Bisphosphonates: Mode of Action and Pharmacology

SUPPLEMENT ARTICLE Bisphosphonates: Mode of Action and Pharmacology R. Graham G. Russell, MD, PhD Botnar Research Centre and Oxford University Institute of Musculoskeletal Sciences, Oxford, United Kingdom The author has indicated he has no financial relationships relevant to this article to disclose. ABSTRACT The profound effects of the bisphosphonates on calcium metabolism were discovered over 30 years ago, and they are now well established as the major drugs used for the treatment of bone diseases associated with excessive resorption. Their principal uses are for Paget disease of bone, myeloma, bone metastases, and osteoporosis in adults, but there has been increasing and successful application in pediatric bone diseases, notably osteogenesis imperfecta. Bisphosphonates are structural analogues of inorganic pyrophosphate but are resistant to enzymatic and chemical breakdown. Bisphosphonates inhibit bone resorption by selective adsorption to mineral surfaces and subsequent internalization by bone-resorbing osteoclasts where they interfere with various biochemical processes. The simpler, non nitrogen-containing bisphosphonates (eg, clodronate and etidronate) can be metabolically incorporated into nonhydrolysable analogues of adenosine triphosphate (ATP) that may inhibit ATP-dependent intracellular enzymes. In contrast, the more potent, nitrogen-containing bisphosphonates (eg, pamidronate, alendronate, risedronate, ibandronate, and zoledronate) inhibit a key enzyme, farnesyl pyrophosphate synthase, in the mevalonate pathway, thereby preventing the biosynthesis of isoprenoid compounds that are essential for the posttranslational modification of small guanosine triphosphate (GTP)-binding proteins (which are also GTPases) such as Rab, Rho, and Rac. The inhibition of protein prenylation and the disruption of the function of these key regulatory proteins explains the loss of osteoclast activity. The recently elucidated crystal structure of farnesyl diphosphate reveals how bisphosphonates bind to and inhibit at the active site via their critical nitrogen atoms. Although bisphosphonates are now established as an important class of drugs for the treatment of many bone diseases, there is new knowledge about how they work and the subtle but potentially important differences that exist between individual bisphosphonates. Understanding these may help to explain differences in potency, onset and duration of action, and clinical effectiveness. www.pediatrics.org/cgi/doi/10.1542/ peds.2006-2023h doi:10.1542/peds.2006-2023h Key Words bone resorption, osteoclasts, bisphosphonates, osteoporosis Abbreviations PPi inorganic pyrophosphate ATP adenosine triphosphate FPP farnesyl diphosphate GGPP geranylgeranyl diphosphate FPPS farnesyl pyrophosphate synthase Accepted for publication Oct 5, 2006 Address correspondence to R. Graham G. Russell, MD, PhD, Botnar Research Centre, Nuffield Department of Orthopaedic Surgery, University of Oxford, Headington, Oxford OX3 7LD, United Kingdom. E-mail: graham.russell@ ndos.ox.ac.uk PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275); published in the public domain by the American Academy of Pediatrics S150 RUSSELL

THE DISCOVERY AND development of the bisphosphonates as a major class of drugs for the treatment of bone diseases was recently reviewed 1 and represents a fascinating story that has its origins in studies of biological calcification processes. There are many books and review articles available that describe the chemistry, pharmacology, and clinical applications of bisphosphonates. 2 5 It had been known since the 1930s that trace amounts of polyphosphates were capable of acting as water softeners by inhibiting the crystallization of calcium salts, such as calcium carbonate, 6 and in the 1960s Fleisch et al 7,8 showed that inorganic pyrophosphate, a naturally occurring polyphosphate and a known byproduct of many biosynthetic reactions in the body, was present in serum and urine and could prevent calcification by binding to newly forming crystals of hydroxyapatite. It was proposed that inorganic pyrophosphate (PPi) might be the body s own natural water softener that normally prevents calcification of soft tissues and regulates bone mineralization. It subsequently became clear that calcification disorders might be linked to disturbances in PPi metabolism. The first example was an inherited disorder, hypophosphatasia, in which lack of alkaline phosphatase is associated with mineralization defects of the skeleton and elevated PPi levels, 9 indicating that alkaline phosphatase is probably the key extracellular enzyme responsible for hydrolyzing pyrophosphate. Attempts to exploit these concepts by using pyrophosphate and polyphosphates to inhibit ectopic calcification in blood vessels, skin, and kidneys in laboratory animals were successful only when the compounds were injected. 10 Orally administered pyrophosphate and polyphosphates were inactive because of their hydrolysis in the gastrointestinal tract. During the search for more stable analogues of pyrophosphate that might also have the antimineralization properties of pyrophosphate but would be resistant to hydrolysis, several different chemical classes were studied. The bisphosphonates (at that time called diphosphonates), characterized by P-C-P motifs, were among these classes. 11 13 Like pyrophosphate, bisphosphonates had high affinity for bone mineral 14 and were found to prevent calcification both in vitro and in vivo but, unlike pyrophosphate, were also able to prevent experimentally induced pathologic calcification when given orally to rats in vivo. 15 This property of being active by mouth was key to their future use in humans. In these early studies bisphosphonates were shown not only to prevent the experimentally induced calcification of many soft tissues, including skin, kidneys, and blood vessels in vivo but, with some of the compounds (eg, etidronate), to also inhibit mineralization of ectopic bone as well of normal calcified tissues such as bone and cartilage. 16 Bisphosphonates seem to prevent calcification by physicochemical mechanisms that produce direct impairment of the calcification process by acting as crystal poisons after adsorption to mineral surfaces rather than by effects on the deposition of matrix. Perhaps the most important step toward the future use of bisphosphonates occurred when we found that bisphosphonates, as we had already shown for PPi, 17 also had the novel property of being able to inhibit the dissolution of hydroxyapatite crystals. 18 This finding led to studies to determine if they might also inhibit bone resorption, which they did in many different experimental models. 19,20 In growing intact rats, the bisphosphonates block the removal of both bone and cartilage, thus retarding the modeling of the metaphysis, which becomes club-shaped and radiologically denser than normal. This effect is the basis of the Schenk model 21 and is a phenomenon of interest in pediatrics because it is also observed in children who are treated with high doses of bisphosphonates. The bisphosphonates are also effective in preventing bone destruction in a number of animal models of human disease, such as immobilization osteoporosis, 22 and the prevention of bone loss associated with ovariectomy. If not given in excess, bisphosphonates do not impair bone growth and can maintain or improve the biomechanical properties of bone in both normal animals and experimental models of osteoporosis. 23 In general, there is a good correlation between potency and structure-activity relationships in vitro and in vivo. 24 In the presence of bisphosphonates, isolated osteoclasts form fewer and smaller erosion cavities on various mineralized matrices in vitro. 25 PHARMACOLOGY AND CELLULAR ACTIONS Etidronate was the first bisphosphonate to be used in humans for fibrodysplasia ossificans progressiva and Paget disease. 26 Once the potential clinical value of bisphosphonates had been appreciated, research efforts were devoted to the development of compounds with a more powerful antiresorptive activity but without a corresponding ability to inhibit mineralization. With compounds such as etidronate there was only a 10- to 100- fold difference between doses that inhibit mineralization compared with doses that reduce bone resorption. Enhancing this window was readily achieved, and many hundreds of bisphosphonates have been synthesized; more than a dozen have been used in humans. With the development of bisphosphonates that were more potent inhibitors of bone resorption, these dose differences widened to several orders of magnitude, which meant that inhibition of skeletal mineralization observed with etidronate ceased to be a major clinical concern. The gradation of potency evaluated in the animal models corresponded quite well with that found in humans, although the differences in potency are much smaller in humans. Bisphosphonates accumulate in bone, so it is impor- PEDIATRICS Volume 119, Supplement 2, March 2007 S151

