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Novel bone antiresorptive approaches Michael W Lark* and Ian E James Inhibition of bone resorption is a mechanism that has been clinically validated as a means to control bone loss in diseases such as postmenopausal osteoporosis. The development of marketable drugs in this area has resulted in significant clinical benefits; however, improvements can still be made. Several novel antiresorptive mechanisms are currently under consideration in the pharmaceutical industry, which will hopefully result in the development of improved bone antiresorptive therapies. Addresses Department of Musculoskeletal Diseases Biology, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA *e-mail: [email protected] Current Opinion in Pharmacology 2002, 2:330–337 1471-4892/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations BMU basic multicellular unit OA osteoarthritis OPG osteoprotegerin PPAR-γγ peroxisome proliferator activated receptor γ PTH parathyroid hormone RA rheumatoid arthritis RANK receptor activator of NF-κB RANK-L RANK ligand SERM selective estrogen receptor modulator vATPase vacuolar type H+-ATPase
Introduction Bone turnover is a complex, multicellular process involving cell recruitment and differentiation, and the activity of highly specialized cells. The mature cells that control bone turnover are the bone-resorbing osteoclasts and the bone-forming osteoblasts. The balance of osteoblastmediated bone formation and osteoclast-mediated bone resorption is what ultimately controls skeletal integrity. Despite the fact that these cells have specialized and opposing activities, it has become generally accepted that they work in concert to maintain normal bone balance. One of the most accepted models of bone turnover is that of the basic multicellular unit (BMU) [1•]. In the BMU model, bone-resorbing osteoclasts remove mineralized bone matrix, which is followed by new bone deposition by bone-forming osteoblasts. During development, the balance of the two processes is in favor of bone formation; whereas, in estrogen-depleted postmenopausal osteoporosis, the balance favors bone resorption. In both of these situations, bone formation and resorption (bone turnover) occurs, but the balance favors one process above the other. These processes are also modulated in several other settings such as fracture repair [2] and in other diseases, such as periodontal disease [3,4], rheumatoid arthritis (RA) [5–9], steroid-induced bone loss [8,9] and bone loss due to metastasis targeted to bone [6,10].
Mature osteoblasts and osteoclasts are derived from different progenitor populations. The osteoblast is derived from a mesenchymal cell precursor, which has also been shown to differentiate into adipocytes and chondrocytes [11–13]. With aging and/or menopause, there is an increase in bone marrow adipocytes [14]. One approach currently under consideration is to drive bone formation in the aging or menopausal population by influencing the osteoblast/adipocyte balance to one of increased numbers of osteoblasts. There are a large number of studies suggesting that the nuclear peroxisome proliferator activated receptor γ (PPAR-γ) may be key to the maintenance of this balance. PPAR-γ antagonists have been shown to drive differentiation towards osteoblasts rather than adipocytes and several groups are attempting to develop PPAR-γ antagonists to increase bone formation [15–17]. The osteoclast, however, is derived from a hematopoetic cell precursor [18]. The factors that drive these differentiation processes are currently being defined. Interestingly, osteoblast progenitors may also influence the differentiation of monocyte-like cells into osteoclasts, again showing the exquisite interplay that controls bone homeostasis [1•]. A bone-forming cell has also been shown to reside within bone tissue in a relatively quiescent state. These cells are called bone-lining cells [1•]. One of the most studied bone formation agents, parathyroid hormone (PTH), has effects on a number of bone-forming cell populations [19]. PTH drives the differentiation of osteoblast precursors, stimulates mature osteoblasts and activates bone-lining cells. PTH also has clear anabolic activity in vivo in animals and humans. There are several conditions in which osteoclast-mediated bone resorption is accelerated. The most widely known of these is postmenopausal osteoporosis, but accelerated bone resorption also contributes significantly to the pathology of diseases such as RA [5–8], metastatic bone disease [6,10], steroid-induced osteopenia [8,9] and possibly even osteoarthritis (OA). In this review, we will briefly describe the various conditions in which bone resorption is accelerated and the novel approaches being considered for therapeutic intervention in this process.
Diseases involving accelerated bone resorption When a woman reaches menopause there is a significant reduction in systemic estrogen levels, which results in an increase in bone resorption. If this increase in bone resorption is allowed to progress without some control, a woman could eventually lose a significant amount of bone mass. In addition to menopause, several other risk factors have been identified that also appear to influence elevated bone turnover, such as smoking and physical inactivity [20]. Postmenopausal osteopenia and osteoporosis have gained considerable attention over the past decade due to the development of improved imaging and biochemical marker
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technologies. In addition, antiresorptive therapies have been developed and used clinically to control bone loss. These include estrogen, selective estrogen receptor modulators (SERMs), calcitonin and bisphosphonates. Unfortunately, all of these therapies have shortcomings associated with them; therefore, further research investment is required to identify new types of antiresorptive therapies. None the less, these therapies have offered us a means to help control bone resorption and to understand its role in bone turnover and maintenance. RA is a disease where there is significant bone loss [5–9]. It is a systemic autoimmune inflammatory disease that results in significant cartilage and bone remodeling. One of the hallmarks of this disease is bone lesions, which are identified using X-rays and are used to monitor disease progression. Current therapeutic approaches involve the control of inflammation using non-steroidal anti-inflammatory drugs, steroids, cytokine-neutralizing agents and ultimately joint replacement. Unfortunately, some steroids stimulate bone resorption and, although they control inflammation, pain and swelling, they can actually stimulate bone loss. Drug discovery efforts in this area are predominantly focused on the identification of agents that can be dosed orally and have both anti-inflammatory and jointpreserving activities. As bone loss is a major feature of this disease and because matrix degradation products of bone and/or cartilage have been shown to be proinflammatory, inhibition of bone resorption in this disease could be extremely beneficial. One of the key secondary events that occurs in cancer patients, particularly those with breast cancer, is the development of secondary metastases in bone. Recently, it has been shown that the tumor cell begins to interact with the bone and, once partially established, it releases factors that stimulate osteoclast differentiation and activation [21]. The resident osteoclasts begin to actively resorb the bone, providing an environment for continued tumor growth. This can result in significant pain to the patient and can ultimately result in bone fracture. Antiresorptive bisphosphonates have been evaluated in this disease and have been shown to prevent tumor-induced bone loss [22]. OA is a disease that involves a small number of joints at any one time. Articular cartilage is lost at the same time as bone is remodeled. This disease is treated with analgesics, anti-inflammatory agents and joint replacement. It is continually debated whether this disease is initiated by changes in the bone or articular cartilage. There is one view that this is a cartilage disease that results in significant bony changes, such as subchondral bone sclerosis and osteophyte formation; the other opinion is that bone changes initiate the disease, resulting in secondary articular cartilage loss. Because drugs targeted to bone resorption are readily available, these compounds have begun to be profiled in models of osteoarthritic bone turnover and cartilage loss. For example, it has been shown
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that calcitonin prevents cartilage degradation, as assessed using biochemical markers in a dog model of OA [23]. In addition, the bisphosphonate zoledronate (Novartis Pharma AG, Basel, Switzerland) has been shown to prevent cartilage destruction in an animal model [24] and also to reduce the levels of cartilage type II collagen degradation fragments in patients with Paget’s disease of bone [25]. Together, these data suggest that inhibition of bone resorption in OA may prevent cartilage loss in this disease.
