Osteomyelitis Treatment with Nanometer-sized Hydroxyapatite Particles as a Delivery Vehicle for a Ciprofloxacin- Bisphosphonate Conjugate; New Fluoroquinolone-bisphosphonate Derivatives Show Similar Binding Affinity to Hydroxyapatite and Improved Antibacterial Activity against Drug-resistant Pathogens

Orthopaedic injuries comprise a majority of combat injuries seen in recent US military conflicts. The development and increased use of modern body armor, which has been correlated with decreased chest and upper abdominal injuries (Mabry et al., 2000), will likely continue to result in an increased relative rate of extremity injuries. These injuries commonly present with significant open bone fractures of the limbs and substantial contamination with bacteria from high energy trauma. Once bone infection is established, one fourth of open fractures will progress to osteomyelitis (OM) (Lew and Waldvogel, 1997; Wolski, 2004; Buxton et al., 2004). We developed a novel local drug-delivery system consisting of ciprofloxacin-bisphosphonate conjugate (E41) bound to calcium phosphate (CP) bone particles. In situ, the conjugate dissociates from CP and maintains its antibacterial activity. This topical granulated antibiotic delivery system reduced infection rates in our in vivo OM model. Current studies contrast two CP homeostatic bone-substitute particles, nanometer-sized hydroxyapatite NanOssTM (Nan), and μ-sized tricalcium phosphate SkeliteTM (TCP) in vitro for binding to E41, and in vivo for E41-CP blockade of rat OM. E41’s binding to both CPs were saturable, and maximal binding was similar. But affinity constants were different (p≤ 0.01), showing a higher E41 affinity for Nan. The amount of E41 able to saturate 50% of Nan was higher than TCP (ca. 30-fold). E41-CP concentrations were contrasted in our post-traumatic S. aureus acute rat OM model, with enumeration of S. aureus in infected tibiae (tibial load). Using the previously determined 95%-Infective inoculum, all untreated controls became infected (examined at 24 h); and both E41-CPs lowered tibial loads at 50% saturation: E41-Nan loads were lower than E41-TCP (p≤ 0.01). Sixteen of 19 E41-Nan tibiae were sterile, vs. 5 of 18 for E41-TCP (p< 0.01). Weak acids, e.g., lactic acid released c.a. thirty-fold more E41 from Nan (8.96 μg CB) than TCP (0.27 μg CB, p ≤ 0.05). These results suggest E41 may be released from Nan in situ due to the low pH of wounds. We conclude that nanometer sized CP particles are comparable to, and perhaps superior than, μ-sized particles, as a local drug-delivery vehicle for bisphosphonate-containing antibiotics. With the concept of treating open fracture wounds with targeted topical drug delivery consisting of an antibiotic-bisphosphonate bound to CP established, our attention has focused on the effectiveness of other fluoroquinolone-bisphosphonate combinations. Gatifloxacin-bisphosphonates (E43) and Moxifloxacinhydroxybisphosphonate (E46) were contrasted to E41 for activity against methicillin-susceptible S.aureus, ciprofloxacin-resistant MRSA, ciprofloxacin-susceptible MRSA, Acinetobacter baumannii and other pathogens. E43 showed greater antimicrobial activity against several Gram-positive and Gram-negative pathogens, e.g., a 16-fold difference with MRSA. In in vitro binding studies binding was similar, and saturable. Maximal binding was: E41, 89.7 % ± 4.74, E43, 76.8 % ± 2.35 and E46, 95.9 % ± 0.13. Kd values were: E41, 1.35 x 10 8 mM (Nan), E43, 1.83 x 10 mM, and E46, 1.72 x 10 mM. Versus E41 and E46, E43 has improved activity against bone-pathogenic S. aureus, and other bacteria, and shows a similar binding affinity to Nan. These investigations report an animal model mimicking severe trauma, for the prevention and treatment of OM, and the creation of antibioticbisphosphonate conjugates, now bound to nanometer size bone particles for the treatment of OM. These dry particles are ideal for topical treatment of open battlefield wounds, providing targeted drug delivery, and the prevention of disease from contaminated wounds. In addition, new flouroquinolone-bisphosphonates provise improved antibacterial activity against drug-resistant bacteria.