The RANKL Pathway and Denosumab




Denosumab (Prolia) is a fully human monoclonal antibody directed against receptor activator of nuclear factor-κB ligand (RANKL), which interferes with the formation, activation, and survival of osteoclasts. It was approved by the Food and Drug Administration in June 2010 as a new treatment for postmenopausal osteoporosis in women who are at high risk for fracture. Given its mechanism of action, it is an antiresorptive therapy that is administered as a 60-mg subcutaneous injection every 6 months. It is the first biologic antiresorptive therapy for osteoporosis, and the first osteoporosis therapy to show efficacy and safety in patients with renal impairment.


Bone remodeling is a lifelong process in which old bone is replaced by new bone. This remodeling occurs in response to mechanical stresses and hormonal changes. This coordinated process of bone resorption and formation renews the skeleton while maintaining its structure. Bone remodeling also helps maintain mineral homeostasis, releasing calcium and phosphorus into the circulation. Bone remodeling occurs within discrete units called basic multicellular units or bone remodeling units (BMUs). These remodeling sites are found on both cortical and trabecular bone surfaces. The remodeling process results in the formation of a new osteon via a coordinated and sequential effort between osteoclasts and osteoblasts. Osteoclasts are tissue-specific macrophages created by the differentiation of monocyte/macrophage precursor cells at or near the bone surface. Their transformation from monocyte/macrophage lineage precursor cells into multinucleated cells is choreographed by a sequence of events that includes proliferation, differentiation, fusion, and activation. Interleukins (IL-1, IL-4, IL-7, IL-11, IL-17), tumor necrosis factor (TNF)-α, transforming growth factor β, prostaglandin E2, and hormones act together to control osteoclasts. Osteoblasts arise from pluripotent mesenchymal stem cells that can also differentiate into chondrocytes, myotubes, and adipocytes.


During the remodeling process a previously quiescent bone surface is activated by unknown signals that attract osteoclast precursors from the circulation to the skeleton, where they join together and form multinucleated cells. Osteocytes are believed to play an important role in initiating bone remodeling by transmitting local signals to osteoblasts and osteoclasts on bone surfaces via a canalicular system. Once formed, these multinucleated preosteoclasts attach to the bone surface, differentiate, and begin to resorb bone matrix, creating bone resorption pits. Once completed, resorption is associated with apoptosis of the osteoclasts and a reversal phase during which cells including preosteoblasts migrate to the surface of the bone. Mature osteoblasts direct the formation of new bone matrix and regulate its mineralization. This phase is followed by the apoptosis of osteoblasts and the incorporation of these cells into the bone as osteocytes or their transformation into bone surface lining cells.


Postmenopausal osteoporosis is a condition of acceleration of the bone remodeling rate. After menopause each remodeling cycle leads to a net loss of bone, due to a small deficiency in the amount of new bone that is formed compared with the amount of old bone that is removed. This small deficit in bone formation can result in trabecular perforations that occur during the resorptive phase, leading to a loss of bone surface on which osteoblasts can form new bone. Inhibition of bone remodeling has therefore become a major target for therapies that treat postmenopausal osteoporosis. This class of therapies includes estrogen, bisphosphonates, calcitonin, selective estrogen receptor modulators or estrogen agonist/antagonists, and denosumab.


The role of osteoprotegerin/RANKL/RANK in bone biology


Breakthroughs in the understanding of osteoclast differentiation and activation came from the analysis of a family of biologically related TNF/TNF-like proteins: osteoprotegerin (OPG), receptor activator of nuclear factor (NF) κB (RANK), and RANK ligand (RANKL), all of which regulate osteoclast function. RANKL is expressed on the surface of marrow stromal cells, activated T cells, and precursors of bone-forming osteoblasts. RANKL accelerates osteoclastogenesis when it binds to its receptor RANK on osteoclast precursor cells to enhance NF-κB and other signaling pathways. This binding of RANKL to RANK promotes osteoclast formation, activation, and survival. OPG is a soluble cytokine receptor that competes with RANK to bind RANKL, thus sequestering RANKL and neutralizing its effects. Thus OPG, which is produced by osteoblasts, is a natural decoy receptor for RANKL.