Bisphosphonates and estrogens — current therapies used to inhibit bone resorption There are currently two well-accepted antiresorptive therapies on the market — estrogens and bisphosphonates. These compounds have very different mechanisms of action and are used in different patient populations. Estrogens and SERMs are predominantly prescribed to treat menopausal symptoms [26]. Estrogens have been associated with an increased risk of breast and uterine cancer, resulting in the search for an equally effective agent with fewer associated risks. SERMs have some effect on postmenopausal symptom relief, as well as collateral benefits on both cardiovascular and cancer risk. There are ongoing efforts to identify SERMs with improved efficacy and with additional collateral benefits. Despite the fact that these agents are not predominantly prescribed for bone loss, they have the benefit of preventing osteoclast-mediated bone resorption and they are registered for treatment of this indication. The second lead class of compounds that inhibits bone resorption are the bisphosphonates. Currently there are two marketed bisphosphonates, risedronate (Actonel®; Proctor & Gamble, Cincinatti, OH, USA and Aventis Pharma, Collegeville, PA, USA) and alendronate (Fosamax®; Merck and Company, Whitehouse Station, NJ, USA) . In addition, both zoledronate and ibandronate are in late-stage clinical development. These compounds all work through a similar mechanism of action, targeting the osteoclast. Specifically, these compounds interfere with the cholesterol biosynthesis pathway within the osteoclast [27]. In addition, they have been reported to cause osteoclastic apoptosis [28]. These compounds are extremely effective at inhibiting osteoclastic bone resorption in both humans and animals; in clinical trials they reduce fracture risk by 40–50% in osteoporotic patients. Unlike the estrogenic compounds, bisphosphonates are used to treat patients with fairly advanced bone loss. In the extreme case, these patients present clinically only when they fracture. With the advent of accessible bone mass measurement and bone resorption biochemical marker technology, patients at risk of fracture are being identified before the first fracture takes place. These individuals are being treated at a much higher frequency with bisphosphonate therapy. Despite the significant efficacy and widespread use of these agents, they have several issues associated with them. The first is variable tolerability and complicated dosing regimes. Bisphosphonates often cause esophageal irritation [29] and need to be taken in an upright position, with significant food restrictions.
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Figure 1 A diagramatic representation of a resorbing osteoclast showing novel antiresorptive targets. The targets for novel antiresorptive therapies are shown in red boxes.
HCO–3 H2O + CO2 Cell nuclei
CA II HCO–3 Cl–
H+ + HCO–3 Osteoclast ClC-7 chloride channel
Vacuolar ATPase Cl–
H+ Vitronectin receptor
Cathepsin K
Vitronectin receptor
Bone
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Recently, companies such as Merck have reformulated their bisphosphonate (alendronate), to be used in a onceweekly regime. In addition, data have been recently published, suggesting that zoledronate will be efficacious if used once yearly [30]. Lastly, data are emerging suggesting that bisphosphonates have an extremely long residence time in bone. High-dose treatment of animals with bisphosphonates results in an increase in microfractures of the bone and reduced bone strength [31]. It is currently unclear if this will be an issue in patients who have an extended exposure to bisphosphonate treatment.
Bone formation therapies Currently, there is substantial focus on the development of bone formation therapies. One such agent — teriparatide (Forteo; Eli Lilly and Company, Indianapolis, IN, USA), PTH1–34 — is in late stage clinical development (discussed in the review by J Fox, this issue, pp 338–344). This compound has shown substantial reduction of fracture risk in osteoporotic women as a stand-alone therapy. However, it is becoming more widely accepted that these types of bone-formation therapies will probably be used in combination with antiresorptive treatments in the future. Therefore, identification of antiresorptives that can be used in combination with bone-formation agents is also the focus of some drug discovery efforts [19]. The ideal antiresorptive for cyclical use with a bone-formation agent would be one that has rapid onset of action and rapid loss in activity. This will allow for the ‘cleaner’ use of various combination paradigms for the treatment of bone loss. On the basis of this discussion, it is clear that there is a significant opportunity for the development of new bone antiresorptive therapies. A compound with the efficacy of a bisphosphonate, but lacking the tolerability issues associated with it and with a rapid onset of action, would be ideal. As the opportunity exists for the discovery of
improved antiresorptive agents, many industry-wide efforts are currently focused on the development of antiresorptives with a novel mechanism of action.
Novel targets The following section describes some of the potential antiresorptive targets that we consider appropriate for drug intervention (Figure 1). The choice of these targets is supported by the phenotypes expressed in the respective murine knockouts (Table 1). The first two targets, the receptor activator of NF-κB ligand (RANK-L) and osteoprotegerin (OPG) are intimately involved in the control of osteoclastogenesis and the activity of mature osteoclasts (Figure 2) and, at the time of writing, are being considered for the treatment of postmenopausal osteoporosis using protein agents. In contrast, the remaining targets discussed below are being considered for treatments using intervention with small-molecule non-peptide inhibitors. RANK-L
RANK-L is a member of the tumor necrosis factor (TNF) cytokine superfamily [32]. It is expressed by osteoblasts and bone marrow stromal cells, and is upregulated in these cells in response to the catabolic agents PTH, 1,25 dihydroxyvitamin D3, interleukin-1, TNF-α and prostaglandin E2. A major breakthrough in the bone field came with the identification that RANK-L, in the presence of macrophage colony-stimulating factor, is the soluble factor that replaces the requirement for an osteoblastic/stromal cell feeder layer in the in vitro osteoclastogenesis co-culture assay. RANK-L binds to the receptor RANK on hematopoietic precursors and induces their proliferation and differentiation into mature boneresorbing osteoclasts [33]. It has been demonstrated that mice injected with RANK-L develop hypercalcemia with a concomitant increase in osteoclast number. Furthermore, targeted deletion of either RANK-L or RANK in mice
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Table 1 Mouse knockout data supporting novel antiresorptive targets. Null mutation
Phenotype
Human disease
References
RANK-L OPG
Severe osteopetrosis: no osteoclasts Severe osteoporosis: increased numbers of osteoclasts Osteopetrosis: normal number of osteoclasts with impaired function Osteopetrosis: normal number of osteoclasts with impaired function Osteopetrosis: normal number of osteoclasts with impaired function Osteopetrosis: increase in osteoclast number that have impaired function
– –
[34] [39]
Pycnodysostosis
[49,50,51]
Cathepsin K Osteoclast vATPase (atp6i) CLC-7 chloride channel
3 integrin knockout
results in a severe osteopetrotic (bone thickening) phenotype in the knockout animals [34–36], characterized by a profound growth retardation, the absence of osteoclasts and marrow spaces and no tooth eruption. The mice also develop a severe chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes.
Human infantile malignant osteopetrosis [43,45] Human infantile malignant osteopetrosis [48] Glanzmann thrombasthenia
[55]
Osteoprotegerin
OPG is a soluble decoy receptor that inhibits osteoclastogenesis by binding to RANK-L, thereby preventing its interaction with RANK on the surface of hematopoietic precursor cells [38]. This has been demonstrated in vitro, where osteoclast differentiation from precursor cells has been blocked in a dose-dependent manner using recombinant OPG. Although this glycoprotein is expressed in a wide variety of tissues, suggesting a role in extra-osseous tissues, the major phenotype that has been described in OPG–/– mutant mice is severe osteoporosis (bone forming) [39]. As would be expected, a profound osteopetrosis is observed in OPG-overexpressing transgenic mice, which is concomitant with a decrease in osteoclast number. These same effects are observed upon administration of recombinant OPG into normal mice.
A recent study by Oyajobi et al. [37] has shown that RANK/RANK-L is an appropriate target for the inhibition of bone resorption both in vitro and in vivo. They demonstrated that a soluble murine RANK construct conjugated to human immunoglobulin Fc protein (muRANK•Fc) could inhibit human-PTH-related protein-induced resorption in fetal rat long-bone cultures. Also, muRANK•Fc, when administered to normal growing mice, resulted in a complete disappearance of osteoclasts from the metaphyses of long bones that was associated with a significant increase in calcified trabeculae. Finally, in a model of humoral hypercalcemia of malignancy, daily administration of muRANK•Fc from the time of tumor implantation profoundly inhibited osteoclastic bone resorption and prevented hypercalcemia. The authors suggest that these data highlight the utility of disrupting RANK-L/RANK signaling as a novel therapeutic approach for the treatment of diseases characterized by bone loss, such as humoral hypercalcemia and skeletal metastases associated with osteolysis.
