An in vitro Assay to Measure Targeted Drug Delivery to Bone Mineral

Abstract

Targeted delivery of drugs to their site of action is a promising strategy to decrease adverse effects and enhance efficacy, but successful applications of this strategy have been scarce. Human bone is a tissue with unique properties due to its high hydroxyapatite mineral content. However, with the exception of bisphosphonates, bone mineral has not been targeted in a successful clinical application of drugs that act on bone, such as anti-resorptive or bone anabolic agents. Herein we present an NMR-based in vitro assay to measure binding affinities of small molecules to hydroxyapatite (HAP) or bone powder. Binding was shown to be specific and competitive, and the assay can be carried out in a direct binding format or in competition mode. A selection of clinically relevant bisphosphonates was ranked by their binding affinity for HAP. The binding affinity decreases in the order: pamidronate > alendronate > zoledronate > risedronate > ibandronate. The differences in binding affinities span a factor of 2.1 between pamidronate and ibandronate, consistent with previous studies. The rank order is very similar with bone powder, although the binding capacity of bone powder is smaller and binding kinetics are slower. A zoledronate derivative that lacks the central hydroxy group binds to HAP with 2.3-fold weaker affinity than zoledronate itself. Any small molecule can be analyzed for its binding to HAP or bone powder, and the binding of common bone-staining agents such as alizarin and its derivatives was confirmed in the new assay. This assay supports a strategy for targeted delivery of drugs to bone by attaching a bone-affinity tag to the active drug substance.

Structural features underlying raloxifene’s biophysical interaction with bone matrix

“…The interaction of compounds with the bone surface has historically been predominantly electrostatic, as in the case of tetracyclines, calcein, and bisphosphonates, and has involved the mineral phase rather than the organic phase. Bisphosphonates, in particular, have been shown to bind avidly to HAP using a NMR assay similar to the one used in this paper [20]. However, our data suggest that the hydrophobic interactions are potentially another important way to induce greater molecule-surface affinity and that they occur through the organic phase (especially collagen) rather than the hydroxyapatite.…”

“…A modified version of a protocol previously described [20] was used. In this version, a quantitative measurement is obtained, by calculating the integral of the NMR signal, which is directly proportional to the concentration of the compound in solution.…”

Structural Requirements for Bisphosphonate Binding on Hydroxyapatite: NMR Study of Bisphosphonate Partial Esters

“…Four clinically used BPs etidronate (1), medronate (8), clodronate (12), and alendronate (15) and their phosphoesters ( Figure 1) were systematically studied to evaluate their bone binding properties. Additionally, we used diacetylated alendronate (18) to compare these changes with another type of potential BP prodrug.…”

Carbamoylphosphonates inhibit autotaxin and metastasis formationin vivo

“…In this work we examined the extent of CPO n ¼ 6, 7 absorption using a recently published method for studying bisphosphonate absorption to hydroxyapatite (HAP) as a model for bone 22 , and compared it with CPO n ¼ 5 which was reexamined. As shown in Table 3, the absorption of CPOs to HAP increases as the lengths of the aliphatic chains increase.…”