Novel insights into actions of bisphosphonates on bone: Differences in interactions with hydroxyapatite
Introduction
Bisphosphonates are now the most widely used drugs for treating diseases associated with increased bone resorption, such as osteoporosis. Following the demonstration that inorganic pyrophosphate was a naturally occurring modulator of bone metabolism, the bisphosphonates, which are metabolically stable analogs of pyrophosphate, were studied as inhibitors of calcification and resorption for the treatment of bone disorders [1], [2], [3]. The earliest studies showed that bisphosphonates could bind to hydroxyapatite (HAP) crystals, and inhibit crystal growth and dissolution similar to pyrophosphate [4], [5]. The ability of bisphosphonates to inhibit bone resorption was initially ascribed to these direct inhibitory effects on mineral dissolution. However, with the development and study of increasingly potent bisphosphonates, it became clear that these physicochemical effects on bone mineral did not completely explain the inhibition of bone resorption, and cellular effects were likely to be involved [6], [7], [8]. It is now known that bisphosphonates also act directly on osteoclasts and interfere with specific intracellular biochemical processes such as isoprenoid biosynthesis and subsequent protein prenylation to inhibit cell activity [9], [10].
The high binding affinities of bisphosphonates for bone may affect many important biological properties of these compounds including uptake and retention on the skeleton, diffusion of the drug within bone, release of adsorbed drug from bone, potential recycling of the desorbed drug back onto bone surfaces, and effects on mineral dynamics and cellular function within bone. The strong binding of bisphosphonates to bone mineral together with the ability to be linked to a gamma-emitting technetium isotope [11] was the basis for the use of bisphosphonates as bone-scanning agents to detect metastases and other bone lesions. Many studies have shown that bisphosphonates have prolonged or persistent effects, both in experimental animals and in humans, which is probably related to the rate and extent of chemical desorption and cell-mediated release from bone [12], [13], [14], [15], [16], [17], [18].
Chemically, the bisphosphonates of medical interest are all characterized by 2 phosphonate groups sharing a common carbon atom (P–C–P). This P–C–P backbone is responsible for the strong affinity for bone mineral and thus facilitates the potent inhibitory effects on bone metabolism in vitro and in vivo. Both phosphonate groups are required, as modifications to one or both reduce affinity for bone mineral. It has been well established that R1 substituents (Table 1) with additional capability to co-ordinate to calcium, such as a hydroxyl (OH) or amino (NH2), can display enhanced chemisorption to mineral, presumably via tridentate binding to calcium [4].
Varying the R2 substituents can result in differences in antiresorptive potency of several orders of magnitude. Many bisphosphonates with an R2 side chain containing a basic primary nitrogen atom in an alkyl chain (e.g., pamidronate and alendronate) are more potent antiresorptive agents than either etidronate or clodronate, whereas compounds with more highly substituted nitrogen moieties in R2 (e.g., ibandronate) can display further increases in antiresorptive potency. The most potent antiresorptive bisphosphonates include those containing a nitrogen atom within a heterocyclic ring (e.g., risedronate and zoledronate). These enhancements in antiresorptive potency resulting from differences in the R2 groups appear to be linked to the biochemical activities of these drugs, particularly inhibition of the farnesyl diphosphate synthase enzyme within the mevalonic acid pathway in osteoclasts [19], [20], [21].
Although earlier studies showed differences in mineral binding among bisphosphonates such as etidronate and clodronate in which R1 was varied, the effects on mineral binding of varying R2 has not been systematically studied with many nitrogen-containing bisphosphonates. In a previous study, differences were found in the HAP adsorption isotherms at high equilibrium concentrations, even among bisphosphonates with the same R1 (OH) [20], [22]. The method used, however, did not allow for calculation of binding affinities.
In this present study, using an HAP crystal growth method, we measured the kinetic binding affinities of 6 bisphosphonates in current clinical use at concentrations more similar to in vivo conditions. We have also studied the effects of these bisphosphonates on other HAP surface properties that are likely to influence the mineral binding of bisphosphonates in vivo, including zeta potential and interfacial tension.
Section snippets
Materials
Six bisphosphonates (clodronate, etidronate, risedronate, ibandronate, alendronate, and zoledronate) were used in this study; their chemical names and structures are listed in Table 1. Clodronate has an R1 substituent of –Cl and the other bisphosphonates tested have –OH as the R1 substituent. All 6 compounds were synthesized and characterized in the Procter & Gamble Pharmaceuticals laboratories utilizing previously published methods [19]. HAP, prepared as described previously, was characterized
HAP growth
All 6 bisphosphonates studied produced concentration-dependent inhibition of HAP crystal growth (Table 2, Fig. 1, [2], Fig. 2). The rank order of inhibitory potency at these concentrations was zoledronate (most potent) ∼ alendronate > ibandronate ∼ risedronate > etidronate > clodronate (least potent). Fig. 1 shows representative plots of titrant volume versus HAP growth over time for risedronate and alendronate. The initial non-linear portion of the titrant curves represents expected titrant
Discussion
The results of this study show that even bisphosphonates that share a common P–C–P structure, with OH at R1, can have significantly different kinetic binding affinities for HAP with a rank order of highest to lowest for the bisphosphonates studied of zoledronate > alendronate > ibandronate = risedronate > etidronate. These differences must be attributed to differences in the R2 side chain. This study also showed that the nature of the R2 side chain influences other surface properties, including
Acknowledgments
We are grateful to Elaine B. Taylor for her assistance in the preparation and editing of the manuscript. We thank the National Institutes of Health (NIDCR) for a grant (DE03223) in partial support of this work.
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