There is significant evidence to suggest that OPG may be useful for the treatment of diseases associated with increased osteoclast activity. For example, it has been demonstrated that OPG blocks ovariectomy-associated bone loss in rats. It induces hypocalcemia effects in nude mice bearing tumors that are associated with humoral hypercalcemia of malignancy. Recent studies have also shown that OPG suppresses the profound bone loss associated with the adjuvant arthritis rat model [40]. Most exciting, however, are
Figure 2 The role of RANK-L and OPG in bone-resorbing osteoclast development. The RANK-L on the surface of the osteoblasts binds the RANK receptor on mononuclear osteoclast precursors to drive osteoclast differentiation. This interaction can be blocked by the soluble decoy receptor OPG. 1,25-D3, 1,25 dihydroxyvitamin D3; IL, interleukin.
Proliferation/differentiation survival
Inhibition of osteoclastogenesis
Upregulation of RANKL PTH,1,25-D3, PGE2, IL-1,TNFα, IL-6 Key:
RANK ligand
Upregulation of OPG PGE2,1,25-D3
RANK receptor
M-CSF
c-fms
OPG
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the data from a clinical study in which a single subcutaneous administration of OPG was dosed to postmenopausal women. This resulted in a rapid and dose-dependent reduction in bone turnover, as assessed by urinary NTx measurement. Furthermore, in the high-dose group (3mg/kg), an 80% reduction in NTx levels was maintained after 5 days and remained below baseline for up to 4 weeks [41•]. Taken together, these data strongly suggest that treatment with OPG will be efficacious in the treatment of a variety of disorders that are characterized by bone loss. However, although subcutaneous administration may be tolerated by patients with RA and bone metastases, it still remains to be seen whether this route of administration will be tolerated by patients with osteoporosis. Osteoclast vATPase
Osteoclast-mediated bone resorption requires the secretion of protons both for the dissolution of the mineral phase of bone and for the provision of the appropriate acidic environment for the digestion of the protein matrix by osteoclast-secreted proteolytic enzymes. This process is mediated by a vacuolar-type H+-ATPase (vATPase) that is present in the ruffled membrane of the resorbing osteoclast. One could speculate, therefore, that selective inhibition of this pump would prevent osteoclast-mediated bone resorption. However, this target presents formidable challenges as the vATPases are complex multicomponent enzymes that are ubiquitously distributed in different cell types. Despite this, the utility of this target was demonstrated in in vitro studies, in which osteoclast-mediated resorption was inhibited by using antisense constructs to two vATPase subunits [42]. More recently, Li et al. [43] described a knockout mouse (the atp6i mouse), in which targeted mutation of the 116 kDa (a3) osteoclast proton pump subunit resulted in severe osteopetrosis. The mice had increased bone density, decreased bone marrow space, no tooth eruption and blindness caused by occlusion of the optical foramina. The mice contained normal numbers of osteoclasts in vivo that had an impaired capacity to resorb dentine in vitro. Importantly, all other organs were reported to be normal and there was a normal acid/base balance in blood and urine. In addition to the mouse studies, two groups [44,45] have also demonstrated that a severe form of osteopetrosis is the result of a mutation(s) in the human 116 kDa proton pump subunit. With a phenotype similar to that of the mutant mice, these individuals have abnormally dense bones, normal or elevated numbers of osteoclasts and a blindness that is secondary to the osteopetrotic process. The macrolide antibiotic bafilomycin A1 is a very potent and specific inhibitor of vATPases and has been shown to inhibit bone resorption in vitro and in vivo. Unfortunately, as it inhibits all vATPases it is highly toxic. However, Gagliardi et al. [46] performed a series of chemical modifications to bafilomycin A1 that indicated that it was possible to obtain selective modulation of different
vATPases. This optimization of the specificity of osteoclast vATPase-selective inhibitors resulted in the discovery of SB-242784, which is a potent inhibitor of resorption in vitro [47]. Importantly, oral administration of this compound led to selective inhibition of the bone loss that is associated with ovariectomy in the rat, but had no effect on urinary acid excretion. The murine knockout data, the human gene mutation data and, ultimately, the small molecule inhibitor data define the osteoclast vATPase as an excellent target for antiresorptive therapy. CLC-7
In the resorbing osteoclast, the CLC-7 chloride channel appears to work in concert with the vATPase to maintain the resorptive process. It functions by short-circuiting the membrane potential that is caused by the electrogenic ATPase and appears to be rate-limiting for vATPase activity in osteoclasts. Recently, Kornak et al. [48•] described both a murine knockout of CLC-7 and an example of a human mutation in the CLC-7 gene. Both developed a very severe osteopetrosis, which suggests that CLC-7 potentially provides another excellent novel target for the inhibition of resorption. Cathepsin K
Cathepsin K is a lysosomal cysteine protease that has the unique ability, together with other cathepsins, to cleave both the helical and telopeptide regions of type I collagen — the most prevalent collagen in bone. Data also suggest that cathepsin K can cleave type II collagen. Initial studies at the mRNA and protein levels suggested that cathepsin K is abundantly and selectively expressed by osteoclasts in bone. Tissue localization studies suggested that it plays an important role in bone resorption and this hypothesis has subsequently been borne out by murine knockout data and the description of a human disorder in which the cathepsin K gene is mutated. The knockout of cathepsin K in mice results in osteopetrosis that is characterized by osteosclerosis [49,50]. There is an increase in trabecular number and in trabecular and cortical thickening in the knockout animals when compared to their wild-type littermates. The mutation also leads to the elimination of all osteoclast-related cathepsin activity and, ultimately, to a reduction in osteoclast-mediated matrix degradation. Pycnodysostosis is the human disease that occurs as a result of mutations in the cathepsin K gene [51]. In this disorder there is an elimination of cathepsin K activity that leads to a reduction in the rate of bone turnover and, ultimately, to poor quality dense bone that is predisposed to fracture. Significant data obtained from antisense DNA and synthetic inhibitor studies have been generated that confirm that cathepsin K is a viable target for the treatment of high bone turnover diseases that are characterized by excessive bone loss. For example, Votta et al. [52] demonstrated
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that peptide aldehyde inhibitors of cathepsin K could significantly inhibit the aggressive bone loss that occurs in the rat adjuvant-induced arthritis model. The preferred animal models for evaluation of antiresorptive activity are in the rat, which has caused problems with compound evaluation for the treatment of human disease. Although rat and human enzymes are highly homologous, they do differ in key residues in the active site, and the development of compounds that inhibit the rat enzyme has proven difficult. Lark et al. [53] described a cathepsin K inhibitor, SB-331750, that was sufficiently active against the rat enzyme to be tested in the ovariectomized rat model of bone loss. This study showed that the inhibitor could prevent bone resorption in vivo and that the inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure. Stroup et al. [54•] recently described a non-human primate model of human postmenopausal osteoporosis, in which the active cathepsin K is identical to the human ortholog and, therefore, can be used to evaluate inhibitors of the human enzyme. In this model, a gonadotropin-releasing hormone agonist is used to render the cynomolgus monkey estrogen deficient, which leads to increased bone turnover. Using this model, Stroup et al. [54•] were able to evaluate a potent and selective cathepsin K inhibitor and show that it could significantly reduce the levels of serum markers of bone resorption (carboxy- and amino-telopeptides) relative to untreated controls. Cathepsin K inhibition resulted in an extremely rapid decrease in bone resorption and when the compound was eliminated in vivo, bone resorption resumed normally, suggesting that this compound could be used in situations where rapid onset or return of resorption may be required.