The OPG/RANKL/RANK system plays a significant role in postmenopausal osteoporosis. Postmenopausal women express higher levels of RANKL on marrow stromal cells or lymphocytes than premenopausal women or postmenopausal women taking estrogen. RANKL expression is inversely correlated with serum levels of 17β-estradiol and positively correlated with levels of bone resorption markers. The activity of c-jun N-terminal kinase, an intracellular signal following RANK activation, can be suppressed by 17β-estradiol.


Glucocorticoid-induced osteoporosis is also characterized by increased bone resorption initially followed by decreased bone formation. Glucocorticoid exposure increases RANKL expression, inhibits OPG production by osteoblasts, and suppresses OPG levels. The OPG/RANKL/RANK system also plays an active role in posttransplant osteoporosis through the use of immunosuppressive drugs. Although levels of soluble RANKL and OPG can be measured in the serum, these values can be influenced by age, renal function, and vascular disease ; thus measurement of soluble RANKL and OPG for diagnosis, risk stratification, or therapeutic monitoring of bone-related diseases is not recommended for routine clinical practice.




Treatment implications


Restoring a balanced RANKL/OPG ratio or blocking RANK binding should prevent osteoclast activation and bone resorption. In a study by Bekker and colleagues, a single subcutaneous dose of OPG caused rapid and sustained inhibition of bone resorption as indicated by bone resorption markers. The injection was well tolerated. It is evident from this study that OPG could potentially have clinical applications as a new antiresorptive therapy to treat osteoporosis. OPG, however, is a fairly large protein, which can be difficult to administer in ways other than by injection, and its short half-life would require monthly dosing to maintain consistent suppression of bone resorption. Another potential risk with OPG is the formation of anti-OPG antibodies, which could cross-react with endogenous OPG, neutralizing its activity. Anti-OPG antibodies were seen in one patient in this study. No negative effects were seen clinically, but safety concerns could arise with chronic dosing. Given these limitations, a RANKL inhibitor with a longer half-life and less immunogenicity would be preferable. Monoclonal antibodies have significantly longer half-lives than Fc fusion proteins (such as the OPG injection), so a fully human monoclonal antibody directed against human RANKL was developed. This monoclonal antibody, denosumab, inhibits RANKL with a high specificity, mimicking the effects of OPG on RANKL ( Fig. 1 ). Denosumab is classified as a highly specific molecule because it does not bind to other members of the TNF family, including TNF-α, TNF-β, TNF-related apoptosis-inducing ligand (TRAIL), or CD40 ligand. Denosumab has been investigated as a therapy to treat women with postmenopausal osteoporosis and to treat men and women undergoing sex hormone ablation therapy for cancer, to prevent loss of bone mass related to these therapies.




Fig. 1


RANKL Antibody/RANKL: activation of osteoclasts.

( Adapted from Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337; with permission.)




Treatment implications


Restoring a balanced RANKL/OPG ratio or blocking RANK binding should prevent osteoclast activation and bone resorption. In a study by Bekker and colleagues, a single subcutaneous dose of OPG caused rapid and sustained inhibition of bone resorption as indicated by bone resorption markers. The injection was well tolerated. It is evident from this study that OPG could potentially have clinical applications as a new antiresorptive therapy to treat osteoporosis. OPG, however, is a fairly large protein, which can be difficult to administer in ways other than by injection, and its short half-life would require monthly dosing to maintain consistent suppression of bone resorption. Another potential risk with OPG is the formation of anti-OPG antibodies, which could cross-react with endogenous OPG, neutralizing its activity. Anti-OPG antibodies were seen in one patient in this study. No negative effects were seen clinically, but safety concerns could arise with chronic dosing. Given these limitations, a RANKL inhibitor with a longer half-life and less immunogenicity would be preferable. Monoclonal antibodies have significantly longer half-lives than Fc fusion proteins (such as the OPG injection), so a fully human monoclonal antibody directed against human RANKL was developed. This monoclonal antibody, denosumab, inhibits RANKL with a high specificity, mimicking the effects of OPG on RANKL ( Fig. 1 ). Denosumab is classified as a highly specific molecule because it does not bind to other members of the TNF family, including TNF-α, TNF-β, TNF-related apoptosis-inducing ligand (TRAIL), or CD40 ligand. Denosumab has been investigated as a therapy to treat women with postmenopausal osteoporosis and to treat men and women undergoing sex hormone ablation therapy for cancer, to prevent loss of bone mass related to these therapies.




