Exebacase in Addition to Daptomycin against MRSA

ABSTRACT

Exebacase is a lysin (cell wall hydrolase) with direct lytic activity against Staphylococcus aureus including methicillin-resistant S. aureus (MRSA). Time-kill analysis experiments illustrated bactericidal activity of exebacase-daptomycin against MRSA strains MW2 and 494. Furthermore, exebacase in addition to daptomycin (10, 6, and 4 mg/kg/day) in a two-compartment ex vivo pharmacokinetic/pharmacodynamic simulated endocardial vegetation model with humanized doses resulted in reductions of 6.01, 4.99, and 2.81 log₁₀ CFU/g (from initial inoculum) against MRSA strain MW2.

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) infections are a major public health concern, with cases exhibiting high morbidity and mortality rates (1–4). Therefore, there is great enthusiasm for novel antibacterial approaches, such as bacteriophages (phages) and their specific components that have developed in concert with bacteria and have evolved adaptations for use against bacterial defenses (5, 6).

Exebacase (previously called CF-301) is an antistaphylococcal cell wall hydrolase and is the first lysin to enter phase 3 clinical trials (7–9). The microbiologic attributes of exebacase include rapid bactericidal activity against S. aureus (planktonic and biofilm bacteria both in vitro and in vivo [10–13]), low propensity for resistance, and synergistic effect when used in addition to conventional antistaphylococcal antibiotics (10, 13–15). Exebacase in addition to daptomycin (DAP) has proven efficacious (8, 10, 14, 16), and the goal of this study was to evaluate the use of exebacase in addition to deescalating humanized doses of DAP, compared to DAP alone, in a two-compartment ex vivo pharmacokinetic/pharmacodynamic (PK/PD) simulated endocardial vegetation (SEV) model.

In order to assess the efficacy of exebacase on the activity of DAP, MIC values were determined in duplicate using the broth microdilution (BMD) method (17). Exebacase was supplied by ContraFect Corporation (Yonkers, NY, USA), and DAP was purchased commercially from Sigma-Aldrich Co. (St. Paul, MN, USA). Cation-adjusted Mueller-Hinton broth (MHB) (equivalent of 50 mg/liter calcium) supplemented with horse serum was used for all of the experiments (8, 18). Of note, the addition of Ca²⁺ and different concentrations of Ca²⁺ present in the media did not change the exebacase MIC. MRSA strains used in this study include 494 (USA 100/ST5), a clinical isolate taken from the bloodstream of a patient with bacterial endocarditis (19), and MW2 (USA400/ST1 [20]) a community-acquired MRSA strain that is well-characterized and whole genome sequenced (Table 1). None of the isolates contain mprF mutations, which are known to be associated with daptomycin- vancomycin cross-resistance (21). Emergence of resistance and selection of less susceptible isolates at each time point (SEV models or last time point of time-kill analysis [TKA]) were checked by plates containing 3× MIC of daptomycin or exebacase and MIC evaluation.

TABLE 1.

MIC values for MRSA 494 and MW2 for DAP alone, exebacase alone, and DAP in the presence of exebacase

MRSA strain	MIC (mg/liter)
	DAP	Exebacase	DAP-exebacase
494	0.125	2	0.0313
MW2	0.125	1	0.0313

To identify the impact of exebacase on DAP activity during the 24-h exposure, TKA experiments were performed at a starting target inoculum of 10⁶ CFU/ml (17, 22). DAP in addition to exebacase (1× the MIC) demonstrated a 4-log reduction in CFU per milliliter for both 494 and MW2, reaching detection limits by the end of a 24-h exposure (Fig. 1). While the exebacase-alone treatment exhibited regrowth by 24 h, DAP in addition to exebacase was below the limit of detection. We did not observe any resistance against either strain with single treatments. We were not able to test the resistance development against combination therapies since there were no colonies on 24-h plates.

FIG 1.

Twenty-four-hour time-kill experiments against strains MW2 (A) and MRSA 494 (B). Standard deviation bars are specified for each time point. Limit of detection, 10².

SEVs were made as reported previously (18). Daptomycin dose deescalation was investigated with doses of 10, 6, and 4 mg/kg/day alone for 4 days and in addition to only one dose of exebacase on the first treatment day. All models and samples were performed in duplicate for reproducibility purposes. To assess the impact of exebacase on the efficacy of DAP, we evaluated the log₁₀ CFU per gram counts of the ex vivo PK/PD SEV models of MW2 as shown in Fig. 2. DAP 4- and 6-mg/kg alone regimens exhibited initial bactericidal activity, accompanied thereafter with regrowth up to 8.04 and 5.25 log₁₀, respectively. The DAP 10-mg/kg regimen showed activity with a final CFU/g of 5.05 log₁₀. Exebacase, consisting of a one-time dose of 0.0031 mg/ml on day 1 only, showed no bactericidal activity or efficacy and displayed CFU per gram counts similar to those of the growth control (GC) up to 96 h. Only one dose of exebacase on the first treatment day in addition to a daily dose of DAP 4, 6, or 10 mg/kg during 96 h showed reductions in CFU/g of 2.33, 1.74, and 2.35 log₁₀ at 96 h, respectively, compared to those of their corresponding DAP alone counterpart. The mean CFU per gram values at the end of exposure (96 h) were significantly different for all DAP in addition to exebacase treatments compared to those of either GC or exebacase alone regimens (P < 0.01). The mean CFU per gram values at the end of 96 h were significantly different for DAP 4 and 10 mg/kg in addition to exebacase compared to those of DAP 4 and 10 mg/kg alone (P = 0.02).

FIG 2.

PK/PD SEV two-compartment models. MW2 models encompassing growth control (GC), exebacase alone, DAP 4 mg/kg (A), DAP 6 mg/kg (B), and DAP 10 mg/kg (C) alone and in addition to exebacase. Brackets in each figure show the Δlog CFU per gram difference between single DAP treatment versus DAP in addition to exebacase treatment at the end of a 96-h exposure time. Standard deviation bars are specified for each time point. Limit of detection, 10².

The targeted and achieved pharmacokinetic parameters in the ex vivo models, including peak concentration (C_max), half-life (t_1/2), and area under the concentration-time curve from 0 to 24 h (AUC_0–24) values for DAP doses 4, 6, and 10 mg/kg/day as well as humanized exebacase exposures of 0.0031 mg/ml (C_max) on day 1, are listed in Table S1 and Fig. S1 in the supplemental material. Emergence of resistance was not observed with any of the regimens.

This study has explored the use of an antistaphylococcal lysin as an efficacious additive to antistaphylococcal antibiotics such as DAP.

While there have been several nonclinical studies of exebacase in rat and rabbit endocarditis models, our ex vivo PK/PD model is unique owing to its capability of simulating humanized doses of exebacase in addition to DAP against a high inoculum of bacterial burden (15, 23). DAP in addition to only 1 dose of exebacase in our model displays enhanced bactericidal activity over clinically relevant dose ranges, which bodes well for its clinical translation (12, 24). The fact that the reduction in CFU per gram was maintained over the duration of the model while the exebacase is no longer present after day 1 can potentially be due to a post-antibiotic sub-MIC effect (PA-SME) of exebacase as reported in prior research (9, 11, 12, 25–27). The PA-SME of exebacase has led to prolonged doubling time, reduction in logarithmic growth phase, and elongated lag phase of the bacteria (11, 25).

Exebacase in addition to DAP therapy was effective, inspiring DAP dose de-escalation in our SEV PK/PD model against the well-categorized strain MW2. This observation may have important clinical implications related to potential dose de-escalation, which may warrant further clinical studies leading to improved patient outcomes.