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number of osteoclasts that formed in vitro from macrophages derived from the knockout mouse. Also, they showed that the closely related integrin, αvβ5 did not substitute for αvβ3 during cytokine stimulation or authentic osteoclastogenesis. However, αvβ3 knockout mice did develop osteosclerosis with age. Interestingly, there was a 3.5-fold increase in osteoclasts in the mutant mice compared to their heterozygote littermates. However, these mutant osteoclasts were unable to excavate pits in whale dentine in in vitro cultures. In addition, significant hypocalcemia was evident in the knockout mice. The authors surmise that the resorptive defect in β3 -deficient osteoclasts may reflect an absence of matrix-derived intracellular signals, as their cytoskeleton is distinctly abnormal and they fail to spread in vitro to form actin rings ex vivo, or to form normal ruffled membranes in vivo. The authors concluded that, although the αvβ3 integrin is not required for osteoclastogenesis, it does appear to be essential for normal osteoclast function. These αvβ3 knockout data are supported by studies using small-molecule inhibitors of this integrin that show efficacy in the ovariectomized rat model of osteoporosis and in the rat adjuvant arthritis model. Lark et al. [56] described a non-peptide RGD mimetic αvβ3 antagonist that inhibited osteoclast resorption and prevented net bone loss in vitro, and inhibited cancellous bone turnover when dosed orally in the ovariectomized rat model. They concluded that, mechanistically, the orally active αvβ3 inhibitor prevented bone loss in vivo by inhibiting osteoclast-mediated bone resorption, ultimately preventing cancellous bone turnover.
These data show unequivocally that inhibition of cathepsin K results in the inhibition of bone resorption both in vitro and in vivo, and that the inhibition of this protease may be a viable approach to the treatment of diseases characterized by excessive bone loss.
In a rat adjuvant arthritis model that is characterized by inflammation and excessive bone loss, Badger et al. [57] were able to demonstrate that symptoms of the adjuvant arthritis were significantly reduced by either prophylactic or therapeutic treatment with the αvβ3 antagonist, SB-273005. Measurements of paw inflammation and of bone, cartilage and soft-tissue structure indicated that this compound exerts a protective effect on joint integrity and thus appears to have disease-modifying properties.
Vitronectin receptor
Conclusions and future prospects
The integrins are a family of heterodimeric receptors that consist of an alpha and a beta chain. Osteoclast adhesion to bone is mediated, at least in part, by the αvβ3 integrin, which binds to the Arg-Gly-Asp trimer (RGD in the singleletter code for amino acids) that is present in some of the bone matrix proteins. Data suggest that the osteoclast forms a tight sealing zone via the αvβ3–RGD interaction that is responsible for the maintenance of the resorptive process. Therefore, disruption of this interaction could provide a potential therapeutic target in diseases marked by excessive bone loss.
The identification and development of novel bone antiresorptive agents is being actively pursued in the pharmaceutical and biotechnological industries as well as in academia, which has resulted in the rapid advancement of the bone biology field. As suggested in this review, there is still significant opportunity for the development of novel bone antiresorptive therapies. With the introduction of bone-forming PTH, it is likely that, in the future, an antiresorptive drug will be used in combination with PTH. As clinical experience is gained with combination therapy, antiresorptives against novel targets may prove to be either more or less beneficial in various therapeutic settings. In addition, the broader therapeutic application is also being investigated for antiresorptive agents. For example, preclinical and clinical data are emerging that suggest that these
To address this, McHugh et al. [55] engineered mice in which the gene for the β3 integrin subunit was deleted. Their data showed that the mutation did not decrease the
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compounds may be useful in the treatment of diseases such as periodontal disease, metastatic bone disease, RA and OA. As clinical experience is gained with antiresorptive therapies, it is likely that broader utility will be seen.
17.
Acknowledgements We would like to thank Simon Blake for his careful reading of this review.
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31. Mashiba T, Turner CH, Hirano T, Forwood MR, Jacob DS, Johnston CC, Burr DB: Effects of high-dose etidronate treatment on microdamage accumulation and biomechanical properties in beagle bone before occurrence of spontaneous fractures. Bone 2001, 29:271-278.
12. Ducy P: Bonekey: and then they were two…progressing along the osteoblast differentiation pathway. URL: http://www.bonekeyibms.org/cgi/content/full/ibmske
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13. Minguell JJ, Erices A, Conget P: Mesenchymal stem cells. Exper Biol Med 2001, 226:507-520.
33. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S et al.: Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998, 93:165-176.
14. Justesen J, Stenderup K, Ebbesen EN, Mosekilde L, Steiniche T, Kassem M: Adipocyte tissue volume in bone marrow is increased with aging and in patients with osteoporosis. Biogerontology 2001, 2:165-171. 15. Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR, Pittenger MF: Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogenactivated protein kinase. J Biol Chem 2000, 275:9645-9652. 16. Gori F, Thomas T, Hicok KC, Spelsberg TC, Riggs BL: Differentiation of human marrow stromal precursor cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation. J Bone Min Res 1999, 14:1522-1535.
34. Kim N, Odgren PR, Kim DK, Marks SC, Choi YW: Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene. Proc Nat Acad Sci USA 2000, 97:10905-10910. 35. Dougall WC, Glaccum M, Charrier K, Rohrbach K, Brasel K, De Smedt T, Daro E, Smith J, Tometsko ME, Maliszewski CR et al.: RANK is essential for osteoclast and lymph node development. Genes Develop 1999, 13:2412-2424. 36. Li J, Sarosi I, Yan XQ, Morony S, Capparelli C, Tan HL, McCabe S, Elliott R, Scully S, Van G et al.: RANK is the intrinsic hematopoietic
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cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Nat Acad Sci USA 2000, 97:1566-1571. 37.
Oyajobi BO, Anderson DM, Traianedes K, Williams PJ, Yoneda T, Mundy GR: Therapeutic efficacy of a soluble receptor activator of nuclear factor kappa B-IgG Fc fusion protein in suppressing bone resorption and hypercalcemia in a model of humoral hypercalcemia of malignancy. Cancer Res 2001, 61:2572-2578.
38. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone et al.: Osteoprotegerin a novel secreted protein involved in the regulation of bone density. Cell 1997, 89:309-319. 39. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu WL, Lacey DL et al.: Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 1998, 12:1260-1268. 40. Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, Capparelli C, Li J, Elliott R, McCabe S et al.: Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999, 402:304-309. 41. Bekker PJ, Holloway D, Nakanishi A, Arrighi M, Leese PT, • Dunstan CR: The effect of a single dose of osteoprotegerin in postmenopausal women. J Bone Min Res 2001, 16:348-360. The authors describe a randomized, double-blind, placebo-controlled, sequential dose escalation study in postmenopausal women and show that a single subcutaneous injection of OPG is effective in rapidly and profoundly reducing bone turnover for a sustained period. 42. Laitala T, Vaananen HK: Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H+-ATPase. J Clin Invest 1994, 93:2311-2318. 43. Li YP, Chen W, Liang YQ, Li E, Stashenko P: Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification. Nature Gen 1999, 23:447-451. 44. Frattini A, Orchard PJ, Sobacchi C, Giliani S, Abinun M, Mattsson JP, Keeling DJ, Andersson AK, Wallbrandt P, Zecca L et al.: Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nature Genet 2000, 25:343-346. 45. Kornak U, Schulz A, Friedrich W, Uhlhaas S, Kremens B, Voit T, Hasan C, Bode U, Jentsch TJ, Kubisch C: Mutations in the a3 subunit of the vacuolar H+-ATPase cause infantile malignant osteopetrosis. Hum Mol Gen 2000, 9:2059-2063. 46. Gagliardi S, Nadler G, Consolandi E, Parini C, Morvan M, Legave MN, Belfiore P, Zocchetti A, Clarke GD, James I et al.: 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienamides novel and selective inhibitors of the vacuolar H+-ATPase of osteoclasts with bone antiresorptive activity. J Med Chem 1998, 41:1568-1573. 47.
