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Abstract

  1. Top of page
  2. Abstract
  3. Background
  4. Bone and the Immune System
  5. Clinical Implications
  6. Clinical Trials
  7. Summary
  8. REFERENCES

This is the first article in a new series entitled “Clinical Aspects of Molecular Research in Rheumatology,” which will appear regularly in Arthritis Care & Research. As with all health care professionals, our readership is increasingly faced with the reality that in medicine today there is simply “too much to know.” This is particularly true for those confronted by the challenges of interpreting the remarkable advances occurring in the fields of cellular and molecular biology as applied to research in rheumatology. This series will present concise reviews written by leaders in the field that are tailored to nonlaboratory-based readers. It is hoped that these articles will provide both an overview of cutting edge science in an interpretable format, and provide direction for those desiring to learn more about the precise topics. We welcome reader feedback and ideas for future topics.

Leonard H. Calabrese, DO

Section Editor

Over the last decade, significant progress has been made in understanding the phenomenon of bone remodeling that involves the coupling of bone resorption with bone formation. This dynamic process allows for maintenance of the size, shape, and strength of bone. The pivotal pathway linking osteoclast-mediated bone resorption and osteoblast-mediated bone formation in the basic multicellular unit consists of 3 factors: RANKL, its receptor RANK, and a soluble inhibitor receptor for RANKL, osteoprotegerin (OPG). RANKL is a member of the tumor necrosis factor (TNF) family, and RANK and OPG are both members of the TNF receptor superfamily.

In addition to the essential role of the RANKL/RANK/OPG pathway for normal bone turnover, basic research efforts have also identified these proteins as the link between the immune system and the pathologic bone resorption that occurs in rheumatoid arthritis (RA), manifesting as bone erosions at the local level, and as osteoporosis at the systemic level. In an example of the rapid acceleration of translational research, therapies based on this cytokine pathway are now under development and in clinical trials only a few short years after the discovery of these molecules. This review will summarize the extraordinary progress made in this field over the last decade.

Background

  1. Top of page
  2. Abstract
  3. Background
  4. Bone and the Immune System
  5. Clinical Implications
  6. Clinical Trials
  7. Summary
  8. REFERENCES

Bone remodeling is the predominant metabolic process regulating bone structure, and the pivotal cell involved in initiation of this process is the osteoclast. The rate of bone remodeling (bone turnover) is a critical factor in normal bone development in the early decades of life as well as in the bone loss resulting in osteoporosis. Bone remodeling is dependent on the activation frequency, level of activity of osteoclasts and osteoblasts, and the termination rate of the basic multicellular units. Osteoclasts are bone-resorbing cells derived from monocyte/macrophage precursors. It has been appreciated for decades that osteoclast and osteoblast activity are linked together within the basic multicellular unit and that regulation of bone remodeling occurs through the osteoblasts. Osteoblasts have receptors for calciotropic hormones and cytokines, and appear to orchestrate the local recruitment and activity of osteoclasts. However, before the last decade, only limited information about the factors involved in stimulation of osteoclast differentiation and activation was available.

Breakthroughs in unraveling the signals involved in osteoclast differentiation and activation occurred when in vitro experiments using cocultures of bone marrow or spleen cells with stromal cells resulted in the generation of osteoclasts (1). This observation suggested that factors produced by stromal cells were responsible for osteoclastogenesis.

Using mutant mice, a number of discoveries regarding regulation of bone mass were made. One of the pivotal advances was the identification of OPG, reported initially in 1997 (2). OPG is a 110-kd disulfide-linked dimer. Mice transgenic for OPG expression developed osteopetrosis, with an observed decrease in mature osteoclast differentiation and bone resorption. OPG-knockout mice developed severe osteoporosis with frequent fractures (3). In vitro, recombinant OPG was found to block osteoclast differentiation from precursor cells in a dose-dependent manner and, in mice, to be a critical regulator of bone mass (4). A subsequent experiment in mice demonstrated that recombinant OPG blocked ovariectomy-associated bone loss. The investigators concluded that OPG could act as a soluble factor in the regulation of bone mass.

