The Inhibition of RANKL Causes Greater Suppression of Bone Resorption and Hypercalcemia Compared with Bisphosphonates in Two Models of Humor
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内分泌学杂志 2005年第8期
Metabolic Disorders Research (S.M., K.W., S.A., F.A., Z.G., M.G., H.L.T., C.C., C.R.D., P.J.K) and Cancer Biology (C.S., B.W.), Amgen, Inc., Thousand Oaks, California 91320-1789
Address all correspondence and requests for reprints to: Dr. Paul J. Kostenuik, Metabolic Disorders Research, M/S 15-2-B, Amgen, Inc., 1 Amgen Center Drive, Thousand Oaks, California 91320-1789. E-mail: paulk@amgen.com.
Abstract
Humoral hypercalcemia of malignancy (HHM) is mediated primarily by skeletal and renal responses to tumor-derived PTHrP. PTHrP mobilizes calcium from bone by inducing the expression of receptor activator for nuclear factor-B ligand (RANKL), a protein that is essential for osteoclast formation, activation, and survival. RANKL does not influence renal calcium reabsorption, so RANKL inhibition is a rational approach to selectively block, and thereby reveal, the relative contribution of bone calcium to HHM. We used the RANKL inhibitor osteoprotegerin (OPG) to evaluate the role of osteoclast-mediated hypercalcemia in two murine models of HHM. Hypercalcemia was induced either by sc inoculation of syngeneic colon (C-26) adenocarcinoma cells or by sc injection of high-dose recombinant PTHrP (0.5 mg/kg, sc, twice per day). In both models, OPG (0.2–5 mg/kg) caused rapid reversal of established hypercalcemia, and the speed and duration of hypercalcemia suppression were significantly greater with OPG (5 mg/kg) than with high-dose bisphosphonates (pamidronate or zoledronic acid, 5 mg/kg). OPG also caused greater reductions in osteoclast surface and biochemical markers of bone resorption compared with either bisphosphonate. In both models, hypercalcemia gradually returned despite clear evidence of ongoing suppression of bone resorption by OPG. These data demonstrate that osteoclasts and RANKL are important mediators of HHM, particularly in the early stages of the condition. Aggressive antiresorptive therapy with a RANKL inhibitor therefore might be a rational approach to controlling HHM.
Introduction
HYPERCALCEMIA IS A significant complication of certain human malignancies, including squamous cell, renal cell, and breast carcinomas. Humoral hypercalcemia of malignancy (HHM) results primarily from the systemic activation of osteoclasts by tumor-derived factors (1, 2, 3). Tumor cell secretion of PTHrP is the principal mechanism of HHM (4, 5). PTHrP acts through PTH/PTHrP receptors in bone and kidney to stimulate osteoclastic bone resorption and calcium reabsorption, respectively (3, 6). The relative calcemic contributions of bone and kidney are difficult to quantify in HHM, although tumors that produce PTHrP appear to have a greater renal contribution to hypercalcemia than tumors that do not produce PTHrP (1, 2, 7). In the absence of effective inhibitors of renal calcium reabsorption, therapy for HHM has focused on antiresorptives (4). Bisphosphonates are effective in many cases of HHM, suggesting that osteoclasts play an important role in mediating hypercalcemia. Bisphosphonates are usually effective at reducing hypercalcemia when preceded by saline hydration (reviewed in Ref. 8). However, an informal meta-analysis of published clinical studies indicates that as many as one quarter of HHM patients fail to achieve normocalcemia after hydration and bisphosphonate therapy (7, 9, 10, 11, 12, 13, 14, 15, 16, 17). Bisphosphonate resistance has been observed in hypercalcemic patients treated with pamidronate (APD) (18) or zoledronic acid (ZOL) (19). Bisphosphonate resistance has been attributed to renal calcium reabsorption (5, 15) and, in some cases, to inadequate inhibition of bone resorption (1, 2, 15).
Animal studies have underscored the difficulty of controlling PTHrP-mediated hypercalcemia with antiresorptives. In rat studies, the infusion of PTHrP caused hypercalcemia that was only partially and transiently inhibited by treatment with a high dose (3 mg/kg) of APD or alendronate (20). It is unclear whether the persistence of hypercalcemia in these bisphosphonate-treated rats was related to residual bone resorption, renal calcemic effects, or both. It is difficult to determine the extent of residual bone resorption after bisphosphonate treatment, because these agents often fail to reduce osteoclast numbers in vivo (21, 22, 23). In some animal and human studies, bisphosphonate treatment has led to paradoxical increases in osteoclast numbers (10, 24, 25, 26, 27). Thus, osteoclast counts are not a reliable surrogate for the determination of bone resorption after bisphosphonate treatment (28), which has confounded attempts to assess the relative contributions of bone vs. kidney in HHM.