Fig. 1


RANKL Antibody/RANKL: activation of osteoclasts.

( Adapted from Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337; with permission.)




Denosumab (Prolia)


Phase 1 Single-Dose, Dose-Escalation Study


A single-dose, placebo-controlled, dose-escalation study with denosumab in postmenopausal women was performed to determine its safety and antiresorptive effect in bone. The mean age ranged from 54 to 63 years and subjects were 7 to 15 years postmenopausal. Bone density was not measured during the study.


After a single subcutaneous dose of denosumab, there was a dose-dependent decrease in bone turnover as reflected by changes in urinary N-terminal telopeptide (NTX)/creatinine and serum NTX. At the higher doses of denosumab, decreases in urine NTX/creatinine were observed as early as 12 hours after the dose: −46% in the placebo group and −77% in the denosumab group. This result suggested that mature and active osteoclastic activity was inhibited almost immediately. The maximum urinary NTX/creatinine reduction was observed at 2 weeks in the 0.01, 0.03, 0.3, and 1.0 mg/kg groups, at 1 month in the 0.1 mg/kg group, and at 3 months in the 3.0 mg/kg group. Therefore, little if any osteoclastic activity remained while denosumab was in the circulation, but this treatment effect was reversible, as indicated by a return toward baseline levels of urinary NTX/creatinine at 2 months in the 0.01 and 0.03 mg/kg groups, at 4 months in the 0.1 mg/kg group, at 6 months in the 0.3 mg/kg group, and at 9 months in the 1.0 and 3.0 mg/kg groups. The serum NTX data confirmed the findings seen with urinary NTX/creatinine. The bone alkaline phosphatase levels remained close to baseline levels in all groups until about 2 weeks post dose, then demonstrated a dose-dependent decrease; this was expected, as denosumab does not primarily interfere with osteoblastic activity. The reduction in levels of markers of bone resorption and formation suggests that denosumab reduced the activation frequency (or birth rate) of BMUs, the cellular units responsible for bone turnover.


None of the subjects discontinued the study because of an adverse event. The incidence of reported infectious events was similar across groups (33% in the placebo group and 38% in the denosumab group overall, with no apparent dose-dependent increase). Partial inhibition of early T-lymphocyte and B-lymphocyte development has been seen in RANKL-deficient mice. There was no clinically significant effect on lymphocyte counts overall (CD3), T cells (CD4, CD8, CD56), or B cells (CD20) in this study. No antidenosumab antibodies were seen in this study.


Phase 2 Study of Postmenopausal Women with Low Bone Mineral Density


This study was performed to assess the efficacy and safety of denosumab through 24 months in postmenopausal women with low bone mineral density (BMD) including those with both osteopenia and osteoporosis. It was a randomized, dose-ranging, placebo-controlled, and active-controlled study involving 8 double-blind treatment groups and one open-label treatment group (alendronate). Participants received one of the following: placebo subcutaneously every 3 months, denosumab 6, 14, or 30 mg subcutaneously every 3 months, denosumab 14, 60, 100, or 210 mg subcutaneously every 6 months alternating with placebo to maintain the blinding or open-label alendronate 70 mg orally once weekly. BMD, bone turnover markers, serum chemistries, hematology assessments, intact parathyroid hormone (PTH) levels, serum denosumab levels, and denosumab-neutralizing antibodies were measured.