Subsequent experiments looking for a ligand for OPG in a complementary DNA library of mouse stromal cells resulted in the 1998 identification of RANKL as the factor inhibited by OPG (5). RANKL is a transmembrane protein that is expressed by osteoblasts/stromal cells that can be enzymatically cleaved into a soluble form. RANKL binds and activates RANK, a transmembrane signaling receptor. RANK is expressed on hematopoietic precursors, and ligand binding with RANKL results in differentiation to mature osteoclasts. In the presence of macrophage colony-stimulating factor 1, RANKL stimulates in monocytes the expression of genes of mature osteoclastic lineage (6). RANKL expression is upregulated by factors such as vitamin D3, interleukin-1 (IL-1), IL-6, TNFα, parathyroid hormone (PTH), and glucocorticoids, all of which have been associated with an increase in bone resorption. In addition to stimulation of osteoclastogenesis, the binding of RANKL to RANK on mature osteoclasts results in prolonged osteoclast survival via suppression of apoptosis. Treatment of mice with recombinant RANKL resulted in hypercalcemia, increased bone resorption, and osteoporosis (4–6). RANKL-knockout mice display severe osteopetrosis, stunted growth, and defective tooth eruption (7).

These experiments established the RANKL/RANK/OPG interaction as essential for osteoclastogenesis and bone remodeling. RANKL probably represents the previously unidentified coupling factor produced by osteoblasts to activate osteoclasts (Figure 1).

thumbnail image

Figure 1. Regulation of osteoclast differentiation, activation, and survival by the RANKL/RANK/OPG pathway. OPG = osteoprotegerin; CFU-M = colony forming unit-macrophage. (Reproduced with permission from Amgen)

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Bone and the Immune System

  1. Top of page
  2. Abstract
  3. Background
  4. Bone and the Immune System
  5. Clinical Implications
  6. Clinical Trials
  7. Summary
  8. REFERENCES

RANKL is also expressed on T cells, and in vitro studies have demonstrated that activated T cells can regulate the development and activation of osteoclasts through RANKL (8). Further experiments demonstrated that activation of osteoclasts by T cells could be blocked by OPG. T cell activation plays a critical role in RA, and osteoclast precursors and mature osteoclasts have been identified at the sites of bone erosion. Using the adjuvant-induced arthritis model in Lewis rats, RANKL has been identified on synovial effector T cells. Administration of OPG completely abolished bone loss and bone erosion in the inflamed joints in a dose-dependent manner but had no impact on the degree of inflammation (9). Bone loss in the control animals was characterized histologically by a marked increase in osteoclast numbers, whereas OPG-treated animals had minimal evidence for osteoclast accumulation. Additionally in the adjuvant-induced arthritis model, OPG administration was able to prevent cartilage destruction, although in other preclinical arthritis models such as collagen-induced arthritis and TNF transgenic mice, OPG has not been protective of cartilage damage (10).

Recent studies of patients with RA have demonstrated that in addition to T cells, monocytes and synovial fibroblasts express RANKL, and RANKL messenger RNA has been found in these cells isolated from RA pannus (11). Patients with RA have also been found to have high levels of RANKL and OPG in their serum (12).

RANKL is also highly expressed in lymph nodes, and RANKL-knockout mice lack lymph nodes (8). In vitro studies have also demonstrated a role for RANKL/RANK in T cell and dendritic cell interactions and have demonstrated that OPG inhibition of RANKL/RANK binding may impact the early maturation of thymocytes (13). Subsequent studies in mice transgenic for OPG did not reveal any impact on the lymph node development and had no impact on the inflammatory response (2). The potential interaction between immune cell activation and RANKL/RANK/OPG signaling will need to be closely monitored in the development programs for any new therapies that might inhibit this pathway.

Clinical Implications

  1. Top of page
  2. Abstract
  3. Background
  4. Bone and the Immune System
  5. Clinical Implications
  6. Clinical Trials
  7. Summary
  8. REFERENCES

Abnormal bone remodeling is the major pathophysiologic abnormality in a number of bone disorders, suggesting a potential role for a therapy that can block osteoclast differentiation and activation. Presently, antiresorptive therapies such as bisphosphonates and hormone replacement therapy have been utilized as treatment for these disorders.