tant to know what happens during long-term administration. From a clinical point of view, it is reassuring that the inhibition of bone resorption reaches a new steadystate level rather than becoming progressively lower, even when the compounds are given continuously. 27 The level of suppression depends on the administered dose and has also been observed in humans. 28 There seems to be no progression of the antiresorptive effect with time, which suggests that the bisphosphonate buried in the bone is inactive for at least as long as it remains buried there. This also means that within the therapeutic-dosage range, there is little risk of a continuous and progressive decrease in bone turnover in the long run that might lead to an increase in bone fragility. An additional important pharmacologic property of bisphosphonates is that the total dose administered is a major determinant of their effects. This has been well studied for ibandronate 29 and zoledronate. 30 In both cases the same inhibition of bone resorption has been documented regardless of whether the bisphosphonate was given in small frequent (eg, daily) doses compared with larger doses given less frequently. This was the basis for the development of intermittent-dosing regimens in humans. The pronounced selectivity of bisphosphonates for bone rather than other tissues is the basis for their value in clinical practice. Their preferential uptake by and adsorption to mineral surfaces in bone bring them into close contact with osteoclasts. During bone resorption, bisphosphonates are probably internalized by endocytosis along with other products of resorption. Many studies have shown that bisphosphonates can affect osteoclastmediated bone resorption in a variety of ways, including effects on osteoclast recruitment, differentiation, and resorptive activity, and may induce apoptosis. Because mature, multinucleated osteoclasts are formed by the fusion of mononuclear precursors of hematopoietic origin, bisphosphonates could also inhibit bone resorption by preventing osteoclast formation, in addition to affecting mature osteoclasts. In vitro, bisphosphonates can inhibit dose-dependently the formation of osteoclast-like cells in long-term cultures of human bone marrow. 31 In organ culture, also, some bisphosphonates can inhibit the generation of mature osteoclasts, possibly by preventing the fusion of osteoclast precursors. 32,33 It is likely that bisphosphonates are selectively internalized by osteoclasts rather than other cell types because of their accumulation in bone and the endocytic activity of osteoclasts. During the process of bone resorption, the subcellular space beneath the osteoclast is acidified by the action of vacuolar-type proton pumps in the ruffled border of the osteoclast membrane. 34 The acidic ph of this microenvironment causes dissolution of the hydroxyapatite bone mineral, whereas the breakdown of the extracellular bone matrix is brought about by the action of proteolytic enzymes, including cathepsin K. Because bisphosphonates adsorb to bone mineral, especially at sites of bone resorption where the mineral is most exposed, 35,36 osteoclasts are the cell type in bone most likely to be exposed to the highest concentrations of free, non mineral-bound bisphosphonate as a result of the release of the bisphosphonate from bone mineral in the low-ph environment beneath osteoclasts. It has been estimated that pharmacologic doses of alendronate that inhibit bone resorption in vivo could give rise to local concentrations as high as 1 mm alendronate in the resorption space beneath an osteoclast, which is much higher than the concentrations of bisphosphonates required to affect osteoclast morphology and cause osteoclast apoptosis in vitro. 37 In contrast to their ability to induce apoptosis in osteoclasts, which contributes to the inhibition of resorptive activity, some experimental studies suggest that bisphosphonates may protect osteocytes and osteoblasts from apoptosis induced by glucocorticoids. 38 Recent evidence suggests that the inhibition of osteocyte apoptosis by bisphosphonates is mediated through the opening of connexion 43 hemichannels and activation of extracellular signal-regulated kinases. 39 The possibility that bisphosphonates used clinically may get access to osteocytes differentially depending on their mineral-binding affinities and inherent structural properties needs to be studied. STRUCTURE-ACTIVITY RELATIONSHIPS AND MECHANISM OF ACTION The features of the bisphosphonate molecule necessary for biological activity were well defined in the early studies. The P-C-P moiety is responsible for the strong affinity of the bisphosphonates for binding to hydroxyapatite and allows for a number of variations in structure on the basis of substitution in the R 1 and R 2 positions on the carbon atom (Fig 1). The ability of the bisphosphonates to bind to hydroxyapatite crystals and to prevent both crystal growth and dissolution was enhanced when the R 1 side chain (attached to the geminal carbon atom of the P-C-P group) was a hydroxyl group (as in etidronate) rather than a halogen atom such as chlorine (as in clodronate). The presence of a hydroxyl group at the R 1 position increases the affinity for calcium (and, thus, bone mineral) because of the ability of bisphosphonates to chelate calcium ions by tridentate rather than bidentate binding. 40 The ability of bisphosphonates to inhibit bone resorption in vitro and in vivo also requires the P-C-P structure. Monophosphonates (eg, pentane monophosphonate) or P-C-C-P or P-N-P compounds are ineffective as inhibitors of bone resorption. Furthermore, the antiresorptive effect cannot be accounted for simply by adsorption of bisphosphonates to bone mineral and prevention of hydroxyapatite dissolution. It became clear that S152 RUSSELL

FIGURE 1 The generic structure of pyrophosphate compared with bisphosphonates and their functional domains. bisphosphonates must inhibit bone resorption by cellular effects on osteoclasts rather than simply by physicochemical mechanisms. After the successful clinical use of clodronate and etidronate in the 1970s and 1980s, more potent antiresorptive bisphosphonates, which had different R 2 side chains but in which R 1 was unaltered, were studied. In particular, bisphosphonates containing a basic primary amino-nitrogen atom in an alkyl chain (as in pamidronate and alendronate) were found to be 10- to 100-fold more potent than etidronate and clodronate. Then, in the 1980s, there was a phase in which synthesis of novel compounds took place specifically to determine their possible effects on calcium metabolism, with the result that compounds highly effective as inhibitors of bone resorption were identified and studied. These compounds, especially those that contain a tertiary amino-nitrogen (such as ibandronate 41 and olpadronate 42 ), were even more potent at inhibiting bone resorption. Among this generation of compounds that were synthesized to optimize their antiresorptive effects, the most potent antiresorptive bisphosphonates were those containing a nitrogen atom within a heterocyclic ring (as in risedronate 43 and zoledronate 44 ), which are up to 10 000-fold more potent than etidronate in some experimental systems (Fig 2). The analysis of structure-activity relationships allowed the spatial features of the active pharmacophore to be defined in considerable detail even before the molecular mechanism of action was fully elucidated. For maximal potency, the nitrogen atom in the R 2 side chain must be a critical distance away from the P-C-P group and in a specific spatial configuration. 45 This principle was used successfully for predicting the features required in the chemical design of new and more active compounds. Although the structure of the R 2 side chain is the major determinant of antiresorptive potency, both phosphonate groups are also required for the drugs to be pharmacologically active. FIGURE 2 Structures of the bisphosphonates used in clinical studies classified according to their biochemical mode of action. PEDIATRICS Volume 119, Supplement 2, March 2007 S153

