Assessment of a bone biopsy technique for tiludronic acid dosage in horses: a preliminary study

Introduction Tiludronic acid as a bisphosphonate has a strong affinity for bone, making difficult the assessment of its PK profile in this deep compartment on living animals. The invasive nature of sample collection remains a limiting factor. This study was carried out in order to assess a bone biopsy technique allowing the repetition of sampling over a long period of time to dose tiludronic acid in equine bone.
Material and Methods Six healthy 4- to 8-year-old Standardbred geldings were treated with tiludronic acid 1 mg/kg in a saline infusion over 30 minutes. The horses were subjected to euthanasia on days 1, 43, 57, 92, 182 and 222 post-treatment, respectively. Bone samples (test samples and larger reference samples) were taken at 4 sites per side and per horse: the lateral aspect of the metacarpal bone III (MCIII) of the forelimb, the 13th rib, the tuber coxae and the cuboïd bone. Test samples were taken with a 5-mm diameter dental drill (Implanteo™, Anthogyr), while larger reference samples were taken around the drill sample sites with an osteotome. All samples were taken immediately after euthanasia. Tiludronic acid concentrations were measured by HPLC with UV detection.
Results The tuber coxae was the easiest site to sample. The sample site of the MCIII was easily accessible but due to the extreme hardness of the bone, the drill sampling was technically difficult to perform. Drill samples obtained from the 13th rib were very small, and the access more limited. Finally, the access to the cuboïd bone required considerable dissection, not performable in vivo. Extraction and dosage of tiludronic acid from the MCIII was difficult for technical reasons in most cases, in drill samples as well as in reference samples, and most (96%) of the values obtained were considered unreliable. This was also true for some samples from the 13th rib and from the cuboïd bone, to a lesser extent (42% of unreliable values for both sites). Moreover, for these two sample sites, less technical problems were encountered for the extraction and dosage of tiludronic acid in drill samples than in reference samples. No extraction or dosage problem was encountered with the tuber coxae samples. The ratio of tiludronic acid concentrations in drill versus reference samples ranged from 73% to 185% (mean: 124%), 65% to 208% (mean: 118%), and 26% to 110% (mean: 62%) respectively in the tuber coxae, 13th rib and cuboïd bone. In all but one horse, the highest concentrations in tiludronic acid were found in the tuber coxae, while the lowest values tended to be in the cuboïd bone at any time post-treatment. Tiludronic acid was still found in all bone samples 7 months after treatment.
Discussion As previously reported with other tools, the drill tested in this study should permit to conveniently perform bone biopsies in the equine tuber coxae. This biopsy site would be the most appropriate for repeated sampling to dose tiludronic acid over time. This would help to design appropriate PK/PD studies with tiludronic acid in horses. This study also further confirms the long persistence of tiludronic acid in equine bone.