Estrogen loss at menopause results in accelerated bone loss characterized by increased activation frequency of basic multicellular units with the development of excessive, poorly filled resorption pits resulting in increased trabecular perforations and loss of biomechanical bone strength. Similar bone loss also occurs in patients treated with hormone ablation therapy (patients with breast cancer and prostate cancer). Androgen-deprivation therapy with gonadotropin-releasing hormone agonist/antagonists in prostate cancer and aromatase inhibitor therapy in breast cancer have both been shown to be effective treatments, but both are associated with increased bone turnover and increased fracture rates related to hormone deficiency.

Metastatic bone disease is associated with increases in bone remodeling and osteoclast activation. Activated osteoclasts are found in lytic bone lesions stimulated by PTH-related protein and IL-1 in the local microenvironment. Presently, bisphosphonates are being utilized for treatment of metastatic bone involvement in multiple myeloma and breast cancer patients with moderate success in reducing bone pain and prolonging patient survival. In a mouse model of multiple myeloma, myeloma cells were found to express RANKL, and OPG treatment was effective at inhibiting osteolysis (14).

Hypercalcemia can be a complication of metastatic malignancy. This is the direct result of osteoclastic bone resorption, which is mediated by RANKL. Presently, bisphosphonates are the treatment of choice for significant hypercalcemia, with most patients responding, but the response may require 3–6 days. Inhibition of RANKL/RANK binding might allow for a more rapid correction of severe hypercalcemia. Indeed, in a recent study, OPG treatment rapidly reversed established hypercalcemia in 2 rat models of humoral hypercalcemia of malignancy, and the rate of suppression was significantly greater than that associated with the bisphosphonates pamidronate and zoledronic acid (15).

Paget's disease is characterized by a marked increase in bone turnover associated with the clinical signs of bone pain and skeletal deformities, potentially resulting in fracture. Laboratory studies demonstrate a profound elevation of osteoclastic bone resorption and osteoblast-associated new bone formation markers. Histologically, numerous large osteoclasts are the initial finding, resulting in localized osteolysis followed by excessive new bone formation by osteoblasts, resulting in an abnormal “mosaic” pattern of bone. Bisphosphonates are the present standard of care, based on their ability to suppress osteoclast-mediated bone resorption. Inhibition of osteoclast activity through the RANKL/RANK pathway might have therapeutic benefit.

In addition to these disorders of bone characterized by local and systemic bone loss, joint damage in RA is mediated by T cell, macrophage, and fibroblast synoviocyte-directed osteoclast bone resorption. Present treatment consists of disease-modifying antirheumatic drugs and biologics inhibiting TNFα and IL-1. Significant clinical response to these treatments, as reflected by an American College of Rheumatology 50% response (16), is found in 30–40% of patients and includes a significant impact on bone erosion and joint space narrowing.

All of the above conditions are characterized by excessive bone resorption. Blocking RANKL/RANK binding could provide significant benefit in all of these disorders based on the results from the preclinical models.

Clinical Trials

  1. Top of page
  2. Abstract
  3. Background
  4. Bone and the Immune System
  5. Clinical Implications
  6. Clinical Trials
  7. Summary
  8. REFERENCES

After the discovery of OPG, various forms of engineered OPG were produced. Capparelli et al reported that a single subcutaneous injection of a fusion protein of human OPG caused a significant and sustained reduction in osteoclast surface as early as day 1, resulting in a progressive increase in bone density in mice (17). Preclinical studies also revealed that the new bone formed had an excellent biomechanical quality. Other OPG constructs demonstrated efficacy in preclinical models of bone loss, including post ovariectomy, experimental bone metastasis, RA, glucocorticoid-induced osteoporosis, and hypercalcemia (15, 18–21).

Native OPG has a short half-life (∼20–30 minutes in rats), which makes it less attractive as a treatment due to the need for multiple injections. Additionally, concerns over immunogenicity exist. It is possible that if antibodies develop to engineered OPG they could cross-react with native OPG and negatively impact bone remodeling.

