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Preclinical evaluation of efficacy and pharmacokinetics of gentamicin containing extracellular‐matrix envelope

Abstract Background Using synthetic antibiotic‐eluting envelope (ABE) is an effective intervention for prevention of cardiovascular implantable electronic device (CIED) infection. The biologic extracellular‐matrix envelope (ECME), may offer potential advantages over the synthetic ABE. To further minimize the risk of infection, the ECME can be hydrated in gentamicin prior to CIED implantation. We aimed to evaluate the efficacy and pharmacokinetics (PK) of gentamicin containing ECME in an animal model. Methods For all experiments, the ECME was hydrated in gentamicin (40 mg/Ml) (treatment) for 2 min. In vitro antimicrobial efficacy against six different bacterial species was assessed. In vivo experiments were conducted using a rabbit model of CIED pocket infection. Serum and ECM gentamicin concentrations were measured. Five different organisms were inoculated into the device pocket of control (ECME hydrated in 0.9% saline) and treatment groups. Macroscopic appearance and colony forming units from CIED, ECME, and tissue were determined. Results No bacteria were recovered from any culture after 12 h of exposure to the gentamicin containing ECME. Serum gentamicin levels dropped below the limit of quantification at 15 h after implant. Gentamicin concentration in the ECME remained relatively stable for up to 7 days. Signs of clinical infection were observed in the control but not in the treatment group. In the presence of gentamicin, statistically significant reduction was demonstrated across all tested bacterial species. Conclusions In this preclinical animal infection model, gentamicin containing ECME was highly effective in reducing bacterial burden in the implant pocket, while systemic exposure after implantation remained low.


F I G U R E 1 Extracellular matrix envelope
infections are of critical importance. 10 However, only a limited number of evidence-based interventions are currently available. As majority of infections develop at the time of device implantation, these strategies are directed at decreasing the bacterial burden at the implant site and include perioperative antistaphylococcal antibiotic prophylaxis, 11 and aseptic techniques. More recently, adjunctive use of a synthetic absorbable antibiotic-eluting envelope (ABE) was shown to significantly reduce the incidence of CIED pocket infections. 12,13 With such approach, the pulse generator is placed into the ABE, stabilizing the device within the surgical pocket; while gradually delivering antibiotics over a period of days.
A second type of CIED envelope constructed from a biologic material is currently available for clinical use (ECM Aziyo Biologics; Silver Spring, MD) ( Figure 1). This envelope consists of a multilaminate sheet of decellularized, noncrosslinked, lyophilized extracellular matrix (ECM), derived from porcine small intestinal submucosa. This biological scaffold provides a substrate for better tissue integration, vascular ingrowth, and more rapid clearance of bacteria. [14][15][16][17][18][19][20][21] Furthermore, some studies suggest intrinsic bactericidal activity. Over time, the ECM envelope remodels into a vascularized pocket that may facilitate implant removal or revision when required. Given its theoretical lessened foreign body response and resistance to infection, the ECM envelope may offer plausible advantages over the synthetic ABE.
To further minimize the risk of CIED infection, this biologic ECM envelope can be hydrated in a gentamicin solution prior to implantation. Gentamicin is an aminoglycoside with broad-spectrum bactericidal activity including the most common organisms identified in CIED infections including Staphylococcus spp. and aerobic Gramnegative organisms. [22][23][24][25] When given as monotherapy, gentamicin has also demonstrated in vitro efficacy against established Staphyloccocal biofilms. 26 However, there are major limitations associated with systemic use of gentamicin therapy including the potential development of nephrotoxicity and otoxocity. 27 Local gentamicin delivery to the site that potentially harbors pathogens, may overcome some of these risks. Earlier studies have demonstrated reduced infection rates with local application of gentamicin containing collagen sponge during CIED implantation. 28,29 Furthermore, Deering et al 30 demonstrated that after hydration of ECM envelope in different antibiotics solution, elution was observed for a period of up to 6 days. In this same study, subsequent experiments performed using an animal model of cardiac device pocket infection showed that the released antibiotics (vancomycin or gentamicin) substantially reduced colonization by two strains of Staphylococcus on CIED devices. However, in these initial studies, only two strains of Gram-positive bacteria were tested in a semiquantitative animal model and pharmacokinetic (PK) studies were not conducted.
In this study, we aimed to evaluate the efficacy and PK of gentamicin containing ECM envelope using an animal model of cardiac device pocket infection.

Animal experiments
All animal studies conformed to the Guide for the Care and Use of Laboratory Animals, and the surgical procedures and animal care were conducted at an independent research organization (Wuxi Apptec,

Serum gentamicin concentrations
For this study, 22 New Zealand White rabbits were implanted with a CIED placed into a small-size ECM envelope in a subcutaneous pocket on the left or right dorsal side. Prior to implantation, as with in vitro studies, the ECM envelope was hydrated in 20 mL of a 40 mg/mL gentamicin solution for 2 min.
To measure the serum gentamicin concentration, postimplantation, four blood samples from the rabbit models were obtained at 15 min; 1, 3, 6, 10, 15 h; and 7 days (Supporting Information Table S1).  Data acquisition and processing was performed using Thermo Xcalibur software (Thermo Fisher Scientific). Gentamicin C1, C1a, and C2 were measured by the LC/MS method, and total gentamicin was calculated as the sum of the three components.