Denosumab treatment for 24 months was associated with significant increases in BMD from baseline compared with placebo. BMD increases in the lumbar spine ranged from 4.13% to 8.89% compared with a −1.18% change from baseline in the placebo group ( P <.001). At 24 months, all doses of denosumab were associated with significant increases from baseline compared with placebo ( P <.001) for BMD of the total hip, distal one-third radius and total body. When the active treatment groups were compared at 24 months, denosumab treatment was associated with similar or greater increases in BMD than alendronate at all 4 skeletal sites with the exception of the 14-mg 6-month dose. Denosumab treatment maintained reductions in serum C-terminal telopeptide (CTX) and urine NTX compared with placebo ( P <.001) and reductions in bone-specific alkaline phosphatase (BSAP) compared with placebo ( P <.002) during the second year of treatment, consistent with reductions seen during the first year of treatment. Statistically significant ( P <.001) median percent reductions from baseline in serum CTX and urine NTX were observed for all doses and time points except the 14-mg 6-month dose group, for which values approached baseline levels at the time points just before the next denosumab dose ( Fig. 2 ).




Fig. 2


Denosumab lowered serum C-telopeptide.

( From Lewiecki EM, Miller PD, McClung MR, et al. Two-year treatment with denosumab (AMG 162) in a randomized phase 2 study of postmenopausal women with low bone mineral density. J Bone Miner Res 2007;22:1832–41; with permission.)


The percentage of subjects who experienced adverse events during the 2-year study period was generally similar among the placebo, denosumab, and alendronate groups. Upper respiratory tract infection was the most common adverse event in the denosumab group (17.4% placebo, 24.2% denosumab, 23.9% alendronate). The incidences of hypertension and urinary tract infection were greater in the denosumab group than in the placebo group. Serious adverse events were reported in 4 (8.7%), 42 (13.4%), and 6 (13.0%) of subjects in the placebo, denosumab, and alendronate groups, respectively. Six cases of serious adverse events of infections associated with hospitalization were observed in the denosumab group (2 cases each of diverticulitis and pneumonia and 1 case each of atypical pneumonia and labyrinthitis). These events were common community-acquired infections that were successfully treated with standard antibiotics during uncomplicated hospital courses. The small study size and the disproportionately greater number of subjects assigned to denosumab treatment make it difficult to determine the clinical relevance of these small differences observed among treatment groups for some of the adverse events. Analysis by exposure-adjusted rates revealed that rates of occurrence of adverse events among all treatment groups did not increase with extended time of exposure to the study drug. Two subjects had transient, nonneutralizing antibodies to denosumab in the first 12 months. No neutralizing antibodies to denosumab were observed during the first or second year of treatment.


These changes in BMD and bone resorption markers with denosumab use over a 2-year period were consistent with a reduction in osteoclast-mediated bone resorption through inhibition of RANKL. Reversal in the suppression of bone turnover markers at the end of each dosing interval with the lower dose of denosumab suggested a reversible effect of denosumab on osteoclasts and their precursors. These results suggested that further study of denosumab to treat postmenopausal osteoporosis was warranted.


This phase 2 study was extended for an additional 24 months with blinded doses of denosumab or placebo administered every 6 months. Denosumab-treated patients who continued the study were reassigned based on their randomization group at enrollment. Patients randomized to the denosumab 6 and 14 mg every 3 months group and 14, 60, and 100 mg every 6 months group received denosumab 60 mg every 6 months, the dose selected for the phase 3 trials. Patients randomized to 30 mg every 3 months received placebo for 12 months then were subsequently treated with denosumab 60 mg every 6 months for 12 months. Patients randomized to the 210 mg every 6 months group received placebo for the rest of the study. The placebo group was maintained for a total of 48 months. The alendronate subjects discontinued therapy after 24 months and were followed. All patients received 1000 mg calcium daily and 400 IU of vitamin D daily. BMD was measured at 36 and 48 months. Markers of bone turnover were collected every 6 months to measure serum CTX, urine NTX, and BSAP. Intact PTH levels were assessed at 36 and 48 months. Hematology assessments, serum chemistries, and measurement of serum denosumab levels were performed at 30, 36, 42, and 48 months. Samples were analyzed for antidenosumab-binding antibodies and denosumab-neutralizing antibodies. Adverse events were recorded at each visit.