Osteoprotegerin (OPG) is a novel antiresorptive agent with a different mechanism of action than bisphosphonates. OPG is a soluble secreted protein of the TNF receptor family (29) that functions as a decoy receptor for receptor activator of NF-B ligand (RANKL) (30). RANKL is a TNF family member that binds to RANK on osteoclasts and their precursors to promote osteoclast differentiation, activation, and survival (30, 31, 32, 33, 34). The very existence of osteoclasts appears to require functional RANK-RANKL interactions, because mice that lack the genes for either RANKL (33) or RANK (35) are virtually devoid of osteoclasts. In rats, a single treatment with human recombinant OPG resulted in a greater than 95% reduction in osteoclast surface (36). Time-course analysis showed that both the suppression and the eventual recovery of osteoclast surface were temporally coordinated with changes in serum levels of recombinant OPG, biochemical markers of bone turnover, bone volume, and bone density (36). Thus, osteoclast surface appears to be a robust and sensitive measure of bone resorption after OPG treatment. This unique relationship could assist in distinguishing the relative contributions of bone and kidney to humoral hypercalcemia.
We have previously demonstrated that OPG delays the onset of hypercalcemia and reverses established hypercalcemia in a syngeneic mouse model of HHM. The sc C-26 colon adenocarcinoma model employed in those studies caused significant elevations in serum PTHrP in association with increased osteoclast surface (37). OPG treatment significantly delayed the onset of hypercalcemia in tumor-bearing mice, and this response was accompanied by a 99% reduction in osteoclast surface. Interestingly, hypercalcemia gradually returned in OPG-treated animals despite this profound reduction in osteoclast surface. Similar phenomena were observed in another murine model of HHM using a different RANKL inhibitor. RANK-Fc, a soluble extracellular fragment of RANK, significantly delayed the onset of hypercalcemia in nude mice bearing a PTHrP-secreting human lung tumor xenograft. Hypercalcemia eventually developed in these mice despite a 95% reduction in osteoclast numbers (38). These observations are consistent with an increasing contribution of renal calcium reabsorption with time in response to tumor-derived PTHrP. The control of hypercalcemia with OPG was previously shown to occur without changes in renal calcium reabsorption (39) or urinary calcium excretion (39, 40). OPG is therefore a suitable molecule to isolate the role of osteoclasts in the pathogenesis of HHM. Tumors may produce other proresorptive cytokines besides PTHrP, so we tested whether direct PTHrP challenge of mice would recapitulate the gradual return of hypercalcemia associated with the C-26 model despite continued osteoclast suppression by OPG. The effects of high-dose bisphosphonates were also examined to explore potential mechanisms of therapeutic resistance to bisphosphonate treatment.
Materials and Methods
Animals
All animal studies were conducted in accordance with federal animal care guidelines and in compliance with the Amgen institutional animal care and use committee. The C-26 studies used male CDF1 mice (10–12 wk old), and the PTHrP study used male BDF1 mice (4 wk old; Charles River Laboratories, Wilmington, MA). All mice were fed standard laboratory rodent chow (Harlan Teklad, Madison, WI) throughout the experiments. Each experimental group consisted of five mice.
C-26 tumor
C-26 cells were obtained from the Tumor Repository of the National Cancer Institute (Bethesda, MD). C-26 tumor cells were passaged sc, and dissected tumors were cut into fragments and cultured at 37 C in 5–6% CO2. Adherent cells were cultured in DMEM (Invitrogen Life Technologies, Inc., Grand Island, NY), 1x penicillin-streptomycin/glutamine (Invitrogen Life Technologies, Inc.), and 1x nonessential amino acids (Invitrogen Life Technologies, Inc.). Cell cultures were harvested by trypsinization (Invitrogen Life Technologies, Inc.) and resuspended in unsupplemented DMEM at a concentration of 2.5 x 106 cells/ml. Mice were injected sc in a shaved area of the right flank with 0.5 x 106 cells in a 200-μl cell suspension.