Visentin L, Dodds RA, Valente M, Misiano P, Bradbeer JN, Oneta S, Liang XG, Gowen M, Farina C: A selective inhibitor of the
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osteoclastic V-H+-ATPase prevents bone loss in both thyroparathyroidectomized and ovariectomized rats. J Clin Invest 2000, 106:309-318. 48. Kornak U, Kasper D, Bosl MR, Kaiser E, Schweizer M, Schulz A, • Friedrich W, Delling G, Jentsch TJ: Loss of the CIC-7 chloride channel leads to osteopetrosis in mice and man. Cell 2001, 104:205-215. The authors show that mice deficient for the ubiquitously expressed ClC-7 chloride channel are osteopetrotic. They also identify CLCN7 mutations in a patient with human infantile malignant osteopetrosis. 49. Saftig P, Hunziker E, Wehmeyer O, Jones S, Boyde A, Rommerskirch W, Moritz JD, Schu P, Vonfigura K: Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsink-deficient mice. Proc Nat Acad Sci USA 1998, 95:13453-13458. 50. Gowen M, Lazner F, Dodds R, Kapadia R, Feild J, Tavaria M, Bertoncello I, Drake F, Zavarselk S, Tellis I et al.: Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralization. J Bone Miner Res 1999, 14:1654-1663. 51. Gelb BD, Shi GP, Chapman HA, Desnick RJ: Pycnodysostosis, a lysosomal disease caused by cathepsin k deficiency. Science 1996, 273:1236-1238. 52. Votta BJ, Levy MA, Badger A, Bradbeer J, Dodds RA, James IE, Thompson S, Bossard MJ, Carr T, Connor JR et al.: Peptide aldehyde inhibitors of cathepsin k inhibit bone resorption both in vitro and in vivo. J Bone Miner Res 1997, 12:1396-1406. 53. Lark MW, Stroup GB, James IE, Dodds RA, Hwang SM, Blake SM, Lechowska BA, Hoffman SJ, Smith BR, Kapadia R et al.: Inhibition of cathepsin K with the small molecule, non-peptide inhibitor, SB 331750, prevents bone matrix resorption in the ovariectomized rat. Bone 2002, in press. 54. Stroup GB, Lark MW, Veber DF, Bhattacharyya A, Blake S, Dare LC, • Erhard KF, Hoffman SJ, James IE, Marquis RW et al.: Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption in vivo in a nonhuman primate. J Bone Miner Res 2001, 16:1739-1746. The authors describe the effects of a small molecule inhibitor of cathepsin K on biochemical markers of resorption. They show an acute and sustained inhibition of resorption when the compound is administered to chemically ovariectomized cynomolgus monkeys. 55. McHugh KP, Hodivala-Dilke K, Zheng MH, Namba N, Lam J, Novack D, Feng X, Ross FP, Hynes RO, Teitelbaum SL: Mice lacking beta 3 integrins are osteosclerotic because of dysfunctional osteoclasts. J Clin Invest 2000, 105:433-440. 56. Lark MW, Stroup GB, Dodds RA, Kapadia R, Hoffman SJ, Hwang SM, James IE, Lechowska B Liang XG, Rieman DJ et al.: Antagonism of the osteoclast vitronectin receptor with an orally active nonpeptide inhibitor prevents cancellous bone loss in the ovariectomized rat. J Bone Miner Res 2001, 16:319-327. 57.
Badger AM, Blake S, Kapadia R, Sarkar S, Levin J, Swift BA, Hoffman SJ, Stroup GB, Miller WH, Gowen M, Lark MW: Diseasemodifying activity of SB 273005, an orally active, nonpeptide alpha(v)beta(3) (vitronectin receptor) antagonist, in rat adjuvantinduced arthritis. Arth Rheum 2001, 44:128-137.
Novel bone antiresorptive approaches Michael W Lark* and Ian E James Inhibition of bone resorption is a mechanism that has been clinically validated as a means to control bone loss in diseases such as postmenopausal osteoporosis. The development of marketable drugs in this area has resulted in significant clinical benefits; however, improvements can still be made. Several novel antiresorptive mechanisms are currently under consideration in the pharmaceutical industry, which will hopefully result in the development of improved bone antiresorptive therapies. Addresses Department of Musculoskeletal Diseases Biology, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA *e-mail: [email protected] Current Opinion in Pharmacology 2002, 2:330–337 1471-4892/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations BMU basic multicellular unit OA osteoarthritis OPG osteoprotegerin PPAR-γγ peroxisome proliferator activated receptor γ PTH parathyroid hormone RA rheumatoid arthritis RANK receptor activator of NF-κB RANK-L RANK ligand SERM selective estrogen receptor modulator vATPase vacuolar type H+-ATPase
Introduction Bone turnover is a complex, multicellular process involving cell recruitment and differentiation, and the activity of highly specialized cells. The mature cells that control bone turnover are the bone-resorbing osteoclasts and the bone-forming osteoblasts. The balance of osteoblastmediated bone formation and osteoclast-mediated bone resorption is what ultimately controls skeletal integrity. Despite the fact that these cells have specialized and opposing activities, it has become generally accepted that they work in concert to maintain normal bone balance. One of the most accepted models of bone turnover is that of the basic multicellular unit (BMU) [1•]. In the BMU model, bone-resorbing osteoclasts remove mineralized bone matrix, which is followed by new bone deposition by bone-forming osteoblasts. During development, the balance of the two processes is in favor of bone formation; whereas, in estrogen-depleted postmenopausal osteoporosis, the balance favors bone resorption. In both of these situations, bone formation and resorption (bone turnover) occurs, but the balance favors one process above the other. These processes are also modulated in several other settings such as fracture repair [2] and in other diseases, such as periodontal disease [3,4], rheumatoid arthritis (RA) [5–9], steroid-induced bone loss [8,9] and bone loss due to metastasis targeted to bone [6,10].
Mature osteoblasts and osteoclasts are derived from different progenitor populations. The osteoblast is derived from a mesenchymal cell precursor, which has also been shown to differentiate into adipocytes and chondrocytes [11–13]. With aging and/or menopause, there is an increase in bone marrow adipocytes [14]. One approach currently under consideration is to drive bone formation in the aging or menopausal population by influencing the osteoblast/adipocyte balance to one of increased numbers of osteoblasts. There are a large number of studies suggesting that the nuclear peroxisome proliferator activated receptor γ (PPAR-γ) may be key to the maintenance of this balance. PPAR-γ antagonists have been shown to drive differentiation towards osteoblasts rather than adipocytes and several groups are attempting to develop PPAR-γ antagonists to increase bone formation [15–17]. The osteoclast, however, is derived from a hematopoetic cell precursor [18]. The factors that drive these differentiation processes are currently being defined. Interestingly, osteoblast progenitors may also influence the differentiation of monocyte-like cells into osteoclasts, again showing the exquisite interplay that controls bone homeostasis [1•]. A bone-forming cell has also been shown to reside within bone tissue in a relatively quiescent state. These cells are called bone-lining cells [1•]. One of the most studied bone formation agents, parathyroid hormone (PTH), has effects on a number of bone-forming cell populations [19]. PTH drives the differentiation of osteoblast precursors, stimulates mature osteoblasts and activates bone-lining cells. PTH also has clear anabolic activity in vivo in animals and humans. There are several conditions in which osteoclast-mediated bone resorption is accelerated. The most widely known of these is postmenopausal osteoporosis, but accelerated bone resorption also contributes significantly to the pathology of diseases such as RA [5–8], metastatic bone disease [6,10], steroid-induced osteopenia [8,9] and possibly even osteoarthritis (OA). In this review, we will briefly describe the various conditions in which bone resorption is accelerated and the novel approaches being considered for therapeutic intervention in this process.