Of the 412 subjects enrolled in the original study, 337 (82%) completed the 24-month study, 307 (75%) of these subjects entered the extension, and 262 (64%) of the original cohort completed the 48 months of treatment. Discontinuation was balanced among groups, with the most common reason for discontinuation being withdrawal of consent (94/412 = 23%). Increases in BMD reached similar levels for all groups that switched to 60 mg every 6 months. Almost all of the subjects who received denosumab treatment for 48 months had increases in BMD (greater than 0%) at the lumbar spine (97.4%) and total hip (95.5%) whereas most subjects in the placebo group lost BMD at these sites (72.4% and 82.8%, respectively).


As mentioned previously, markers of bone turnover (serum CTX, urine NTX, and BSAP) were rapidly reduced by denosumab treatment and remained substantially decreased over the entire 48 months of the study. During the last 24 months of the study, markers of bone turnover were measured at 6-month intervals just before the next dose, which corresponded to the nadir of the serum denosumab levels. Only a small percentage of denosumab-treated patients (≤6.8%) had serum CTX levels below the quantifiable limit of the assay (0.049 ng/mL) at 30, 36, 42, and 48 months. In the denosumab 210 mg every 6 months treatment group that discontinued therapy at 24 months, the levels of serum CTX and urine NTX returned to values near baseline and were not significantly different from placebo.


For the 30 mg every 3 months group that was assigned to placebo at 24 months for 12 months and then retreated with denosumab 60 mg every 6 months for 12 months, BMD increased to a similar degree to that observed after initial denosumab treatment. By month 48, the lumbar spine BMD had increased 9% and the total hip BMD had increased 3.9% from baseline. Markers of bone turnover rapidly decreased to values below baseline after retreatment with denosumab. At 42 and 48 months, values of markers of bone turnover were similar to those of the continuous treatment groups.


To further evaluate the effect of discontinuing denosumab therapy on bone remodeling, data for the 210 mg and 30 mg every 3 months treatment group were combined because both of these groups stopped denosumab at the end of the 24 months. Despite the increase in serum CTX levels after discontinuation of denosumab, most patients (42/62 = 68%) had serum CTX levels within the range for postmenopausal women as defined by the assay manufacturer (mean = 0.439 ng/mL). At month 48, all except one subject had serum CTX levels below the upper range limit (1.351 ng/mL) for this assay. The clinical consequences of the increase in levels of markers of bone turnover and the decrease in BMD after discontinuing denosumab therapy are unknown.


Most patients experienced an adverse event over the 48 months of the study. The most common adverse events reported were upper respiratory tract infections, arthralgia, and back pain. Serious adverse events were reported by 10.9% (5/46) subjects in the placebo group, 17.8% (56/314) subjects in the denosumab group, and 17.4% (8/46) subjects in the alendronate group. Overall, the incidence of malignancy was balanced among the treatment groups: 4.3% (2/46) in the placebo group, 4.8% (15/314) in the denosumab group, and 4.3% (2/46) in the alendronate group. The overall incidence of infections was similar for all treatment groups: 67.4% (31/46) for placebo, 66.2% (208/314) for denosumab, and 69.6% (32/46) for alendronate. None of the placebo or alendronate-treated patients developed infections requiring hospitalization; however, 3.2% (10/314) of the denosumab-treated patients did. All of the infections were common community-acquired infections, and no opportunistic infections were reported. Infections were treated with standard antibiotics and hospitalizations were uncomplicated. Four deaths (gastric cancer, adenocarcinoma, brain cancer, and cerebral vascular accident) occurred in the 314 denosumab-treated patients. No deaths occurred in the smaller placebo or alendronate groups. No patients experienced clinically relevant changes in chemistry or hematology values. No patient experienced symptomatic hypocalcemia, and no patients developed neutralizing antibodies to denosumab.


This study was not powered to assess reduction in fracture risk with denosumab compared with placebo or alendronate. Clinical fractures were reported as adverse events. Clinical fractures occurred in 10.9% (5/46) of the placebo subjects, 10.5% (33/314) of the denosumab subjects, and 6.5% (3/46) of the alendronate subjects. There was no increase in fracture incidence in the groups that discontinued denosumab treatment, but the size of these groups was small and the follow-up period was short. Further studies need to be performed to determine if a limited period of increased bone remodeling after discontinuing denosumab therapy increases the risk of fracture.