In summary, studies of the relationships between bisphosphonate structure and antiresorptive potency suggested that the ability of bisphosphonates to inhibit bone resorption depend on 2 separate properties of the bisphosphonate molecule. The 2 phosphonate groups, together with a hydroxyl group at the R 1 position, 46 impart high affinity for bone mineral and act as a bone hook, which allows rapid and efficient targeting of bisphosphonates to bone mineral surfaces. Once localized within bone, the structure and three-dimensional conformation of the R 2 side chain (as well as the phosphonate groups in the molecule) determine the biological activity of the molecule and influence the ability of the drugs to interact with specific molecular targets. Our understanding of what these molecular targets might be has become much clearer as a result of recent work. Over the years there have been many efforts to explain how bisphosphonates work on cells, especially via inhibitory effects on enzymes (eg, by direct or indirect inhibition of the osteoclast proton-pumping H ATPase, 47,48 phosphatases, or lysosomal enzymes 49,50 ). Because osteoclasts are highly endocytic, bisphosphonate present in the resorption space is likely to be internalized by endocytosis and thereby affect osteoclasts directly. 25 The uptake of bisphosphonates by osteoclasts in vivo has been confirmed by using radiolabeled 51 and fluorescently labeled alendronate, which was internalized into intracellular vacuoles. After cellular uptake, a characteristic morphologic feature of bisphosphonatetreated osteoclasts is the lack of a ruffled border, the region of invaginated plasma membrane facing the resorption cavity. Bisphosphonates also disrupt the cytoskeleton of the osteoclast. 52 Early explanations for these effects invoked the inhibition of protein kinases or phosphatases that regulate cytoskeletal structure, such as protein tyrosine phosphatases. 53 56 However, a more likely mechanism by which the cytoskeleton may be affected involves loss of function of small GTPases such as Rho and Rac. Since the early 1990s there has been a systematic effort by our group and others to elucidate the molecular mechanisms of action of bisphosphonates, 5,57,58 and we have proposed that bisphosphonates can be classified into at least 2 major groups with different modes of action (Fig 3). The first group comprises the non nitrogen-containing bisphosphonates that perhaps most closely resemble pyrophosphate, such as clodronate and etidronate, and these can be metabolically incorporated into nonhydrolyzable analogues of adenosine triphosphate (ATP) by reversing the reactions of aminoacyl transfer RNA synthetases. 59 The resulting metabolites contain the P-C-P moiety in place of the, -phosphate groups of ATP, thus resulting in nonhydrolyzable (AppCp) nucleotides. 60 63 It is likely that intracellular accumulation of these metabolites within osteoclasts inhibits their function and may cause osteoclast cell death. The AppCp-type metabolites of bisphosphonates are cytotoxic when internalized and cause similar changes in morphology to those observed in clodronate-treated cells, possibly by interference with mitochondrial ATP translocases. 64 Overall, this group of bisphosphonates, therefore, seem to act as prodrugs, being converted to active metabolites after intracellular uptake by osteoclasts in vivo. In contrast, the second group contains the more potent, nitrogen-containing bisphosphonates such as alendronate, risedronate, and zoledronate. Members of this group interfere with other metabolic reactions, notably in the mevalonate biosynthetic pathway, and affect cellular activity and cell survival by interfering with protein prenylation and, therefore, the signaling functions of key regulatory proteins. These mechanisms have been reviewed in detail elsewhere. 1 The mevalonate pathway is a biosynthetic route responsible for the production of cholesterol, other sterols, and isoprenoid lipids such as isopentenyl diphosphate (also known as isopentenyl pyrophosphate), as well as farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP). FPP and GGPP are required for the posttranslational modification (prenylation) of small GTPases such as Ras, Rab, Rho, and Rac, which are prenylated at a cysteine residue in characteristic C-terminal motifs. 65 Small GTPases are important signaling proteins that regulate a variety of cell processes important for osteoclast function, including cell morphology, cytoskeletal arrangement, membrane ruffling, trafficking of vesicles, and apoptosis. 66 69 Prenylation is FIGURE 3 Two distinct molecular mechanisms of action of bisphosphonates (BPs) that both inhibit osteoclasts (see text for details). GTP indicates guanosine triphosphate. S154 RUSSELL

required for the correct function of these proteins because the lipid prenyl group serves to anchor the proteins in cell membranes and may also participate in protein-protein interactions. 70 Many observations point to the importance of the mevalonate pathway for osteoclast function and strengthen the proposal that the nitrogen-containing bisphosphonates inhibit osteoclastic bone resorption predominantly by inhibition of this pathway. These bisphosphonates inhibit the synthesis of mevalonate metabolites including FPP and GGPP, and thereby impair the prenylation of proteins, 71 and cause alteration of function of small GTPases. There is a strong structureactivity relationship such that changes to the structure of the nitrogen-containing R 2 side chain or to the phosphonate groups, which alter antiresorptive potency, also influence the ability to inhibit protein prenylation to a corresponding degree. 72 An important verification of the critical importance of this pathway has come from showing that the addition of intermediates of the mevalonate pathway (such as FPP and GGPP) could overcome bisphosphonate-induced apoptosis and other events in many cell systems. Another prediction was that if inhibition of the mevalonate pathway could account for the antiresorptive effects of bisphosphonates, then the statin drugs should also inhibit bone resorption. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, one of the first steps in the mevalonate pathway. They proved to be even more potent than bisphosphonates at inhibiting osteoclast formation and bone resorption in vitro, 73,74 an effect that could also be overcome by the addition of geranylgeraniol (which can be used for protein geranylgeranylation) but not farnesol (which is used for protein farnesylation). Hence, it seems that although nitrogen-containing bisphosphonates can prevent both farnesylation and geranylgeranylation of proteins (probably by inhibiting enzymes required for synthesis of FPP and GGPP), loss of geranylgeranylated proteins in osteoclasts is of greater consequence than loss of farnesylated proteins. This is consistent with the known role of geranylgeranylated proteins such as Rho, Rac, and Rab in processes that are fundamental to osteoclast formation and function (eg, cytoskeletal rearrangement, membrane ruffling, and vesicular trafficking 75 ), and further work has confirmed this, particularly the importance of Rab proteins. The comparison between bisphosphonates and statins is interesting. The statins are widely used as cholesterollowering drugs (they are able to lower cholesterol biosynthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase). Despite several studies, there is no substantial evidence that statins have effects on bone when used clinically, perhaps because they are selectively taken up by the liver rather than bone, which is the converse of the case for bisphosphonates. Therefore, this is an excellent example of how drug specificity is achieved by highly selective tissue targeting. The exact enzymes of the mevalonate pathway that are inhibited by individual bisphosphonates have been partially elucidated. Several enzymes of the pathway use isoprenoid diphosphates as a substrate (isopentenyl pyrophosphate isomerase, FPP synthase, GGPP synthase, squalene synthase) and, thus, are likely to have similar substrate-binding sites. Thus, if nitrogen-containing bisphosphonates act as substrate analogues of an isoprenoid diphosphate, it is possible that these bisphosphonates will inhibit more than 1 of the enzymes of the mevalonate pathway. Early studies revealed that incadronate and ibandronate, but not other bisphosphonates, are inhibitors of squalene synthase, an enzyme in the mevalonate pathway that is required for cholesterol biosynthesis. 76,77 Inhibition of squalene synthase, however, would not lead to inhibition of protein prenylation. However, it is now clear that farnesyl pyrophosphate synthase (FPPS) is a major site of action of the nitrogencontaining bisphosphonates (N-bisphosphonates). 78 FPPS catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. There is a strong relationship among individual bisphosphonates between inhibition of bone resorption and inhibition of FPPS, with the most potent bisphosphonates having IC 50 values (concentration that inhibits response by 50%) in the nanomolar range. 79 Modeling studies have provided a molecular rationale for bisphosphonate binding to FPPS. 80 Our recent studies using protein crystallography, enzyme kinetics, and isothermal titration calorimetry led to the first published high-resolution radiograph structures of the human enzyme in complexes with risedronate and zoledronate. 81 These agents bind to the dimethylallyl/ geranyl pyrophosphate ligand pocket and induce a conformational change. The interactions of the N-bisphosphonate cyclic nitrogen with Thr201 and Lys200 suggest that these inhibitors achieve potency by positioning their nitrogen in a proposed carbocation 82 binding site. This explains how the nitrogen moiety is so important to the potency of these bisphosphonates. Kinetic analyses reveal that inhibition is competitive with geranyl pyrophosphate and is of a slow, tight-binding character, which indicates that isomerization of an initial enzymeinhibitor complex occurs after binding of the N-bisphosphonate. Taken together, these observations clearly indicate that bisphosphonates can be grouped into 2 classes: those that can be metabolized into nonhydrolyzable analogues of ATP (the least potent bisphosphonates) and those that are not metabolized but can inhibit protein prenylation (the potent, nitrogen-containing bisphosphonates). The identification of 2 such classes may help to explain some of the other pharmacologic differences between the 2 classes. PEDIATRICS Volume 119, Supplement 2, March 2007 S155