Diseases involving accelerated bone resorption When a woman reaches menopause there is a significant reduction in systemic estrogen levels, which results in an increase in bone resorption. If this increase in bone resorption is allowed to progress without some control, a woman could eventually lose a significant amount of bone mass. In addition to menopause, several other risk factors have been identified that also appear to influence elevated bone turnover, such as smoking and physical inactivity [20]. Postmenopausal osteopenia and osteoporosis have gained considerable attention over the past decade due to the development of improved imaging and biochemical marker
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technologies. In addition, antiresorptive therapies have been developed and used clinically to control bone loss. These include estrogen, selective estrogen receptor modulators (SERMs), calcitonin and bisphosphonates. Unfortunately, all of these therapies have shortcomings associated with them; therefore, further research investment is required to identify new types of antiresorptive therapies. None the less, these therapies have offered us a means to help control bone resorption and to understand its role in bone turnover and maintenance. RA is a disease where there is significant bone loss [5–9]. It is a systemic autoimmune inflammatory disease that results in significant cartilage and bone remodeling. One of the hallmarks of this disease is bone lesions, which are identified using X-rays and are used to monitor disease progression. Current therapeutic approaches involve the control of inflammation using non-steroidal anti-inflammatory drugs, steroids, cytokine-neutralizing agents and ultimately joint replacement. Unfortunately, some steroids stimulate bone resorption and, although they control inflammation, pain and swelling, they can actually stimulate bone loss. Drug discovery efforts in this area are predominantly focused on the identification of agents that can be dosed orally and have both anti-inflammatory and jointpreserving activities. As bone loss is a major feature of this disease and because matrix degradation products of bone and/or cartilage have been shown to be proinflammatory, inhibition of bone resorption in this disease could be extremely beneficial. One of the key secondary events that occurs in cancer patients, particularly those with breast cancer, is the development of secondary metastases in bone. Recently, it has been shown that the tumor cell begins to interact with the bone and, once partially established, it releases factors that stimulate osteoclast differentiation and activation [21]. The resident osteoclasts begin to actively resorb the bone, providing an environment for continued tumor growth. This can result in significant pain to the patient and can ultimately result in bone fracture. Antiresorptive bisphosphonates have been evaluated in this disease and have been shown to prevent tumor-induced bone loss [22]. OA is a disease that involves a small number of joints at any one time. Articular cartilage is lost at the same time as bone is remodeled. This disease is treated with analgesics, anti-inflammatory agents and joint replacement. It is continually debated whether this disease is initiated by changes in the bone or articular cartilage. There is one view that this is a cartilage disease that results in significant bony changes, such as subchondral bone sclerosis and osteophyte formation; the other opinion is that bone changes initiate the disease, resulting in secondary articular cartilage loss. Because drugs targeted to bone resorption are readily available, these compounds have begun to be profiled in models of osteoarthritic bone turnover and cartilage loss. For example, it has been shown
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that calcitonin prevents cartilage degradation, as assessed using biochemical markers in a dog model of OA [23]. In addition, the bisphosphonate zoledronate (Novartis Pharma AG, Basel, Switzerland) has been shown to prevent cartilage destruction in an animal model [24] and also to reduce the levels of cartilage type II collagen degradation fragments in patients with Paget’s disease of bone [25]. Together, these data suggest that inhibition of bone resorption in OA may prevent cartilage loss in this disease.
Bisphosphonates and estrogens — current therapies used to inhibit bone resorption There are currently two well-accepted antiresorptive therapies on the market — estrogens and bisphosphonates. These compounds have very different mechanisms of action and are used in different patient populations. Estrogens and SERMs are predominantly prescribed to treat menopausal symptoms [26]. Estrogens have been associated with an increased risk of breast and uterine cancer, resulting in the search for an equally effective agent with fewer associated risks. SERMs have some effect on postmenopausal symptom relief, as well as collateral benefits on both cardiovascular and cancer risk. There are ongoing efforts to identify SERMs with improved efficacy and with additional collateral benefits. Despite the fact that these agents are not predominantly prescribed for bone loss, they have the benefit of preventing osteoclast-mediated bone resorption and they are registered for treatment of this indication. The second lead class of compounds that inhibits bone resorption are the bisphosphonates. Currently there are two marketed bisphosphonates, risedronate (Actonel®; Proctor & Gamble, Cincinatti, OH, USA and Aventis Pharma, Collegeville, PA, USA) and alendronate (Fosamax®; Merck and Company, Whitehouse Station, NJ, USA) . In addition, both zoledronate and ibandronate are in late-stage clinical development. These compounds all work through a similar mechanism of action, targeting the osteoclast. Specifically, these compounds interfere with the cholesterol biosynthesis pathway within the osteoclast [27]. In addition, they have been reported to cause osteoclastic apoptosis [28]. These compounds are extremely effective at inhibiting osteoclastic bone resorption in both humans and animals; in clinical trials they reduce fracture risk by 40–50% in osteoporotic patients. Unlike the estrogenic compounds, bisphosphonates are used to treat patients with fairly advanced bone loss. In the extreme case, these patients present clinically only when they fracture. With the advent of accessible bone mass measurement and bone resorption biochemical marker technology, patients at risk of fracture are being identified before the first fracture takes place. These individuals are being treated at a much higher frequency with bisphosphonate therapy. Despite the significant efficacy and widespread use of these agents, they have several issues associated with them. The first is variable tolerability and complicated dosing regimes. Bisphosphonates often cause esophageal irritation [29] and need to be taken in an upright position, with significant food restrictions.
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Figure 1 A diagramatic representation of a resorbing osteoclast showing novel antiresorptive targets. The targets for novel antiresorptive therapies are shown in red boxes.
HCO–3 H2O + CO2 Cell nuclei
CA II HCO–3 Cl–
H+ + HCO–3 Osteoclast ClC-7 chloride channel
Vacuolar ATPase Cl–
H+ Vitronectin receptor
Cathepsin K
Vitronectin receptor
Bone
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Recently, companies such as Merck have reformulated their bisphosphonate (alendronate), to be used in a onceweekly regime. In addition, data have been recently published, suggesting that zoledronate will be efficacious if used once yearly [30]. Lastly, data are emerging suggesting that bisphosphonates have an extremely long residence time in bone. High-dose treatment of animals with bisphosphonates results in an increase in microfractures of the bone and reduced bone strength [31]. It is currently unclear if this will be an issue in patients who have an extended exposure to bisphosphonate treatment.
Bone formation therapies Currently, there is substantial focus on the development of bone formation therapies. One such agent — teriparatide (Forteo; Eli Lilly and Company, Indianapolis, IN, USA), PTH1–34 — is in late stage clinical development (discussed in the review by J Fox, this issue, pp 338–344). This compound has shown substantial reduction of fracture risk in osteoporotic women as a stand-alone therapy. However, it is becoming more widely accepted that these types of bone-formation therapies will probably be used in combination with antiresorptive treatments in the future. Therefore, identification of antiresorptives that can be used in combination with bone-formation agents is also the focus of some drug discovery efforts [19]. The ideal antiresorptive for cyclical use with a bone-formation agent would be one that has rapid onset of action and rapid loss in activity. This will allow for the ‘cleaner’ use of various combination paradigms for the treatment of bone loss. On the basis of this discussion, it is clear that there is a significant opportunity for the development of new bone antiresorptive therapies. A compound with the efficacy of a bisphosphonate, but lacking the tolerability issues associated with it and with a rapid onset of action, would be ideal. As the opportunity exists for the discovery of
improved antiresorptive agents, many industry-wide efforts are currently focused on the development of antiresorptives with a novel mechanism of action.
