Authors: N Webb*, K. Richelsoph, J. Steenbergen, W. Haggard
Title: HPLC Determination of Elution Behavior of Daptomycin Loaded Calcium Sulfate Pellets
Addresses: University of Memphis Biomedical Engineering Department, 330 Engineering Technology Bldg., Memphis, TN 38152
Purpose: This study investigated how to best incorporate daptomycin (Cubist Pharmaceuticals; Lexington, MA) within calcium sulfate for optimum local release of this antibiotic from a degradable bone graft substitute.
Methods: Calcium sulfate hemihydrate powder was mixed with a potassium sulfate solution (4% K2SO4 by weight in DI water), with one part solution to four parts powder (by weight). After mixing the powder and potassium sulfate solution for two minutes, daptomycin was added, at one part daptomycin to 20 parts powder. The pellets were cast in a silicone elastomer mold (4.8mm x 3.3mm) and allowed to cure for 24 hours. Average pellet weight after 24 hours was approximately 78 mg. Elution characteristics were determined for each group of pellets. Three elution samples consisting of 8 pellets each were tested. Each sample consisted of 8 pellets in 20 ml of phosphate buffered saline (PBS), kept at 37°C for the duration of the test. Aliquots of the eluent (1 ml) were collected for each sample on days 1, 2, 5, 7, 10, 14, 21, and 28. The pellets were dried at 40oC for one hour and their weights were recorded. The PBS was replaced at each time interval, and the pellets were returned to the elution vessels at 37oC. Aliquots were frozen at - 4°C until the elution was completed. All aliquots were tested using HPLC (Varian ProStar, C8 column, 0.45% NH4H2PO4 mobile phase) for daptomycin concentration.
Results: Daptomycin inhibited the conversion of calcium sulfate hemihydrate into the hardened dihydrate form. For this reason, an accelerant of potassium sulfate was required to produce a suitable pellet. The addition of daptomycin appears to cause initial average pellet weight to decrease by about 20mg, compared to pellets loaded with tobramycin. A slight weight gain in the daptomycin pellets was experienced during the first 24 hours, which was followed by a steady weight loss during the remainder of the test. The concentration of the daptomycin eluate at 24 hours [Fig 1] was 10 times higher than that of an earlier study with vancomycin [ORS Trans., 2004, p.1063]. Concentration data were normalized for pellet weight and represented by Fig 1.
Discussion: Calcium sulfate pellets have been used to deliver antibiotic agents [ORS Trans., 2004, p.1063]. Previously, when producing antibiotic loaded pellets, saline, acting as an accelerant, was mixed with calcium sulfate hemihydrate powder. When incorporating daptomycin into calcium sulfate, however, a saline solution does not adequately accelerate the reaction and an alternative accelerant, potassium sulfate, was utilized.This study presents preliminary data showing that daptomycin can be delivered using modified calcium sulfate pellets. Incorporation of a potassium sulfate solution is required to convert the hemihydrate structure to a dihydrate structure. Initial studies were conducted using 4% potassium sulfate, leading to pellets that exhibited an elution profile as presented in Figure 1. Future studies will investigate altering the percentage of potassium sulfate to optimize set times and elution profiles. This preliminary study has demonstrated the potential for local delivery of daptomycin in a degradable bone graft substitute for musculoskeletal applications.