Bisphosphonates (BPs) are commonly used drugs for managing bone loss or bone resorption in skeletal diseases, such as post-menopausal osteoporosis, Paget’s disease, and bone cancer. In 2014, the FDA approved clodronate disodium (CLO) and tiludronate disodium for treating navicular syndrome in horses over 4 years old. However, concerns have arisen regarding the extra-label use of CLO, particularly in juvenile individuals subjected to exercise, such as racehorses, where BPs may impact bone metabolism by affecting bone modeling/remodeling. The overall objective of this dissertation was to determine the effects of CLO in equine joint tissues in vitro and CLO effects in vivo in juvenile, exercising sheep as a model for juvenile horses. A prior publication found that CLO reaches the synovial fluid when administered intramuscularly. Therefore, an initial in vitro study exposed equine cartilage explants, chondrocytes, and synoviocytes to recombinant equine interleukin-1β (reqIL-1β) to determine the effects of CLO in joint tissues. The results confirmed that reqIL-1β increased the release of inflammatory markers from joint tissues (e.g., GAG, IL-6, PGE2, NO, P < 0.05), yet CLO did not reduce the inflammatory effects of reqIL-1β. Hence, this in vitro study established that CLO was neither cytotoxic nor cytoprotective to joint tissues. Sheep have served as models for assessing the impact of BPs in human medicine and have also been used as a suitable model for studying exercising horses. Before using sheep as a model for intramuscular CLO administration, we determined the pharmacokinetics (PK) and plasma protein binding (PPB) of CLO in sheep and compared them to horses. Sheep PK parameters were similar to those previously published for horses, particularly Cmax and AUCall. Unbound fractions of CLO differed by less than 1.4-fold between sheep and horses. Having established a dose that resulted in PK values similar to those in horses, we subjected the sheep to a novel exercise protocol, using a high-speed exerciser. The sheep adapted quickly to the exercise protocol and tolerated it well with no evidence of significant lameness. Finally, the juvenile sheep were divided into four groups: CLO on day 0, CLO on day 84, CLO on days 0 and 84, and a control group (saline). Physical examinations and lameness evaluations were measured every 14 days and blood was collected every 28 days for the measurement of serum bone biomarkers. Bone was harvested from the tuber coxae halfway through the study and again at euthanasia. In addition, samples of lumbar vertebrae and fused metacarpal III+IV were obtained at euthanasia for bone microstructure analysis and biomechanical testing. No measurable effects of CLO on the sheep skeletons were detected. Serum bone formation markers, bone-specific alkaline phosphatase (BALP) and procollagen type I amino-terminal propeptide, increased over time. Serum bone resorption marker, carboxy-telopeptide of type I collagen cross-links (CTX-I), decreased at several time points consistent with the exercise stimulus. Male sheep had decreases in compressional stress and increases in modulus of elasticity of the fourth lumbar spine in comparison to female sheep, likely due the low levels of sex hormones in the castrated males. The relatively low dose of CLO used in large animals compared to humans may explain the lack of skeletal effects. Previous BPs studies on horses have reported improvements in lameness without evidence of reduced bone resorption, suggesting that analgesic effects may occur without significant changes in bone microstructure or serum markers. Future research should focus on the potential analgesic effects of CLO at low doses which could provide palliative care without significant effects on bone metabolism.
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