Novel targets The following section describes some of the potential antiresorptive targets that we consider appropriate for drug intervention (Figure 1). The choice of these targets is supported by the phenotypes expressed in the respective murine knockouts (Table 1). The first two targets, the receptor activator of NF-κB ligand (RANK-L) and osteoprotegerin (OPG) are intimately involved in the control of osteoclastogenesis and the activity of mature osteoclasts (Figure 2) and, at the time of writing, are being considered for the treatment of postmenopausal osteoporosis using protein agents. In contrast, the remaining targets discussed below are being considered for treatments using intervention with small-molecule non-peptide inhibitors. RANK-L
RANK-L is a member of the tumor necrosis factor (TNF) cytokine superfamily [32]. It is expressed by osteoblasts and bone marrow stromal cells, and is upregulated in these cells in response to the catabolic agents PTH, 1,25 dihydroxyvitamin D3, interleukin-1, TNF-α and prostaglandin E2. A major breakthrough in the bone field came with the identification that RANK-L, in the presence of macrophage colony-stimulating factor, is the soluble factor that replaces the requirement for an osteoblastic/stromal cell feeder layer in the in vitro osteoclastogenesis co-culture assay. RANK-L binds to the receptor RANK on hematopoietic precursors and induces their proliferation and differentiation into mature boneresorbing osteoclasts [33]. It has been demonstrated that mice injected with RANK-L develop hypercalcemia with a concomitant increase in osteoclast number. Furthermore, targeted deletion of either RANK-L or RANK in mice
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Table 1 Mouse knockout data supporting novel antiresorptive targets. Null mutation
Phenotype
Human disease
References
RANK-L OPG
Severe osteopetrosis: no osteoclasts Severe osteoporosis: increased numbers of osteoclasts Osteopetrosis: normal number of osteoclasts with impaired function Osteopetrosis: normal number of osteoclasts with impaired function Osteopetrosis: normal number of osteoclasts with impaired function Osteopetrosis: increase in osteoclast number that have impaired function
– –
[34] [39]
Pycnodysostosis
[49,50,51]
Cathepsin K Osteoclast vATPase (atp6i) CLC-7 chloride channel
3 integrin knockout
results in a severe osteopetrotic (bone thickening) phenotype in the knockout animals [34–36], characterized by a profound growth retardation, the absence of osteoclasts and marrow spaces and no tooth eruption. The mice also develop a severe chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes.
Human infantile malignant osteopetrosis [43,45] Human infantile malignant osteopetrosis [48] Glanzmann thrombasthenia
[55]
Osteoprotegerin
OPG is a soluble decoy receptor that inhibits osteoclastogenesis by binding to RANK-L, thereby preventing its interaction with RANK on the surface of hematopoietic precursor cells [38]. This has been demonstrated in vitro, where osteoclast differentiation from precursor cells has been blocked in a dose-dependent manner using recombinant OPG. Although this glycoprotein is expressed in a wide variety of tissues, suggesting a role in extra-osseous tissues, the major phenotype that has been described in OPG–/– mutant mice is severe osteoporosis (bone forming) [39]. As would be expected, a profound osteopetrosis is observed in OPG-overexpressing transgenic mice, which is concomitant with a decrease in osteoclast number. These same effects are observed upon administration of recombinant OPG into normal mice.
A recent study by Oyajobi et al. [37] has shown that RANK/RANK-L is an appropriate target for the inhibition of bone resorption both in vitro and in vivo. They demonstrated that a soluble murine RANK construct conjugated to human immunoglobulin Fc protein (muRANK•Fc) could inhibit human-PTH-related protein-induced resorption in fetal rat long-bone cultures. Also, muRANK•Fc, when administered to normal growing mice, resulted in a complete disappearance of osteoclasts from the metaphyses of long bones that was associated with a significant increase in calcified trabeculae. Finally, in a model of humoral hypercalcemia of malignancy, daily administration of muRANK•Fc from the time of tumor implantation profoundly inhibited osteoclastic bone resorption and prevented hypercalcemia. The authors suggest that these data highlight the utility of disrupting RANK-L/RANK signaling as a novel therapeutic approach for the treatment of diseases characterized by bone loss, such as humoral hypercalcemia and skeletal metastases associated with osteolysis.
There is significant evidence to suggest that OPG may be useful for the treatment of diseases associated with increased osteoclast activity. For example, it has been demonstrated that OPG blocks ovariectomy-associated bone loss in rats. It induces hypocalcemia effects in nude mice bearing tumors that are associated with humoral hypercalcemia of malignancy. Recent studies have also shown that OPG suppresses the profound bone loss associated with the adjuvant arthritis rat model [40]. Most exciting, however, are
Figure 2 The role of RANK-L and OPG in bone-resorbing osteoclast development. The RANK-L on the surface of the osteoblasts binds the RANK receptor on mononuclear osteoclast precursors to drive osteoclast differentiation. This interaction can be blocked by the soluble decoy receptor OPG. 1,25-D3, 1,25 dihydroxyvitamin D3; IL, interleukin.
Proliferation/differentiation survival
Inhibition of osteoclastogenesis
Upregulation of RANKL PTH,1,25-D3, PGE2, IL-1,TNFα, IL-6 Key:
RANK ligand
Upregulation of OPG PGE2,1,25-D3
RANK receptor
M-CSF
c-fms
OPG
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the data from a clinical study in which a single subcutaneous administration of OPG was dosed to postmenopausal women. This resulted in a rapid and dose-dependent reduction in bone turnover, as assessed by urinary NTx measurement. Furthermore, in the high-dose group (3mg/kg), an 80% reduction in NTx levels was maintained after 5 days and remained below baseline for up to 4 weeks [41•]. Taken together, these data strongly suggest that treatment with OPG will be efficacious in the treatment of a variety of disorders that are characterized by bone loss. However, although subcutaneous administration may be tolerated by patients with RA and bone metastases, it still remains to be seen whether this route of administration will be tolerated by patients with osteoporosis. Osteoclast vATPase
Osteoclast-mediated bone resorption requires the secretion of protons both for the dissolution of the mineral phase of bone and for the provision of the appropriate acidic environment for the digestion of the protein matrix by osteoclast-secreted proteolytic enzymes. This process is mediated by a vacuolar-type H+-ATPase (vATPase) that is present in the ruffled membrane of the resorbing osteoclast. One could speculate, therefore, that selective inhibition of this pump would prevent osteoclast-mediated bone resorption. However, this target presents formidable challenges as the vATPases are complex multicomponent enzymes that are ubiquitously distributed in different cell types. Despite this, the utility of this target was demonstrated in in vitro studies, in which osteoclast-mediated resorption was inhibited by using antisense constructs to two vATPase subunits [42]. More recently, Li et al. [43] described a knockout mouse (the atp6i mouse), in which targeted mutation of the 116 kDa (a3) osteoclast proton pump subunit resulted in severe osteopetrosis. The mice had increased bone density, decreased bone marrow space, no tooth eruption and blindness caused by occlusion of the optical foramina. The mice contained normal numbers of osteoclasts in vivo that had an impaired capacity to resorb dentine in vitro. Importantly, all other organs were reported to be normal and there was a normal acid/base balance in blood and urine. In addition to the mouse studies, two groups [44,45] have also demonstrated that a severe form of osteopetrosis is the result of a mutation(s) in the human 116 kDa proton pump subunit. With a phenotype similar to that of the mutant mice, these individuals have abnormally dense bones, normal or elevated numbers of osteoclasts and a blindness that is secondary to the osteopetrotic process. The macrolide antibiotic bafilomycin A1 is a very potent and specific inhibitor of vATPases and has been shown to inhibit bone resorption in vitro and in vivo. Unfortunately, as it inhibits all vATPases it is highly toxic. However, Gagliardi et al. [46] performed a series of chemical modifications to bafilomycin A1 that indicated that it was possible to obtain selective modulation of different
