did not show an inoculum effect.