This study was extended for an additional 4 years to permit continued evaluation of efficacy and safety of continuous denosumab exposure for up to 8 years. An interim analysis from that extension has been published, representing up to 6 years of exposure to denosumab. BMD was measured at the lumbar spine, total hip, femoral neck, and one-third radius at study entry and years 5 and 6. Bone turnover markers (serum CTX, urine NTX, and serum BSAP) were measured after an overnight fast and before the next denosumab dose, and an additional draw for CTX was performed at 1 month after the dose in year 1 and year 5 of the extension study. All patients in the extension study received denosumab 60 mg every 6 months (including the placebo and off-alendronate groups) but were grouped for analysis purposes according to the treatment regimens received during the 48-month study. Because this extension study did not have a control group, the data from the original 48-month study served as the comparator for both efficacy and safety measures.


Two hundred subjects entered the extension study and 178 completed the 6-year assessment. Six years of continuous treatment was associated with mean BMD increases of 13.3%, 6.1%, and 5.6% at the lumbar spine, total hip, and femoral neck. Even the subjects who had not received continuous denosumab treatment showed similar increases in BMD at the lumbar spine, total hip, and femoral neck. All subjects demonstrated continued increases in BMD over a 6-year period, without evidence of a plateau, which is not seen with other antiresorptive therapies. The mechanism to explain this finding is not known, but at least 3 hypotheses have been discussed. One hypothesis is that denosumab closes the remodeling space and prolongs remodeling with subsequent increases in mineralization over time. Another is that denosumab causes greater reductions in bone resorption and longer remodeling time compared with alendronate, independent of bone surface available for remodeling, resulting in fewer new bone remodeling units and simultaneous filling-in of preexisting resorption cavities. The third hypothesis suggests that the increase in CTX seen at the end of the dosing interval allows for some degree of remodeling, which mineralizes after the next denosumab dose.


At year 6, serum CTX remained below the 48-month study baseline with a median reduction of 54.8% compared with baseline. To determine the differential effects of short-term and long-term denosumab therapy on the magnitude of reduction in CTX, the CTX values at 1 month after dose in years 1 and 5 were compared with those at baseline in the 48-month study, which showed median reductions of 89.3% and 91.2%, respectively. Median reductions in CTX just before the next dose for these intervals were 72.1% in year 1 and 47.5% in year 5. All subjects demonstrated reductions in CTX and BSAP, independent of prior treatment assignment. Both markers of bone turnover remained within the premenopausal reference range when measured in the study extension. The gradual increase in CTX over time with continuous denosumab exposure may reflect discrete changes in the degree of RANKL expression. Further investigation is needed to test this hypothesis.


One hundred and sixty-six subjects (83%) reported at least one adverse event. The 3 most common adverse events of upper respiratory infection (13.5%), arthralgia (11.5%), and back pain (9%) were similar to those seen in the 48 month study. Twenty-six subjects (13%) experienced a serious adverse event. Malignancy was reported in 3.5% of subjects: 1 metastatic cancer of unknown origin, 1 breast carcinoma in situ, 1 breast cancer, 2 lung cancers, and 1 colon cancer. Three infections were associated with hospitalization: pneumonia, endocarditis with staphylococcal septicemia, and diverticulitis. Three deaths occurred during the extension study: one due to unknown causes, one due to liver cancer, and one due to chronic obstructive pulmonary disease. Nine subjects suffered at least one fracture during the extension study. Sites of the fractures included fibula, foot, rib, humerus, hand, radius, thoracic spine, and tibia. There were no reports of delayed fracture healing or nonunion of fractures. No clinically relevant changes in blood chemistries were observed. No subjects developed antibodies to denosumab during the extension study. Thus, the overall safety profile in this ongoing study extension did not change over time. Denosumab was well tolerated and effective through 6 years of continuous treatment.