vATPases. This optimization of the specificity of osteoclast vATPase-selective inhibitors resulted in the discovery of SB-242784, which is a potent inhibitor of resorption in vitro [47]. Importantly, oral administration of this compound led to selective inhibition of the bone loss that is associated with ovariectomy in the rat, but had no effect on urinary acid excretion. The murine knockout data, the human gene mutation data and, ultimately, the small molecule inhibitor data define the osteoclast vATPase as an excellent target for antiresorptive therapy. CLC-7
In the resorbing osteoclast, the CLC-7 chloride channel appears to work in concert with the vATPase to maintain the resorptive process. It functions by short-circuiting the membrane potential that is caused by the electrogenic ATPase and appears to be rate-limiting for vATPase activity in osteoclasts. Recently, Kornak et al. [48•] described both a murine knockout of CLC-7 and an example of a human mutation in the CLC-7 gene. Both developed a very severe osteopetrosis, which suggests that CLC-7 potentially provides another excellent novel target for the inhibition of resorption. Cathepsin K
Cathepsin K is a lysosomal cysteine protease that has the unique ability, together with other cathepsins, to cleave both the helical and telopeptide regions of type I collagen — the most prevalent collagen in bone. Data also suggest that cathepsin K can cleave type II collagen. Initial studies at the mRNA and protein levels suggested that cathepsin K is abundantly and selectively expressed by osteoclasts in bone. Tissue localization studies suggested that it plays an important role in bone resorption and this hypothesis has subsequently been borne out by murine knockout data and the description of a human disorder in which the cathepsin K gene is mutated. The knockout of cathepsin K in mice results in osteopetrosis that is characterized by osteosclerosis [49,50]. There is an increase in trabecular number and in trabecular and cortical thickening in the knockout animals when compared to their wild-type littermates. The mutation also leads to the elimination of all osteoclast-related cathepsin activity and, ultimately, to a reduction in osteoclast-mediated matrix degradation. Pycnodysostosis is the human disease that occurs as a result of mutations in the cathepsin K gene [51]. In this disorder there is an elimination of cathepsin K activity that leads to a reduction in the rate of bone turnover and, ultimately, to poor quality dense bone that is predisposed to fracture. Significant data obtained from antisense DNA and synthetic inhibitor studies have been generated that confirm that cathepsin K is a viable target for the treatment of high bone turnover diseases that are characterized by excessive bone loss. For example, Votta et al. [52] demonstrated
Novel bone antiresorptive approaches Lark and James
that peptide aldehyde inhibitors of cathepsin K could significantly inhibit the aggressive bone loss that occurs in the rat adjuvant-induced arthritis model. The preferred animal models for evaluation of antiresorptive activity are in the rat, which has caused problems with compound evaluation for the treatment of human disease. Although rat and human enzymes are highly homologous, they do differ in key residues in the active site, and the development of compounds that inhibit the rat enzyme has proven difficult. Lark et al. [53] described a cathepsin K inhibitor, SB-331750, that was sufficiently active against the rat enzyme to be tested in the ovariectomized rat model of bone loss. This study showed that the inhibitor could prevent bone resorption in vivo and that the inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure. Stroup et al. [54•] recently described a non-human primate model of human postmenopausal osteoporosis, in which the active cathepsin K is identical to the human ortholog and, therefore, can be used to evaluate inhibitors of the human enzyme. In this model, a gonadotropin-releasing hormone agonist is used to render the cynomolgus monkey estrogen deficient, which leads to increased bone turnover. Using this model, Stroup et al. [54•] were able to evaluate a potent and selective cathepsin K inhibitor and show that it could significantly reduce the levels of serum markers of bone resorption (carboxy- and amino-telopeptides) relative to untreated controls. Cathepsin K inhibition resulted in an extremely rapid decrease in bone resorption and when the compound was eliminated in vivo, bone resorption resumed normally, suggesting that this compound could be used in situations where rapid onset or return of resorption may be required.
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number of osteoclasts that formed in vitro from macrophages derived from the knockout mouse. Also, they showed that the closely related integrin, αvβ5 did not substitute for αvβ3 during cytokine stimulation or authentic osteoclastogenesis. However, αvβ3 knockout mice did develop osteosclerosis with age. Interestingly, there was a 3.5-fold increase in osteoclasts in the mutant mice compared to their heterozygote littermates. However, these mutant osteoclasts were unable to excavate pits in whale dentine in in vitro cultures. In addition, significant hypocalcemia was evident in the knockout mice. The authors surmise that the resorptive defect in β3 -deficient osteoclasts may reflect an absence of matrix-derived intracellular signals, as their cytoskeleton is distinctly abnormal and they fail to spread in vitro to form actin rings ex vivo, or to form normal ruffled membranes in vivo. The authors concluded that, although the αvβ3 integrin is not required for osteoclastogenesis, it does appear to be essential for normal osteoclast function. These αvβ3 knockout data are supported by studies using small-molecule inhibitors of this integrin that show efficacy in the ovariectomized rat model of osteoporosis and in the rat adjuvant arthritis model. Lark et al. [56] described a non-peptide RGD mimetic αvβ3 antagonist that inhibited osteoclast resorption and prevented net bone loss in vitro, and inhibited cancellous bone turnover when dosed orally in the ovariectomized rat model. They concluded that, mechanistically, the orally active αvβ3 inhibitor prevented bone loss in vivo by inhibiting osteoclast-mediated bone resorption, ultimately preventing cancellous bone turnover.
These data show unequivocally that inhibition of cathepsin K results in the inhibition of bone resorption both in vitro and in vivo, and that the inhibition of this protease may be a viable approach to the treatment of diseases characterized by excessive bone loss.
In a rat adjuvant arthritis model that is characterized by inflammation and excessive bone loss, Badger et al. [57] were able to demonstrate that symptoms of the adjuvant arthritis were significantly reduced by either prophylactic or therapeutic treatment with the αvβ3 antagonist, SB-273005. Measurements of paw inflammation and of bone, cartilage and soft-tissue structure indicated that this compound exerts a protective effect on joint integrity and thus appears to have disease-modifying properties.
Vitronectin receptor
Conclusions and future prospects
The integrins are a family of heterodimeric receptors that consist of an alpha and a beta chain. Osteoclast adhesion to bone is mediated, at least in part, by the αvβ3 integrin, which binds to the Arg-Gly-Asp trimer (RGD in the singleletter code for amino acids) that is present in some of the bone matrix proteins. Data suggest that the osteoclast forms a tight sealing zone via the αvβ3–RGD interaction that is responsible for the maintenance of the resorptive process. Therefore, disruption of this interaction could provide a potential therapeutic target in diseases marked by excessive bone loss.
The identification and development of novel bone antiresorptive agents is being actively pursued in the pharmaceutical and biotechnological industries as well as in academia, which has resulted in the rapid advancement of the bone biology field. As suggested in this review, there is still significant opportunity for the development of novel bone antiresorptive therapies. With the introduction of bone-forming PTH, it is likely that, in the future, an antiresorptive drug will be used in combination with PTH. As clinical experience is gained with combination therapy, antiresorptives against novel targets may prove to be either more or less beneficial in various therapeutic settings. In addition, the broader therapeutic application is also being investigated for antiresorptive agents. For example, preclinical and clinical data are emerging that suggest that these
To address this, McHugh et al. [55] engineered mice in which the gene for the β3 integrin subunit was deleted. Their data showed that the mutation did not decrease the
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Musculoskeletal
compounds may be useful in the treatment of diseases such as periodontal disease, metastatic bone disease, RA and OA. As clinical experience is gained with antiresorptive therapies, it is likely that broader utility will be seen.
17.
Acknowledgements We would like to thank Simon Blake for his careful reading of this review.
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