AMG 162 was developed by Amgen Pharmaceuticals (Thousand Oaks, CA) as an inhibitor of RANKL. AMG 162 is a fully human monoclonal IgG2 antibody with a high affinity (Kd 3 × 10−12 M) and specificity for human RANKL. It does not cross-react with TNFα, TNFβ, TRAIL, or CD40 ligand. In contrast to bisphosphonates, it does not adhere to bone. In preclinical models, single-dose administration of AMG 162 produced a rapid and significant reduction in bone resorption as measured by serum N-telopeptide (NTX). No significant safety signals were seen in the preclinical models. The initial trial of AMG 162 in humans was a placebo-controlled, dose-ranging study in 105 healthy postmenopausal women (22). Doses ranged from 0.01 to 3 mg/kg administered as a subcutaneous injection. Mean urinary NTX/creatinine decreased within 12 hours after dosing for all treatment cohorts and remained below baseline for the duration of a 6-month followup period in all groups except the 0.03-mg/kg group. The maximal mean decrease in urinary NTX (84.2%) was in the 3.0-mg/kg cohort on day 85. Pharmacokinetic analysis demonstrated that absorption was rapid. Clearance was nonlinear, with a volume of distribution similar to plasma volume. No significant safety concerns were reported in that study, and no subjects withdrew due to an adverse event. No significant laboratory changes, including serum calcium levels, were noted in the AMG 162 group, and no antiidiotypic antibodies were seen.

Based on the results of this dose-ranging study, a phase 2, placebo-controlled study was conducted in 412 postmenopausal women with low bone mineral density (T score ≤ −1.8). Preliminary data from the initial 12 months of this study have been presented (23, 24). The dosages of AMG 162 varied from 6–30 mg administered subcutaneously every 3 months, or 14–210 mg administered every 6 months. Alendronate (70 mg once weekly) was evaluated in this study as an open-label active comparator. AMG 162 (≥60 mg every 6 months) was able to maintain serum levels with adequate suppression of bone turnover in the majority of subjects for 6 months, and no drug accumulation was seen with repeat administration, and no change in pharmacokinetics occurred.

A treatment effect of AMG 162 occurred as early as 1 month, and at 6 months a 3.8–6.0% improvement in lumbar spine bone mineral density (BMD) was seen, compared with a 5% improvement for the alendronate and 0.5% for placebo. For the total hip, an improvement of 2.0–3.3% in BMD was seen in the AMG 162–treated cohort compared with a 2.0% improvement for the alendronate-treated group and 0.2% for the placebo-treated group. No detrimental effect on cortical bone BMD was observed for AMG 162. No significant difference between the every-3-months and every-6-months treatment was noted. A dose-dependent decrease in bone resorption markers was observed as early as 72 hours, which was sustained but partially reversible (at a dosage of 60 mg every 6 months) by 6 months.

No significant safety issues were reported in this trial. One patient had transient lowering of albumin-adjusted serum calcium level, without any clinical sequelae. Only one patient developed transient anti–AMG 162 antibodies.

A large phase III trial evaluating AMG 162 in patients with postmenopausal osteoporosis to determine treatment impact on fracture risk is ongoing. This trial uses the standard design for regulatory approval evaluating vertebral fracture risk reduction. Other ongoing trials will evaluate AMG 162 in patients with multiple myeloma, patients with metastatic bone disease, and patients receiving hormone deprivation therapy. Additionally, a placebo-controlled trial is ongoing, evaluating AMG 162 in patients with RA with erosive disease to determine the impact on prevention of radiographic progression.

Summary

  1. Top of page
  2. Abstract
  3. Background
  4. Bone and the Immune System
  5. Clinical Implications
  6. Clinical Trials
  7. Summary
  8. REFERENCES

This monoclonal antibody to RANKL represents potentially another exciting triumph of the scientific process with the rapid movement from the “bench” discovery to “clinical trial evaluation” to future “patient care.” The possibility of receiving a subcutaneous injection every 6 months to affect a reduction in the incidence of new fractures or to prevent joint erosion would be a significant advance in the management of these bone diseases. However, many questions remain. Will these gains in bone density and decreases in bone turnover result in fracture risk reduction? With continued administration, will the gains in bone density persist or will tachyphylaxis develop? What impact will antiidiotypic antibodies play on efficacy and safety? Will long-term treatment be associated with immunologic abnormalities or issues with hypocalcemia? The clinical trials are in progress, and we look forward to the answers to these questions.

REFERENCES

  1. Top of page
  2. Abstract
  3. Background
  4. Bone and the Immune System
  5. Clinical Implications
  6. Clinical Trials
  7. Summary
  8. REFERENCES
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