CLINICAL APPLICATIONS OF BISPHOSPHONATES After it was shown that bisphosphonates inhibited experimentally induced calcification and bone resorption, their potential application to clinical disorders was obvious, but it took many years for them to become well established. The earliest clinical applications of bisphosphonates included use of etidronate as an inhibitor of calcification in fibrodysplasia ossificans progressiva (formerly known as myositis ossificans) and in patients who had undergone total hip replacement surgery to prevent subsequent heterotopic ossification and improve mobility. 83 One of the other early clinical uses of bisphosphonates was as agents for bone imaging, bone scanning, for which they still remain outstandingly useful for detecting bone metastases and other bone lesions. The application of pyrophosphate and simple bisphosphonates as bone-scanning agents depends on their strong affinity for bone mineral, particularly at sites of increased bone turnover, and their ability to be linked to a -emitting technetium isotope. 84,85 The most impressive clinical application of bisphosphonates has been as inhibitors of bone resorption, especially for diseases in which no effective treatment existed previously. Thus, bisphosphonates became the treatment of choice for a variety of bone diseases in which excessive osteoclast activity is an important pathologic feature, including Paget disease of bone, metastatic and osteolytic bone disease, and hypercalcemia of malignancy, as well as osteoporosis. The clinical pharmacology of bisphosphonates is characterized by low intestinal absorption ( 1% 4%) but highly selective localization and retention in bone. Significant adverse effects of bisphosphonates are minimal. 86 88 Although there are more similarities than differences between individual compounds and each bisphosphonate is potentially capable of treating any of the disorders of bone resorption in which they are used, in practice different compounds have come to be favored for the treatment of different diseases. There are currently at least 10 bisphosphonates (etidronate, clodronate, tiludronate, pamidronate, alendronate, risedronate, zoledronate, and ibandronate and, to a limited extent, olpadronate and neridronate) that have been registered for various clinical applications in various countries. To a major extent, the diseases in which they are used reflects the history of their clinical development and the degree of commercial interest in and sponsorship of the relevant clinical trials. Paget disease was the first clinical disorder in which a dose-dependent inhibition of bone resorption could be demonstrated by using bisphosphonates in humans. 89,90 Bisphosphonates have become the most important drugs used in the treatment of Paget disease. 91 For many years pamidronate given by intravenous infusion was used extensively, 92 but the newer and more potent bisphosphonates can produce even more profound suppression of disease activity than was possible with the bisphosphonates available in previous years. 93,94 The latest advance is with zoledronate, 95 which, when given as a single 5-mg infusion, produced a greater and longerlasting suppression of excess bone turnover than even oral risedronate given at 30 mg/day over 2 months, hitherto one of the most effective treatments. In terms of commercial success, the use of bisphosphonates in oncology has been preeminent. Many cancers in humans are associated with hypercalcemia (raised blood calcium) and/or increased bone destruction. Bisphosphonates are remarkably effective in the treatment of bone problems associated with malignancy 96 and are now the drugs of choice. 97 99 Clinical trials that investigate the benefit of bisphosphonate therapy use a composite end point defined as a skeletal-related or bone event, which typically includes pathologic fracture, spinal cord compression, radiation or surgery to bone, and hypercalcemia of malignancy. Bisphosphonates significantly reduce the incidence of these events in myeloma 100 and in patients with breast cancer metastases 101,102 and in metastatic prostate cancer, 103 lung cancer, renal cell carcinoma, and other solid tumors. The goals of treatment for bone metastases are also to prevent disease-related skeletal complications, palliate pain, and maintain quality of life. Zoledronate, 104 pamidronate, clodronate, and ibandronate 105,106 have demonstrated efficacy compared with placebo. There is the important possibility that the survival of patients may be prolonged 107 109 in some groups of patients. Recently, osteonecrosis of the jaw 110 was identified as a potential complication of high-dose bisphosphonate therapy in malignant diseases. The other area of outstanding commercial success with bisphosphonates has been in the therapy of osteoporosis, which is a major public health problem. 111,112 Up until the 1990s, there were few treatments for osteoporosis. As a drug class the bisphosphonates have emerged in the past few years as the leading effective treatments for postmenopausal and other forms of osteoporosis. Etidronate was the first of these, 113 115 followed by alendronate 116 118 and then risedronate. 119,120 All have been approved as therapies in many countries and can increase bone mass and reduce fracture rates at the spine by 30% to 50% and at other sites in postmenopausal women. 121 The reduction in fractures may be related not only to the increase in bone mass arising from the inhibition of bone resorption and reduced activation frequency of bone-remodeling units but also to enhanced osteon mineralization. 122 These bisphosphonates also prevent bone loss associated with glucocorticosteroid administration. 123,124 Among the newer bisphosphonates, ibandronate 125 was introduced recently as a once-monthly tablet. In addition to formulations to be taken by mouth weekly or S156 RUSSELL

monthly, new routes of administration are being studied, especially periodic (eg, 3 monthly) injections with ibandronate and once-yearly treatment with zoledronate. 126 This has the attraction of delivering a defined dose without the variability associated with oral administration as well as avoiding potential gastrointestinal intolerance. If these approaches are accompanied by greater compliance and convenience, they are likely to become popular methods of treatment. Other clinical issues under consideration with bisphosphonates include the choice of therapeutic regimen (eg, the use of intermittent dosing rather than continuous, intravenous versus oral therapy), the optimal duration of therapy, the combination with other drugs such as teriparatide, and their extended use in related indications (eg, glucocorticosteroid-associated osteoporosis, male osteoporosis, childhood osteopenic disorders, arthritis, and other disorders). Therefore, there is much that needs to be done to improve the way in which existing drugs can be used and to introduce new ones. In pediatrics, pamidronate has proved remarkably effective in increasing bone in children with the inherited brittle-bone disorder, osteogenesis imperfecta. 127,128 SOME CURRENT ISSUES WITH BISPHOSPHONATES: BONE ARCHITECTURE, STRUCTURE, AND STRENGTH, AND ON BONE HEALING AND FRACTURE REPAIR Many experimental and clinical studies show that bisphosphonates conserve bone architecture and strength. 129 133 However, there have been concerns about whether the use of prolonged high doses of bisphosphonates may impair bone turnover to such an extent that bone strength is impaired. High doses in animals are associated with increased microdamage 134,135 and even fractures. 136 It has been suggested that bisphosphonates might prevent naturally occurring microscopic cracks in bone from healing. There have been isolated reports of adynamic bone associated with bisphosphonate usage, 137 but long-term use of the bisphosphonates in the therapy of osteoporosis seems to be safe. 138 Case reports of induction of osteopetrosis-like lesions in children who were treated with excessive doses of pamidronate have been published. 139 A question often asked is whether bisphosphonates inhibit fracture repair. By reducing bone turnover one might expect bisphosphonates to interfere with fracture healing. However, a recent long-term study in a beagle dog model that simulated fracture repair has demonstrated that ibandronate treatment did not adversely affect normal bone healing. 140 Studies of repair processes after creating drill-hole defects in dogs also showed no impairment with ibandronate. 141 Several other recent studies raised the intriguing possibility that bisphosphonates may enhance fracture repair and related processes. 142 In studies of the osseointegration of metal implants in ovariectomized rats, treatment with ibandronate resulted in improved osseointegration rather than impairment of the healing process. 143 Potential applications of bisphosphonates in orthopedics include protection against loosening of prostheses, 144 better integration of biomaterials and implants, improved healing in distraction osteogenesis, 145 and conserving bone architecture after osteonecrosis 146,147 and in Perthes disease. 148 There are potentially important differences between clinically useful bisphosphonates regarding their potency and duration of action. Efficacy is closely related to affinity for bone mineral and ability to inhibit FPP synthase. Recent studies showing that there are marked differences among bisphosphonates in binding to hydroxyapatite 149 may explain the variations in retention and persistence of effect that have been observed in animal and clinical studies. In the case of zoledronic acid, in particular, the remarkable magnitude of effect and prolonged duration of action can be explained in part by these new observations. In explaining the long duration of action, it has been proposed that there is continual FIGURE 4 Bisphosphonate (BP) uptake and detachment from bone surfaces: effect of binding affinity on recirculation of bisphosphonate on and off bone surfaces. The differences in mineral-binding affinity may affect distribution into different bone compartments and persistence of drug action at bone surfaces. (Adapted from Nancollas GH, Tang R, Phipps RJ, et al. Bone. 2006;38:617 627.) PEDIATRICS Volume 119, Supplement 2, March 2007 S157

