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Author(s): Samuel B. Adams, Jr., MD*, Mohammed F. Shamji, MD, Dana L. Nettles, MS, and Lori, A. Setton, PhD; Duke University Medical Center, Durham, NC

Title: Sustained Release of Vancomycin and Cefazolin from Injectable Elastin-Like Polypeptide Depots

Purpose: Locally administered antibiotic depots have been used for prophylaxis and treatment of musculoskeletal infections. The purpose of this study was to evaluate sustained release of vancomycin and cefazolin from an injectable, polypeptide drug depot.

Methods: Elastin-like polypeptides (ELPs) are non-immunogenic, biodegradable proteins with an amino acid sequence mimicking that of human elastin. While soluble in PBS at room temperature, ELPs undergo a hydrophobic collapse at body temperature that promotes consolidation and entrapment of other molecules (e.g., drugs). The kinetics of antibiotic drug release, as well as bioactivity of released drug, were evaluated following release from an ELP drug depot. Constructs of cross-linked ELPs (400Ql volume, 60-80 mg, n=70) were loaded with 0, 5, 7.5, 10, or 20 mg of either vancomycin or cefazolin. The constructs were incubated in PBS at 37?XC to promote the hydrophobic phase transition and the supernatant was exchanged every 24 hours out to day 14, and intermittent periods to maintain a dilute supernatant out to day 28 for vancomycin and day 10 for cefazolin. Antibiotic concentration in all supernatants, and in the final construct, was measured via UV-VIS spectrophotometry. Bioactivity of the eluted antibiotics from selected samples was performed via the Bauer-Kirby disk diffusion method using Bacillus subtilis (ATCC 6633). Statistical analyses of these data were performed using two way ANOVA to test for an effect of drug:ELP molar loading ratio and time on antibiotic concentration in the supernatant. The dynamic viscosity of the cross-linked ELP was also determined to evaluate the suitability of the formulation for injection.

Results: Vancomycin was slowly released from the ELP drug depots, with no sample reaching over 90% of drug release at 28 days (Figure 1). Vancomycin concentration in the collected supernatant was above the reported MIC (1.5-3Qg/ml) of vancomycin against S. aureus on all days. Vancomycin fractional release was shown to decrease with decreasing amount of initial loaded vancomycin (p<0.01), increasing ELP concentration (p<0.01), and decreasing ratio of cross-linker to ELP (p<0.01). The majority of cefazolin was released by 3 days (Figure 2), with evidence of lower fractional release with increased ELP concentration (p<0.01) and an increased cross-linker:ELP ratio (p<0.01). Although the cefazolin samples demonstrated a faster release rate, no sample was found to have a concentration below the reported MIC (0.25-1Qg/ml) for cefazolin against S. aureus. All vancomycin samples tested, and all cefazolin samples from days 1-3 and the amount remaining in the samples at day 10 demonstrated bioactivity against B. subtilis (bioactivity of the released cefazolin from days 4-10 was not detectable due to the sensitivity of the bioassay). The dynamic viscosity of the ELP at a rate of 10-1 seconds was 0.37 Pa-s, similar to other injectable solutions. Discussion; These data show the potential of ELP to serve as injectable antibiotic drug delivery depots that may provide sustained release of antibiotics effective for the treatment or prophylaxis of musculoskeletal infections. Prior work has demonstrated that in situ forming ELP drug depots are associated with a 25-fold increase in the half-life of small molecular weight protein (~ 50 kDa) clearance from the joint space, as compared to similar sized proteins that remain soluble and do not form depots. Both vancomycin and cefazolin are considerably smaller molecules (~ 1 kDa) and thus associated with additional challenges of more rapid clearance and more rapid diffusion from drug depots. In this study, vancomycin loaded ELP constructs demonstrated sustained release and biological activity for at least 28 days. Cefazolin loaded ELP constructs demonstrated sustained release for at least 10 days, and biological activity for the released drug over the first 3 days. These results suggest that the ELP may serve as an effective carrier for the entrapment of smaller molecules, while providing for the advantages of injectable delivery, retention of drug activity, and both non-immunogenicity and biodegradability of the drug carrier.

Musculoskeletal Infecton Society
Musculoskeletal Infecton Society