Phase 3 Study of Postmenopausal Women with Low Bone Mass (DEFEND)


The Denosumab Fortifies Bone Density (DEFEND) trial evaluated the efficacy and safety of denosumab over a 2-year period compared with a placebo control in a population of postmenopausal women with low bone density but not osteoporosis. This trial was a 2-year randomized, double-blind, placebo-controlled study performed in North America. An extension phase is ongoing. The primary end point was percent change in lumbar spine BMD at 24 months. Additional end points were percent change in volumetric BMD of the distal radius by quantitative computed tomography (QCT); percent change in BMD for the total hip, one-third radius, and total body; hip structural analysis; percent change in markers of bone turnover; and safety. Subjects were randomly assigned to receive denosumab subcutaneously at a dose of 60 mg every 6 months or placebo. Randomization was stratified by time since onset of menopause, that is, less than or equal to 5 years or more than 5 years.


A total of 332 subjects were enrolled in the study. Time since onset of menopause was 5 years or less in 162 subjects and more than 5 years in 170 subjects. Completion rate was 86%, with withdrawal of consent as the most common reason for study discontinuation. The lumbar spine BMD increase for the denosumab group overall was 6.5% at month 24 compared with −0.6% for placebo. BMD increased rapidly with significant increases seen as early as 1 month compared with placebo. The BMD increases at 24 months for the denosumab group overall were 3.4% at the total hip, 1.4% at the one-third radius, and 2.4% for the total body compared with changes of −1.1%, −2.1%, and −1.4%, respectively, in the placebo group overall. QCT analysis of the distal forearm showed that denosumab significantly ( P <.01) increased total volumetric BMD at the distal forearm for both strata and the strata combined compared with placebo at 24 months. Denosumab treatment significantly increased BMD, cross-sectional area, cross-sectional moment of inertia, section modulus, and average cortical thickness relative to placebo at all 3 cross sections but had no significant effect on the outer diameter. These results suggest that the modulation of bone remodeling by denosumab may result in a pattern of effects on cortical bone that may be beneficial. Whether this observation will result in greater fracture reduction in cortical bone compared with other antiresorptive therapy is unknown.


Markers of bone resorption were rapidly reduced by denosumab treatment. Levels of C-terminal telopeptide of collagen type I (CTX-I) reached a nadir at 1 month with a median reduction of 89% from baseline in the denosumab treatment group overall, compared with a 3% decrease in the placebo group overall ( P <.0001). Continued suppression of CTX-I was maintained on denosumab treatment, with reductions from baseline of 63% to 88% observed at the remaining study visits. Denosumab treatment also reduced levels of the bone formation marker, procollagen type 1 amino-terminal propeptide (P1NP), which declined more gradually than CTX-I.


The overall incidence of adverse events during the 24 months was similar between the placebo and denosumab groups. The most common adverse events in both treatment groups were arthralgia, nasopharyngitis, and back pain, with more subjects on denosumab reporting sore throat and rashes. Serious adverse events were reported in 9 subjects in the placebo group (5.5%) and 18 subjects in the denosumab group (11%) ( P = .074). The higher incidence of serious adverse events in the denosumab group was primarily due to a larger number of subjects who had infections treated as hospital inpatients (8 denosumab subjects, 1 placebo subject). The overall incidence of infections reported as adverse events was balanced between the two groups (61% placebo subjects, 60% denosumab subjects). The types of infections reported in the hospitalized subjects were common infections such as pneumonia, diverticulitis, sepsis, pyelonephritis, cellulitis, appendicitis, urinary tract infections in the denosumab subjects, and lobar pneumonia in the placebo subject. No opportunistic infections were seen. Hospitalizations were characterized by uncomplicated courses and successful treatment with standard antibiotics. Malignancies were reported in 1 subject in the placebo group (B-cell lymphoma) and in 4 subjects in the denosumab group (breast carcinoma in situ, mycosis fungoides, uterine cancer, and ovarian cancer) ( P = .215). No deaths occurred during the study. Three subjects (2%) in the placebo group and 2 subjects (1%) in the denosumab group developed nonneutralizing antidenosumab antibodies. Data from this study suggested that denosumab administered subcutaneously at a dose of 60 mg every 6 months was suitable for further evaluation of its ability to reduce osteoporotic-related fractures.