Efficacy of gentamicin containing ECM envelope
To analyze the antibacterial performance of gentamicin containing ECM envelope, in vivo experiments were conducted using an estab-   When 0 CFU were recovered from the treatment group (complete kill), Log 10 (CFU/mL) < 1.0 was used to calculate the log reduction.
treatment group (gentamicin hydrated) and control group (normal saline hydrated).

Low risk of systemic exposure to gentamicin
The serum gentamicin concentrations in rabbit following implantation of ECM envelope at each time point are presented in

Gentamicin concentration in the ECM envelope remains stable for up to 7 days
Gentamicin concentration in the ECM envelope expressed as microgram per gram of explanted device is shown in

Significant reductions in bacterial colonization by gentamicin ECM envelope
In all studies, no target microorganisms were recovered from any sample in the environmental control group, indicating excellent asepsis and elimination of cross-contamination. Representative gross photographs of devices at removal are shown in Figure 3. Signs of clinical infection were observed in the control (saline) group, but not in the gentamicin group.
The results of quantitative bacterial colony counts are presented in

DISCUSSION
The results of the current study demonstrate that gentamicin, deliv-   Note. The bold values represent those with a >3 log reduction as above.

Advantages of local gentamicin delivery
A ≥3-log reduction constitutes a bactericidal effect whereas a <3-log reduction is defined as a bacteriostatic effect.
Staphylococcus spp. demonstrated that a 3-log kill (99.9% reduction in CFU/mL) was achieved across a six-fold range of concentrations (note that the effectiveness between concentration groups was not statistically different). 34 Combined, these studies suggest that a 3-log reduction is the clinically relevant threshold for supporting antimicrobial claims for an implant material coated or impregnated with gentamicin. Previous in vitro elution study 30 and also current in vivo PK study demonstrated that the gentamicin was released from the ECM envelope for at least 7 days. Therefore, the local antibiotic concentration was maintained above MIC level for a relatively long period of time, which cannot be simply achieved by antibiotic wash during the procedure.
Although CIED infections due to Gram-negative bacteria represent a small proportion of cases, they mainly present as pocket infections and majority are caused by P. aeruginosa. 46 In a randomized clinical trial evaluating the efficacy of ABE, when analyzing the microbiology of breakthrough pocket infections, a sizable number of those cases was due to Gram-negative bacteria, predominantly Pseudomonas spp. 47 Therefore, gentamicin containing ECM envelope may in theory be more effective than rifampin/minocycline combination in preventing pocket infections due to Gram-negative bacteria.
Importantly, the results in animal studies may not necessarily translate to humans, and therefore, further clinical studies are needed to confirm efficacy of gentamicin containing ECM envelope in reducing the risk of major CIED infections.

Low risk of systemic exposure to gentamicin
Importantly, in the rabbits evaluated in the current study, serum gentamicin levels peaked early after implantation of the ECM envelope (∼1 h), then rapidly declined to below the lower limit of detection (and below the recommended safety trough concentration of 2 mg/L 27 after 15 h, suggesting that the risk of systemic exposure to gentamicin is low following implantation of the gentamicin containing ECM envelope. The noncompartmental analysis identified PK parameters of serum gentamicin that are consistent with previous reports, including a terminal half-life of approximately 2 h, indicating that systemically absorbed gentamicin is rapidly cleared from the blood. 48 These findings align with previous clinical studies, in which gentamicin delivered via collagen ECM or directly to the surgical site reached concentrations in local tissues that exceeded 300 mg/L, whereas systemic exposure was limited to approximately 1-2 mg/L. 36,49 It is worth noting that the blood volume of a rabbit is approximately 3% of the blood volume of a human (∼0.15 L in rabbits and 5 L in humans). 50,51 Therefore, the concentration of gentamicin expected in the systemic circulation of a patient implanted with one gentamicin containing ECM envelope would be much lower than the levels observed in this rabbit study, and probably undetectable. However, low systemic exposure to gentamicin may be more relevant in older patients and those with pre-existing renal failure. Studies in human subjects are needed to further determine the safety of gentamicin containing ECM envelope in these patients.

Limitations
The translation of experiments conducted in animal models into clinical practice requires further investigation. However, the animal model of cardiac device pocket infection here described has been used earlier to investigate the safety and efficacy of minocycline/rifampin synthetic ABE. 32 Notably, the results of such animal experiments were later confirmed in a prospective randomized clinical trial. 12 Although microscopic analysis of the biofilm present on the device surfaces was not performed in this study, vortexing/sonication is considered a surrogate. Conventional swabs and tissue cultures of the pocket would also be valuable to further confirm the results obtained by vortexing/sonication.

CONCLUSIONS
The results of this preclinical animal infection model demonstrate high, sustained local gentamicin concentrations and excellent bacterial killing in vivo following implantation of the gentamicin containing ECM envelope, with a low risk of systemic exposure to gentamicin.

CONFLICT OF INTEREST
Dr. Sohail reports receiving funds from TYRX Inc. and Medtronic for prior research unrelated to this study administered according to a sponsored research agreement between Mayo Clinic and study sponsor that prospectively defined the scope of the research effort and corresponding budget; and honoraria/consulting fees from Medtronic Inc.