Author(s): Nate Webb*, Jonathan McCanless, Kelly Richelsoph, Judith Steenbergen, Warren O. Haggard; Memphis, TN
Title: Characterization of Daptomycin Loaded Calcium Sulfate Pellets: Clinical and Laboratory Pellets
Purpose: This study investigated the elution and activity of daptomycin (Cubist Pharmaceuticals; Lexington,MA) released from calcium sulfate, a degradable bone graft substitute.
Methods: Pellet Manufacture:Calcium sulfate (CaSO4) hemihydrate powder was mixed with aqueous potassium sulfate (K2SO4) at 4 wt%.The mixing ratio was with one part solution to four parts CaSO4 (by weight). After mixing the CaSO4 and K2SO4 solution for two minutes, lyophilized daptomycin was added, at one part daptomycin to 20 parts CaSO4. Pellets were cast in a silicone elastomer mold (4.8 mm diameter x 3.3 mm height) and allowed to cure for approximately one hour before demolding. Three groups were allowed to set for 24 hours before testing. One group was mixed, allowed to set for 24 hours, gamma sterilized, then evaluated (These pellets were labeled laboratory pellets). CaSO4 from commercially available OsteosetR (Wright Medical Technology, Arlington, TN) kits were also used for elution testing. CaSO4 was mixed with 4% K2SO4 solution for 1 minute followed by 1 minute of undisturbed curing. Daptomycin was added at a ratio of two vials per kit, follwed by one minute of mixing. The pellets were cast, de-molded at 15 minutes, and testing began at 30 minutes (This method was pursued to simulate clinical usage for the clinical pellets). Elution:Elution characteristics were determined for each group of pellets. Three elution samples per group were tested. Each sample consisted of 8 pellets in 20 ml of phosphate buffered saline (PBS) kept at 37?XC 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 (commercial kit was tested at days 1, 2, 3, 4, 5, 7, 11, 15, 21, and 28). PBS solution was completely refreshed at each time interval, and the pellets were returned to the elution vessels at 37oC. Aliquots were frozen prior to testing. Activity:Two sets of tubes were prepared with 1.8 ml of Mueller Hinton II (MHII) broth, supplemented with CaCl2 at 50 ìg/ml. Aliquots of 200 ìl from each sample of the laboratory pellet were added to the broth. Eight tubes of 20 £gl of a known daptomycin concentration in PBS (0.15, 0.31, 0.62, 1.25, 2, 5, 10, and 20 £gg/ml) and one tube with no daptomycin (20 £gl of PBS) served as controls. Tubes were inoculated with 20 ìl of S. aureus (daptomycin MIC approximately 0.15 ug/ml) or S. epidermidis (daptomycin MIC approximately 0.31 ug/ml), incubated at 37oC for 24 hours, and the absorbance at 530 nm (A530) was recorded. The spectrophotometer was adjusted to zero using a blank consisting of 1.8 ml MHII broth and 200 £gl of PBS.
Results: Daptomycin was found to inhibit the conversion of calcium sulfate hemihydrate into the hardeneddihydrate form in an earlier study. For this reason, an accelerant (potassium sulfate) was required to produce a suitable pellet. Elution behavior was similar to previous elution experiments. The elution profile shows an initial release of a large amount of daptomycin at the beginning of the experiment, with a more constant release of daptomycin at the end of the elution study [Figure 2]. The initial release of daptomycin for the sterilized pellets were slightly less (257 ug/ml) than that of the non-sterilized pellets (avg. 459 ug/ml). After day one, the amount released was similar between sterilized and non-sterilized pellets. The clinical pellets showed less elution for the first day, and slightly more elution after day 10 [Figure 3]. Daptomycin recovery for both the laboratory pellets and the clinical pellets were similar (35% and 30%, respectively). Inhibition of growth of both S. aureus and S. epidermidis with the eluates from the laboratory pellets drawn at all days was found [Figure 1 has S. aureus data]. Some inhibition was observed for all time points measured.
Discussion: The data determined that the eluates remain active after elution from calcium sulfate. Thedata also indicates gamma sterilization of the pellets does not greatly effect the elution or activity of the antibiotic, however, more test groups to verify this would be needed. Low estimated recovery of the daptomycin was observed. Additional investigations in the use of DI water as a substitute for PBS may increase recovered daptomycin. Experiments in this area are ongoing. The clinical pellets manufactured from a clinical kit eluted similarly to the pellets made with the standard laboratory process of 24 hour set. Daptomycin recovery was similar for both types of pellets. This preliminary investigation demonstrates that a CaSO4 pellet loaded with daptomycin can be successfully manufactured in an operating room setting. Higher standard deviation for bacteria growth [Figure 1] can be seen on days 10 and 14, due to a high growth of bacteria (approx 50% of control) in one of the samples for each day. Before the test was started, and unknown contaminant was observed on the 10 day aliquot. This may be the cause of the observed differences for those days. Calcium sulfate pellets have previously been used to deliver antibiotic agents. This in vitro study presents preliminary data showing that daptomycin can be delivered using a modified calcium sulfate pellet. The eluates from this modified pellet remain active after delivery. This preliminary study has demonstrated the potential for local delivery of daptomycin in a degradable bone graft substitute for musculoskeletal applications.