The purpose of the 2-year extension study of this phase 3 trial was to determine the effects of denosumab discontinuation on bone density and bone turnover markers. One hundred and twenty-eight subjects discontinued denosumab and 128 continued on placebo. After discontinuation of denosumab, levels of CTX increased to levels 40% to 60% above pretreatment values for the first 6 months of the extension study, then gradually returned to baseline by month 48 (month 24 of the extension study). Bone density also decreased in the off-treatment group, reaching baseline BMD after 12 months off-denosumab and remaining stable over the next 12 months. Thus, this study demonstrated reversibility of bone turnover markers and BMD on discontinuation of denosumab after 24 months.


Fractures in this study were captured as adverse events, as the extension study was not powered to evaluate fracture risk reduction. The incidence of nonvertebral fractures during the off-treatment study period was placebo 4 (3.1%) and off-treatment denosumab 4 (3.1%). One vertebral fracture occurred in the denosumab off-treatment group. It can be postulated that even though BMD decreased and CTX levels increased off-treatment, the fact that there was not an increase in fractures during this period of time and that the BMD and CTX values returned to baseline suggests that this 2-year off-treatment period has no long-term consequences.


Phase 3 Study of Denosumab for Prevention of Fractures in Postmenopausal Women with Osteoporosis


The Fracture Reduction Evaluation of Denosumab in Osteoporosis Every 6 Months (FREEDOM) trial was an international, randomized, placebo-controlled trial comparing subcutaneous injections of either 60 mg of denosumab or placebo every 6 months for 36 months. The primary end point was new vertebral fracture. Secondary end points were time to the first nonvertebral fracture and time to the first hip fracture. Randomization was stratified according to 5-year age groups. Women were eligible for inclusion if they were between the age of 60 and 90 years with a BMD T-score of less than −2.5 but greater than −4 at the lumbar spine or total hip, and did not have any severe or no more than 2 moderate vertebral fractures.


A total of 7876 women were enrolled in the study, 3935 in the placebo group and 3933 in the denosumab group. Baseline characteristics were similar between the two study groups. The mean BMD T-scores were −2.8 at the lumbar spine, −2.2 at the femoral neck, and −1.9 at the total hip. Approximately 24% of women had a prevalent vertebral fracture. Eighty-two percent (6478/7868) of subjects completed all 36 months of the study and 76% (5979/7868) received all of the injections.


The 36-month incidence of new radiographic vertebral fractures was 7.2% (264/3691) in the placebo group and 2.3% (86/3702) in the denosumab group, representing a 68% relative risk reduction ( P <.001). The reduction in risk was similar during each year of the trial ( Fig. 3 ). The reduction in clinical vertebral fractures (69%) and multiple new vertebral fractures (61%) was similar ( P <.001 for both comparisons). Denosumab reduced the risk of nonvertebral fracture, with a cumulative incidence of 8% in the placebo group compared with 6.5% in the denosumab group for a relative risk reduction of 20% (hazard ratio 0.80; 95% confidence interval [CI] 0.67–0.95; P = .01). Denosumab also decreased the risk of hip fracture, with a cumulative incidence of 1.2% in the placebo group compared with 0.7% in the denosumab group for a relative risk reduction of 40% (hazard ratio 0.60; 95% CI 0.37–0.97; P = .04). After 36 months compared with placebo, denosumab was associated with a relative increase in BMD of 9.2% (95% CI 8.2–10.1) at the lumbar spine and 6% (95% CI 5.2–6.7) at the total hip. Denosumab decreased serum CTX levels by 86% at 1 month, by 72% at 6 months before treatment was readministered, and by 72% at 36 months compared with placebo. Levels of P1NP were 18%, 50%, and 76% compared with placebo at the same time points.


Oct 1, 2017 | Posted by in RHEUMATOLOGY | Comments Off on The RANKL Pathway and Denosumab
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