recycling of bisphosphonate off and back onto the bone surface. This notion is supported by observations that bisphosphonates can be found in plasma and urine many months after dosing (Fig 4). There are numerous examples of bisphosphonates having effects on cells and tissues outside the skeleton. The effects on osteoclast precursors, tumor cells, macrophages, and, -T cells are examples and in all cases are probably explained by sufficient bisphosphonates entering cells to inhibit the mevalonate pathway. A wellrecognized adverse effect of the nitrogen-containing bisphosphonates is that they cause an acute-phase response in vivo, 150,151 which can lead to induction of fever and flu-like symptoms in patients. These effects are transient and occur predominantly on first exposure to the drug, especially with intravenous administration. The mechanism has been attributed to release of proinflammatory cytokines, and the mechanism has been further unraveled by showing that it involves selective receptor-mediated activation of, -T cells, leading to their proliferation and activation. 152 The bisphosphonate effect involves the mevalonate pathway in vitro and can be overcome by using statins. 153 Another interesting aspect of these nonskeletal effects are the observations made on protozoan parasites, the growth of which can be inhibited by bisphosphonates acting on FPPS. 154,155 In the future, other clinical indications ripe for future study include the prevention of bone loss and erosions in rheumatoid arthritis, possible applications in other joint diseases, the reduction of bone loss associated with periodontal disease, and loosening of joint prostheses. The recent elucidation of the likely mode of action of bisphosphonates within cells opens up the possibility of exploiting the subtle and potentially important differences between the classes of bisphosphonates and individual compounds. REFERENCES 1. Russell RGG. Bisphosphonates: from bench to bedside. Ann N Y Acad Sci. 2006;1068:367 401 2. Bijvoet O, Fleisch H, Canfield RE, Russell RGG, eds. Bisphosphonates on Bone. Amsterdam, Holland: Elsevier Science; 1995 3. Fleisch H. Bisphosphonates in Bone Disease: From the Laboratory to the Patient. 4th ed. San Diego, CA: Academic Press; 2000 4. Green JR. Bisphosphonates: preclinical review. Oncologist. 2004;9(suppl 4):3 13 5. Rogers MJ. From molds and macrophages to mevalonate: a decade of progress in understanding the molecular mode of action of bisphosphonates. Calcif Tissue Int. 2004;75:451 461 6. Fleisch H, Neuman WF. Mechanisms of calcification: role of collagen, polyphosphates, and phosphatase. Am J Physiol. 1961;200:1296 1300 7. Fleisch H, Bisaz S. Isolation from urine of pyrophosphate, a calcification inhibitor. Am J Physiol. 1962;203:671 675 8. Fleisch H, Russell RGG, Straumann F. Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis. Nature. 1966;212:901 903 9. Russell RG. Excretion of inorganic pyrophosphate in hypophosphatasia. Lancet. 1965;10:461 464 10. Schibler D, Russell RGG, Fleisch H. Inhibition by pyrophosphate of aortic calcification induced by vitamin D 3 in rats. Clin Sci. 1968;35:363 372 11. Fleisch H, Russell RGG, Bisaz S, Casey PA, Muehlbauer RC. The influence of pyrophosphate analogues (diphosphonates) on the precipitation and dissolution. Calcif Tissue Res. 1968; (suppl):10 10A 12. Francis MD, Russell RGG, Fleisch H. Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vivo. Science. 1969;165:1264 1266 13. Fleisch H, Russell RGG, Francis MD. Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture in vivo. Science. 1969;165:1262 1264 14. Jung A, Bisaz S, Fleisch H. The binding of pyrophosphate and two diphosphonates by hydroxyapatite crystals. Calcif Tissue Res. 1973;11:269 280 15. Fleisch H, Russell RGG, Bisaz S, Muehlbauer RC, Williams DA. The inhibitory effect of phosphonates on the formation of calcium phosphate crystals in vitro and on aortic and kidney recalcification in vivo. Eur J Clin Invest. 1970;1:12 18 16. Schenk R, Merz WA, Muhlbauer R, Russell RGG, Fleisch H. Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl 2 MDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif Tissue Res. 1973;11:196 214 17. Fleisch H, Maerki J, Russell RGG. Effect of pyrophosphate on dissolution of hydroxyapatite and its possible importance in calcium homeostasis. Proc Soc Exp Biol Med. 1966;122:317 320 18. Russell RGG, Muhlbauer RC, Bisaz S, Williams DA, Fleisch H. The influence of pyrophosphate, condensed phosphates, phosphonates and other phosphate compounds on the dissolution of hydroxyapatite in vitro and on bone resorption induced by parathyroid hormone in tissue culture and in thyroparathyroidectomised rats. Calcif Tissue Res. 1970;6: 183 196 19. Trechsel U, Stutzer A, Fleisch H. Hypercalcemia induced with an arotinoid in thyroparathyroidectomized rats: a new model to study bone resorption in vivo. J Clin Invest. 1987;80: 1679 1686 20. Stutzer A, Fleisch H, Trechsel U. Short- and long-term effects of a single dose of bisphosphonates on retinoid-induced bone resorption in thyroparathyroidectomized rats. Calcif Tissue Int. 1988;43:294 299 21. Schenk R, Eggli P, Fleisch H, Rosini S. Quantitative morphometric evaluation of the inhibitory activity of new aminobisphosphonates on bone resorption in the rat. Calcif Tissue Int. 1986;38:342 349 22. Muehlbauer RC, Russell RGG, Williams DA, Fleisch H. The effects of diphosphonates, polyphosphates, and calcitonin on immobilisation osteoporosis in rats. Eur J Clin Invest. 1971; 1:336 344 23. Ferretti JL. Effects of biosphosphonates on bone biomechanics. Bijvoet O, Fleisch HA, Canfield RE, Russell RGG, eds. In: Bisphosphonates on Bone. Amsterdam, Holland: Elsevier Science; 1995:211 229 24. Shinoda H, Adamek G, Felix R, Fleisch H, Schenk R, Hagan P. Structure-activity relationships of various bisphosphonates. Calcif Tissue Int. 1983;35:87 99 25. Flanagan AM, Chambers TJ. Dicholormethylenebisphosphonate (C12MBP) inhibits bone resorption through injury to osteoclasts that resorb C12 MBP-coated bone. Bone Miner. 1989;6:33 43 26. Smith R, Russell RGG, Bishop M. Disphosphonates and Page s disease of bone. Lancet. 1971;1(7706):945 947 27. Reitsma PH, Bijvoet OLM, Verlinden-Ooms H, van der Wee- S158 RUSSELL

