PSV-4 Clodronate re-release in response to controlled exercise and bone stressors in juvenile horses

Abstract Concerns over the extra-label use of bisphosphonates in skeletally immature horses has extended to include its likely re-release from bone into circulation in response to stressors. Therefore, the objective of this study was to be first to quantify the re-release of clodronate in response to controlled stressors. Yearling Quarter horses (n = 32) were stratified by age, body weight (BW), sex, and initial bone optical density into four treatment groups for a 168-d trial. Treatments consisted of control (CON; n = 8), single-dose (1X; d 84; n = 8), 2-dose (2X; d 0 and 84; n = 8), and four-dose groups (4X; d 0, 42, 84, and 126; n = 8). All horses received iso-volumetric intramuscular injections of either 1.8 mg/kg BW clodronate disodium (OSPHOSÒ) or saline (placebo) on d 0, 42, 84, and 126. Horses were housed in individual stalls (3.6 m × 7.2 m). Diets were formulated to meet nutrient requirements including concentrate offered every 12-h and ad libitum coastal bermudagrass (Cynodon dactylon) hay and water. Horses were exercised 5 d/wk with a progressive workload, and maximum exercise intensity was reached on d 120, verified via heart rate and blood lactate. On d 120, blood samples were collected prior to the start of the exercise bout (pre), and 0-, 0.5-, 1-, 3-, 12-, and 24-h post exercise. Both tuber coxae of each horse were biopsied on d 168 with blood samples collected prior to (pre), and 0, 12, 24, 48, 72, 168, and 336 h post biopsy. Clodronate was quantified in plasma from exercise and biopsy samples using liquid chromatography tandem mass spectrometry. Exercise and biopsy serum were analyzed for cortisol, a marker of stress, while biopsy serum was analyzed for substance P, a pain marker. Data were analyzed using SAS PROC MIXED. Exercise did not result in clodronate re-release as there was no treatment x time interaction or time effect (P ≥ 0.44), but clodronate was dose-dependently greater in treated groups throughout the exercise collection period (P < 0.01). A treatment x time interaction (P ≤ 0.01) was observed for clodronate concentrations surrounding bone biopsy in which concentrations decreased at 168 h post biopsy before increasing at 336 h in 4X horses. Substance P increased over time (P < 0.01) following biopsy, regardless of treatment. Despite decreasing over time (P ≤ 0.01), there were similarly no treatment differences (P ≥ 0.46) nor treatment ´ time interactions (P ≥ 0.78) for cortisol following either exercise or bone insult. In conclusion, submaximal exercise did not result in re-release of clodronate; however, direct bone insult disrupted clodronate concentrations in horses receiving 4 administrations of bisphosphonate despite no treatment impact on stress or pain indicators.