Pals LJA. Kinetic studies of bone and mineral metabolism during treatment with (3-amino-1-hydroxy-propylidene)- 1,1-bisphosphonate (APD) in rats. Calcif Tissue Int. 1980;32: 145 157 28. Garnero P, Shih WJ, Gineyts E, Karpf DB, Delmas PD. Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to alendronate treatment. J Clin Endocrinol Metab. 1994;79:1693 1700 29. Bauss F, Russell RG. Ibandronate in osteoporosis: preclinical data and rationale for intermittent dosing. Osteoporos Int. 2004; 15:423 433 30. Gasser JA, Green JR. Long-term protective effect of a single IV injection of zoledronic acid on cancellous bone structure and cortical bone in ovariectomized rats. Bone. 2002;30(suppl): 41S 31. Hughes DE, MacDonald BR, Russell RG, Gowen M. Inhibition of osteoclast-like cell formation by bisphosphonates in longterm cultures of human bone marrow. J Clin Invest. 1989;83: 1930 1935 32. Boonekamp PM, van der Wee-Pals LJA, van Wijk-van Lennep MLL, Wil Thesing C, Bijvoet OLM. Two modes of action of bisphosphonates on osteoclastic resorption of mineralized matrix. Bone Miner. 1986;1:27 39 33. Lowik CWGM, van der Pluijm G, van der Wee-Pals LJA, Bloys van Teslong-de Groot H, Bijvoet OLM. Migration and phenotypic transformation of osteoclast precursors into mature osteoclasts: the effect of a bisphosphonate. J Bone Miner Res. 1988;3:185 192 34. Suda T, Nakamura I, Jimi E, Takahashi N. Regulation of osteoclast function. J Bone Miner Res. 1997;12:869 879 35. Masarachia P, Weinreb M, Balena R, Rodan GA. Comparison of the distribution of 3 H-alendronate and 3 H-etidronate in rat and mouse bones. Bone. 1996;19:281 290 36. Azuma Y, Sato H, Oue Y, et al. Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia models. Bone. 1995;16: 235 245 37. Sato M, Grasser W. Effects of bisphosphonates on isolated rat osteoclasts as examined by reflected light microscopy. J Bone Miner Res. 1990;5:31 40 38. Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas Sc, Bellido T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest. 1999;104:1363 1374 39. Plotkin LI, Aguirre JI, Kousteni S, Manolagas SC, Bellido T. Bisphosphonates and estrogens inhibit osteocyte apoptosis via distinct molecular mechanisms downstream of extracellular signal-regulated kinase activation. J Biol Chem. 2005;280: 7317 7325 40. Ebrahimpour A, Francis MD. Bisphosphonate therapy in acute and chronic bone loss: physical chemical considerations in bisphosphonate-related therapies. In: Bijvoet O, Fleisch HA, Canfield RE, Russell RGG eds. Bisphosphonates on Bones. Amsterdam, Holland: Elsevier Science; 1995:125 136 41. Muehlbauer RC, Bauss F, Schenk R, et al. BM 21.0955, a potent new bisphosphonate to inhibit bone resorption. J Bone Miner Res. 1991;6:1003 1011 42. Papapoulos SE, Hoekman K, Lowik CWGM, Vermeij P, Bijvoet OLM. Application of an in vitro model and a clinical protocol in the assessment of the potency of a new bisphosphonate. J Bone Miner Res. 1988;4:775 781 43. Goa KL, Balfour JA. Risedronate. Drugs Aging. 1998;13:83 91; discussion 92 44. Green JR, Mueller K, Jaeggi KA. Preclinical pharmacology of CGP42 446, a new, potent, heterocyclic bisphosphonate compound. J Bone Miner Res. 1994;9:745 751 45. Ebetino FH, Bayless AV, Amburgey J, Ibbotson KJ, Dansereau S, Ebrahimpour A. Elucidation of a pharmacore for the bisphosphonate mechanism of bone antiresorptive activity. Phosphorus Sulfur Silicon Relat Elem. 1996;109/110:217 220 46. van Beek E, Lowik C, Oue I, Papapoulos S. Dissociation of binding and antiresorptive properties of hydroxybisphosphonates by substitution of the hydroxyl with an amino group. J Bone Miner Res. 1996;11:1492 1497 47. David P, Nguyen H, Barbier A, Baron R. The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H( )-ATPase. J Bone Miner Res. 1996;11:1498 1507 48. Carano A, Teitelbaum SA, Konsek JD, Schlesinger PH, Blair HC. Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro. J Clin Invest. 1990; 85:456 461 49. Felix R, Russell RGG, Fleisch H. The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes. Biochim Biophys Acta. 1976;429:429 438 50. Lerner UH, Larsson A. Effects of four bisphosphonates on bone resorption, lysosomal enzyme release, protein synthesis, and mitotic activities in mouse calvarial bones in vitro. Bone. 1987;8:179 189 51. Sato M, Grasser W, Endo N, et al. Bisphosphonate action: alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest. 1991;88:2095 2105 52. Murakami H, Takahashi N, Sasaki T, et al. A possible mechanism of the specific action of bisphosphonates on osteoclasts: tiludronate preferentially affects polarized osteoclasts having ruffled borders. Bone. 1995;17:137 144 53. Schmidt A, Rutledge SJ, Endo N, et al. Alendronate inhibition of protein tyrosine phosphatase activity. Proc Natl Acad Sci USA. 1995;93:3068 3073 54. Endo N, Rutledge SJ, Opas EE, Vogel R, Rodan GA, Schmidt A. Human protein tyrosine phosphatases: alternative splicing and inhibition by bisphosphonates. J Bone Miner Res. 1996;11: 535 543 55. Opas EE, Rutledge SJ, Golub E, et al. Alendronate inhibition of protein-tyrosine-phosphatase-meg1. Biochem Pharmacol. 1997;54:721 727 56. Murakami H, Takahashi N, Tanaka S, et al. Tiludronate inhibits protein tyrosine phosphatase activity in osteoclasts. Bone. 1997;20:399 404 57. Rogers MJ, Watts DJ, Russell RGG, et al. Inhibitory effects of bisphosphonates on growth of amoebae of the cellular slime mold Dictyostelium discoideum. J Bone Miner Res. 1994;9: 1029 1039 58. Rogers MJ, Xiong X, Brown RJ, et al. Structure-activity relationships of new heterocycle-containing bisphosphonates as inhibitors of bone resorption and as inhibitors of growth of Dictyostelium discoideum amoebae. Mol Pharmacol. 1995;47: 398 402 59. Rogers MJ, Brown RJ, Hodkin V, Russell RGG, Watts DJ. Bisphosphonates are incorporated into adenine nucleotides by human aminoacyl-trna synthetase enzymes. Biochem Biophys Res Commun. 1996;224:863 869 60. Rogers MJ, Ji X, Russell RGG, et al. Incorporation of bisphosphonates into adenine nucleotides by amoebae of the cellular slime mould Dictyostelium discoideum. Biochem J. 1994;303: 303 311 61. Frith JC, Monkkonen J, Blackburn GM, Russell RGG, Rogers MJ. Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5, dichloromethylene triphosphate, by mammalian cells in vitro. J Bone Miner Res. 1996;12:1358 1367 62. Frith JC, Monkkonen J, Auriola S, Monkkonen H, Rogers MJ. The molecular mechanism of action of the antiresorptive and anti-inflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and PEDIATRICS Volume 119, Supplement 2, March 2007 S159

causes osteoclast and macrophage apoptosis. Arthritis Rheum. 2001;44:2201 2210 63. Rogers MJ, Xiong X, Ji X, et al. Inhibition of growth of Dictyostelium discoideum amoebae by bisphosphonates is dependent on cellular uptake. Pharm Res. 1997;14:625 630 64. Lehenkari PP, Kellinsalmi M, Napankangas JJJP, et al. Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable adenine-containing metabolite. Mol Pharmacol. 2002; 61:1255 1262 65. Zhang FL, Casey PJ. Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem. 1996; 65:241 269 66. Ridley AJ, Hall A. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992;70:389 399 67. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The small GTP-binding protein, rac, regulates growth factorinduced membrane ruffling. Cell. 1992;70:401 410 68. Zerial M, Stenmark H. Rab GTPases in vesicular transport. Curr Opin Cell Biol. 1993;5:613 620 69. Zhang D, Udagawa N, Nakamura I, et al. The small GTP-biding protein, Rho p21, is involved in bone resorption by regulating cytoskeletal organization in osteoclasts. J Cell Sci. 1995;108: 2285 2292 70. Marshall C. Protein prenylation: a mediator of protein-protein interactions. Science. 1993;259:1865 1866 71. Luckman SP, Hughes DE, Coxon FP, Russell RGG, Rogers MJ. Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTPbinding proteins, including Ras. J Bone Miner Res. 1998;13: 581 589 72. Luckman SP, Coxon FP, Ebetino FH, Russell RGG, Rogers MJ. Heterocycle-containing bisphosphonates cause apoptosis and inhibit bone resorption by preventing protein prenylation: evidence from structure-activity relationships in J774 macrophages. J Bone Miner Res. 1998;13:1668 1678 73. Staal A, Frith JC, French MH, et al. The ability of statins to inhibit bone resorption is directly related to their inhibitory effect on HMG-CoA reductase activity. J Bone Miner Res. 2003; 18:88 96 74. Fisher JE, Rogers MJ, Halasy JM, et al. Mechanism of action of alendronate: geranylgeraniol, an intermediate of the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro. Proc Natl Acad Sci U S A. 1999;96:133 138 75. Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998; 279:509 514 76. Amin D, Cornell SA, Perrone MH, Bilder GE. 1-hydroxy-3- (methylpentylamino)-propylidene-1,1-bisphosphonic acid as a potent inhibitor of squalene synthase. Drug Res. 1996;46: 759 762 77. Amin D, Cornell SA, Gustafson SK, et al. Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis. J Lipid Res. 1992;33: 1657 1663 78. van Beek E, Pieterman E, Cohen L, Lowik C, Papapoulos S. Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo. Biochem Biophys Res Commun. 1999;255:491 494 79. Dunford JE, Thompson K, Coxon FP, et al. Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogencontaining bisphosphonates. J Pharmacol Exp Ther. 2001;296: 235 242 80. Ebetino FH, Roze CN, McKenna CE, et al. Molecular interactions of nitrogen-containing bisphosphonates within farnesyl diphosphate synthase. J Organometallic Chem. 2005;690: 2679 2687 81. Kavanagh K, Guo K, Dunford J, et al. Human farnesyl diphosphate synthase (FDPS): crystal structure and molecular interactions with nitrogen-containing bisphosphonates. Proc Natl Acad Sci U S A. 2006;103:7829 7834 82. Martin MB, Arnold W, Heath HT 3rd, Urbina JA, Oldfield E. Nitrogen-containing bisphosphonates as carbocation transition state analogs for isoprenoid biosynthesis. Biochem Biophys Res Commun. 1999;263:754 758 83. Smith R. Ectopic calcification and ossification. In: Bijovoet O, Fleisch HA, Canfield RE, Russell RGG, eds. Bisphosphonates on Bone. Amsterdam, Holland: Elsevier Science; 1995:433 443 84. Fogelman I, Bessent RG, Turner JF, Citrin DL, Boyce IT, Greig WR. The use of whole-body retention of Tc-99m diphosphonate in the diagnosis of metabolic bone disease. J Nucl Med. 1978;19:270 275 85. Francis MD, Fogelman I. 99mTc-diphosphonate uptake mechanisms on bone. In: Fogleman I, ed. Bone Scanning in Clinical Practice. London, United Kingdom: Springer; 1987:1 6 86. Adami S, Zamberlan N. Adverse effects of bisphosphonates: a comparative review. Drug Saf. 1996;14:158 170 87. van Staa TP. Post-marketing survey with ICT-etidronate therapy. Rev Contemp Pharmacol. 1998;9:277 286 88. DeGroen PC, Lubbe DF, Hirsch LJ, et al. Esophagitis associated with the use of alendronate. N Engl J Med. 1996;335: 1016 1021 89. Russell RGG, Smith R, Preston C, Walton RJ, Woods CF. Diphosphonates in Paget s disease. Lancet. 1974;1(7863): 894 898 90. Delmas PD, Meunier PJ. The management of Paget s disease of bone. N Engl J Med. 1997;336:558 566 91. Douglas DL, Russell RGG, Preston CJ, et al. Effect of dicholormethylene diphosphonate in Paget s disease of bone and in hypercalcaemia due to primary hyperparathyroidism or malignant disease. Lancet. 1980;1(8177):1043 1047 92. Coukell AJ, Markham A. Pamidronate: a review of its use in the management of osteolytic bone metastases, tumourinduced hypercalcaemia and Paget s disease of bone. Drugs Aging. 1998;12:149 168 93. Singer FR, Clemens TL, Eusebio RE, Bekker PJ. Risedronate, a highly effective oral agent in the treatment of patients with severe Paget s disease. J Clin Endocrinol Metab. 1998;83: 1906 1910 94. Siris ES, Chines AA, Altman RD, et al. Risedronate in the treatment of Paget s disease of bone: an open label, multicenter study. J Bone Miner Res. 1998;13:1032 1038 95. Reid IR, Miller P, Lyles K, et al. Comparison of a single infusion of zoledronic acid with risedronate for Paget s disease. N Engl J Med. 2005;353:898 908 96. Ross JR, Saunders Y, Edmonds PM, et al. A systematic review of the role of bisphosphonates in metastatic disease. Health Technol Assess. 2004;8(4):1 176 97. Paterson AD, Kanis JA, Cameron EC, et al. The use of dichloromethylene diphosphonate for the management of hypercalcaemia in multiple myeloma. Br J Haematol. 1983;54: 121 132 98. Ralston SH, Gallacher SJ, Patel U, Fraser WD, Dryburgh FJ, Boyle IT. Comparison of three bisphosphonates in cancerassociated hypercalcaemia. Lancet. 1989;2(8673):1180 1182 99. Body JJ. Hypercalcemia of malignancy. Semin Nephrol. 2004; 24:48 54 100. Sirohi B, Powles R. Multiple myeloma. Lancet. 2004;363: 875 887 S160 RUSSELL

101. Pavlakis N, Schmidt R, Stockler M. Bisphosphonates for breast cancer. Cochrane Database Syst Rev. 2005;(3):CD003474 102. Coleman RE. Bisphosphonates in breast cancer. Ann Oncol. 2005;16:687 695 103. Parker CC. The role of bisphosphonates in the treatment of prostate cancer. BJU Int. 2005;95:935 938 104. Smith MR. Zoledronic acid to prevent skeletal complications in cancer: corroborating the evidence. Cancer Treat Rev. 2005; 31(suppl 3):19 25 105. Bell R. Efficacy of ibandronate in metastatic bone disease: review of clinical data. Oncologist. 2005;10(suppl 1):8 134 106. Body JJ, Diel IJ, Lichinitzer M, et al. Oral ibandronate reduces the risk of skeletal complications in breast cancer patients with metastatic bone disease: results from two randomised, placebo-controlled phase III studies. Br J Cancer. 2004;90: 1133 1137 107. McCloskey EV, MacLennan ICM, Drayson M, Chapman C, Dunn J, Kanis JA. A randomized trial of the effect of clodronate on skeletal morbidity in multiple myeloma. MRC Working Party on Leukeaemia in Adults. Br J Haematol. 1998; 100:317 325 108. Berenson JR, Lichtenstein A, Porter L, et al. Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. N Engl J Med. 1996;334:488 493 109. Diel IJ, Solomayer EF, Costa SD, et al. Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Engl J Med. 1998;339:357 363 110. Maerevoet M, Martin C, Duck L. Osteonecrosis of the jaw and bisphosphonates [published correction appears in N Engl J Med. 2005;353:2728]. N Engl J Med. 2005;353:99 102 111. Carmona RH. Bone Health and Osteoporosis: A Report of the Surgeon General. Rockville MD: US Department of Health and Human Services, Office of the Surgeon General; 2004. Available at: www.hhs.gov/surgeongeneral/library/bonehealth/ content.html. Acessed December 13, 2006 112. Russell RGG. Pathogenesis of osteoporosis. In: Hochberg MC, Silman AJ, Smolen SJ, Weinblatt ME, Weisman MH, eds. Rheumatology. 3rd ed. St Louis, MO: Mosby; 2003:2075 2147 113. Watts NB, Harris ST, Genant HK, et al. Intermittent cyclical etidronate treatment of postmenopausal osteoporosis. N Engl J Med. 1990;323:73 79 114. Storm T, Thamsborg G, Steinriche T, et al. Effect of intermittent cyclical etidronate therapy on bone mass and fracture rate in women with postmenopausal osteoporosis. N Engl J Med. 1990;322:1265 1271 115. van Staa TP, Abenhain L, Cooper C. Use of cyclical etidronate and prevention of non-vertebral fractures. Br J Rheumatol. 1998;37:87 94 116. Liberman UA, Weiss SR, Broll J, et al. Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. The Alendronate Phase III Osteoporosis Treatment Study Group. N Engl J Med. 1995;333: 1437 1443 117. Black DM, Cummings SR, Karpf DB, et al. Randomised trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Fracture Intervention Trial Research Group. Lancet. 1996;348:1535 1541 118. Bone HG, Hosking D, Devogelaer JP, et al. Ten years experience with alendronate for osteoporosis in postmenopausal women. N Engl J Med. 2004;350:1189 1199 119. Reginster J, Minne HW, Sorenson OH, et al. Randomized trial of the effects of risedronate on vertebral fractures in women with established postmenopausal osteoporosis. Vertebral Efficacy With Risedronate Therapy (VERT) Study Group. Osteoporos Int. 2000;11:83 91 120. McClung MR, Geusens P, Miller PD, et al. Effect of risedronate on the risk of hip fracture in elderly women. Hip Intervention Program Study Group. N Engl J Med. 2001;344: 333 340 121. Boonen S, Laan RF, Barton IP, Watts NB. Effect of osteoporosis treatments on risk of non-vertebral fractures: review and meta-analysis of intention-to-treat studies. Osteoporos Int. 2005;16:1291 1298 122. Chavassieux PM, Arlot ME, Reda C, Wei L, Yates AJ, Meunier PJ. Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis. J Clin Invest. 1997;100:1475 1480 123. Adachi JD, Bensen WG, Brown J, et al. Intermittent etidronate therapy to prevent corticosteroid-induced osteoporosis. N Engl J Med. 1997;337:381 387 124. Saag KG, Emkey K, Schnitzer TJ, et al. Alendronate for the prevention and treatment of glucocorticoid-induced osteoporosis. Glucocorticoid-Induced Osteoporosis Intervention Study Group. N Engl J Med. 1998;339:292 299 125. Chesnut CH, Skag A, Christiansen C, et al. Effects of oral ibandronate administered daily or intermittently on fracture risk in postmenopausal osteoporosis. J Bone Miner Res. 2004; 19:1241 1249 126. Reid IR, Brown JP, Burckhardt P, et al. Intravenous zoledronic acid in postmenopausal women with low bone mineral density. N Engl J Med. 2002;346:653 661 127. Glorieux FH, Bishop NJ, Plotkin H, Chabot G, Lanoue G, Travers R. Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. N Engl J Med. 1998;339: 947 952 128. Rauch F, Glorieux FH. Osteogenesis imperfecta, current and future medical treatment. Am J Med Genet C Semin Med Genet. 2005;139:31 37 129. Borah B, Ritman EL, Dufresne TE, et al. The effect of risedronate on bone mineralization as measured by microcomputed tomography with synchrotron radiation: correlation to histomorphometric indices of turnover. Bone. 2005;37: 1 9 130. Borah B, Dufresne TE, Chmielewski PA, Gross GJ, Prenger MC, Phipps RJ. Risedronate preserves trabecular architecture and increases bone strength in vertebra of ovariectomized minipigs as measured by three-dimensional microcomputed tomography. J Bone Miner Res. 2002;17:1139 1147 131. Borah B, Dufresne TE, Chmielweski PA, Johnson TD, Chines A, Manhart MD. Risedronate preserves bone architecture in postmenopausal women with osteoporosis as measured by three-dimensional microcomputed tomography. Bone. 2004; 34:736 746 132. Lalla S, Hothorn LA, Haag N, Bader R, Bauss F. Lifelong administration of high doses of ibandronate increases bone mass and maintains bone quality of lumbar vertebrae in rats. Osteoporos Int. 1998;8:97 103 133. Bauss F, Lalla S, Endele R, Hothorn LA. Effects of treatment with ibandronate on bone mass, architecture, biomechanical properties, and bone concentration of ibandronate in ovariectomized aged rats. J Rheumatol. 2002;29:2200 2208 134. MashibaT, Mori S, Burr DB, et al. The effects of suppressed bone remodeling by bisphosphonates on microdamage accumulation and degree of mineralization in the cortical bone of dog rib. J Bone Miner Metab. 2005;23(suppl):36 42 135. Mashiba T, Hirano T, Turner CH, Forwood MR, Johnston CC, Burr DB. Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces some biomechanical properties in dog rib. J Bone Miner Res. 2000;15: 613 620 136. Flora L, Hassing GS, Cloyd GG, Bevan JA, Parfitt AM, Villanueva AR. The long-term skeletal effects of EHDP in dogs. Metab Bone Dis Relat Res. 1981;3:389 300 137. Odvina CV, Zerwekh JE, Rao DS, Malouf N, Gottschalk FA, PEDIATRICS Volume 119, Supplement 2, March 2007 S161

Pak CY. Severely suppressed bone turnover: a potential complication of alendronate therapy. J Clin Endocrinol Metab. 2005;90:1294 1301 138. Ott SM. Long-term safety of bisphosphonates. J Clin Endocrinol Metab. 2005;90:1897 1899 139. Whyte MP, Wenkert D, Clements KL, McAlister WH, Mumm S. Bisphosphonate-induced osteopetrosis. N Engl J Med. 2004; 349:457 463 140. Monier-Faugere MC, Geng Z, Paschalis EP, et al. Intermittent and continuous administration of the bisphosphonate ibandronate in ovariohysterectomized beagle dogs: effects on bone morphometry and mineral properties. J Bone Miner Res. 1999;14:1768 1778 141. Bauss F, Schenk RK, Hort S, Muller-Beckmann B, Sponer G. New model for simulation of fracture repair in full-grown beagle dogs: model characterization and results from a longterm study with ibandronate. J Pharmacol Toxicol Methods. 2004;50:25 34 142. Little DG, McDonald M, Bransford R, Godfrey CB, Amanat N. Manipulation of the anabolic and catabolic responses with OP-1 and zoledronic acid in a rat critical defect model. J Bone Miner Res. 2005;20:2044 2052 143. Kurth AH, Eberhardt C, Muller S, Steinacker M, Schwarz M, Bauss F. The bisphosphonate ibandronate improves implant integration in osteopenic ovariectomized rats. Bone. 2005;37: 204 210 144. Wilkinson JM, Eagleton AC, Stockley I, Peel NF, Hamer AJ, Eastell R. Effect of pamidronate on bone turnover and implant migration after total hip arthroplasty: a randomized trial. J Orthop Res. 2005;23:1 8 145. Little DG, Smith NC, Williams PR, et al. Zoledronic acid prevents osteopenia and increases bone strength in a rabbit model of distraction osteogenesis. J Bone Miner Res. 2003;18: 1300 1307 146. Little DG, Peat RA, Mcevoy A, Williams PR, Smith EJ, Baldock PA. Zoledronic acid treatment results in retention of femoral head structure after traumatic osteonecrosis in young Wistar rats. J Bone Miner Res. 2003;18:2016 2022 147. Lai KA, Shen WJ, Yang CY, Shao CJ, Hsu JT, Lin RM. The use of alendronate to prevent early collapse of the femoral head in patients with nontraumatic osteonecrosis: a randomized clinical study. J Bone Joint Surg Am. 2005;87:2155 2159 148. Little DG, McDonald M, Sharpe IT, Peat R, Williams P, McEvoy T. Zoledronic acid improves femoral head sphericity in a rat model of Perthes disease. J Orthop Res. 2005;23:862 868 149. Nancollas GH, Tang R, Phipps RJ, et al. Novel insights into actions of bisphosphonates on bone: differences in interactions with hydroxyapatite. Bone. 2006;38:617 627 150. Sauty A, Pecherstorfer M, Zimmeroth I, et al. Interleukin-6 and tumor necrosis factor alpha levels after bisphosphonates treatment in vitro and in patients with malignancy. Bone. 1996;18:133 139 151. Schweitzer DH, Oostendorp-vande Ruit M, van der Pluijm G, Lowik CWGM, Papapoulos SE. Interleukin-6 and the acute phase response during treatment of patients with Paget s disease with the nitrogen-containing bisphosphonate dimethylaminohydroyxpoproylidene bisphosphonate. J Bone Miner Res. 1995;10:956 962 152. Sanders JM, Ghosh S, Chan JM, et al. Quantitative structureactivity relationships for gammadelta T cell activation by bisphosphonates. J Med Chem. 2004;47:375 384 153. Thompson K, Rogers MJ. Statins prevent bisphosphonateinduced gamma, delta T-cell proliferation and activation in vitro. J Bone Miner Res. 2004;19:278 288 154. Gabelli SB, McLellan JS, Montalvetti A, Oldfield E, Docampo R, Amzel LM. Structure and mechanism of the farnesyl diphosphate synthase from Trypanosoma cruzi: implications for drug design. Proteins. 2005;62:80 88 155. Szabo CM, Matsumura Y, Fukura S, et al. Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates and diphosphates: a potential route to new bone antiresorption and antiparasitic agents. J Med Chem. 2002;45:2185 2196 S162 RUSSELL

Bisphosphonates: Mode of Action and Pharmacology R. Graham G. Russell Pediatrics 2007;119;S150 DOI: 10.1542/peds.2006-2023H Updated Information & Services References Subspecialty Collections Permissions & Licensing Reprints including high resolution figures, can be found at: http://pediatrics.aappublications.org/content/119/supplement_2/s150 This article cites 147 articles, 15 of which you can access for free at: http://pediatrics.aappublications.org/content/119/supplement_2/s150 #BIBL This article, along with others on similar topics, appears in the following collection(s): Pharmacology http://www.aappublications.org/cgi/collection/pharmacology_sub Therapeutics http://www.aappublications.org/cgi/collection/therapeutics_sub Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: http://www.aappublications.org/site/misc/permissions.xhtml Information about ordering reprints can be found online: http://www.aappublications.org/site/misc/reprints.xhtml

Bisphosphonates: Mode of Action and Pharmacology R. Graham G. Russell Pediatrics 2007;119;S150 DOI: 10.1542/peds.2006-2023H The online version of this article, along with updated information and services, is located on the World Wide Web at: http://pediatrics.aappublications.org/content/119/supplement_2/s150 Pediatrics is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since 1948. Pediatrics is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, 60007. Copyright 2007 by the American Academy of Pediatrics. All rights reserved. Print ISSN: 1073-0397.