OPG vs. APD in a C-26 tumor model
Total serum calcium levels and body weights were determined daily starting 12 d after C-26 tumor inoculation. Mice were bled retroorbitally, and total serum calcium was determined with a Hitachi 717 serum chemistry analyzer (Roche, Indianapolis, IN). Normal serum calcium ranges were determined from 35 measurements of PBS-treated control mice. Normocalcemia was defined as the mean of these measurements [8.33 mg/dl (2.02 mmol/liter)] ± 2 SD [0.56 mg/dl (0.14 mmol/liter)]. Treatments were performed when an individual tumor-bearing mouse achieved a serum calcium level of 11 mg/dl or greater (2.75 mmol/liter; designated d 0). APD (Novartis, East Hanover, NJ) was suspended in PBS and injected once into the tail vein at a dose of 1.5, 5, or 15 mg/kg. The recombinant OPG used in these studies was a human OPG construct fused to the Fc portion of human IgG1 (29). OPG was suspended in PBS and injected once into the tail vein at doses of 0.2, 1, and 5 mg/kg. Blood was drawn from the retroorbital sinus daily for the next 5 d to monitor total serum calcium. Mice were then killed by isofluorane inhalation. The left tibia was harvested for histomorphometry.
OPG vs. ZOL in a C-26 tumor model
C-26 tumor cells were injected sc, and blood ionized calcium levels were monitored using a calcium/pH analyzer (model 634, Chiron Diagnostics, Norwood, MA). The normocalcemic range for this study, as determined by 35 measurements of PBS-treated control mice, was 1.29 ± 0.08 mmol/liter (mean ± 2 SD). Ten days after tumor inoculation, C-26 tumor-bearing mice were significantly hypercalcemic (mean ± 1 SD, 1.71 ± 0.35 mmol/liter). OPG (5 mg/kg) or ZOL (5 mg/kg) was injected once (sc) into tumor-bearing mice; tumor-free control mice were injected with PBS. Blood ionized calcium was monitored regularly for the next 96 h. Terminal serum was collected by cardiac puncture for analysis of tartrate-resistant acid phosphatase-5b (TRAP-5b), and the left tibia was harvested for histomorphometry. Serum TRAP-5b (Immunodiagnostic Systems, Inc., Fountain Hills, AZ) was measured in duplicate in accordance with the manufacturer’s instructions.
OPG vs. APD in PTHrP model of hypercalcemia
Mice were challenged twice daily (morning and evening, by sc injection), with 0.5 mg/kg PTHrP-(1–34) (Bachem, Torrance, CA). Control mice received PBS injections twice daily. Retroorbital blood was drawn exactly 3 h after each morning challenge to monitor changes in blood ionized calcium. Blood ionized calcium levels were determined using a calcium/pH analyzer (model 634, Chiron Diagnostics). The normocalcemic range for this study, as determined from 35 measurements of PBS-treated control mice, was 1.21 ± 0.08 mmol/liter (mean ± 2 SD). After 3 d of challenges, PTHrP-treated mice were significantly hypercalcemic (1.43 ± 0.01 mol/liter). At this time (d 0), mice were given a single iv injection of OPG (5 mg/kg), APD (5 mg/kg), or PBS. Mice continued to be challenged in the morning and evening with PTHrP or PBS. Retroorbital blood samples were obtained exactly 3 h after each morning or evening challenge to monitor blood ionized calcium. Terminal blood samples were drawn when the mice were killed (96 h after treatment) for clinical chemistry analysis. The left tibia was harvested for histomorphometry.
Tissue processing and histomorphometry
Tibiae were fixed in phosphate-buffered zinc formalin, decalcified in formic acid, embedded in paraffin, and sectioned as previously described (29). Sections were stained for TRAP activity (leukocyte acid phosphatase kit, Sigma-Aldrich Corp., St. Louis, MO) and counterstained with hematoxylin. The area of analysis consisted of 2 mm2 cancellous bone immediately distal to the proximal growth plate. Histomorphometry was performed by tracing the cancellous bone surface with the aid of a digital video camera attached to a microscope. Analysis was performed using Osteomeasure bone analysis software (Osteometrics, Inc., Decatur, GA). Osteoclasts were scored based on contact with the bone surface and TRAP-positive staining. Osteoblasts were scored based on contact with bone surface and plump cuboidal morphology.
Statistical analyses
The statistical significance of differences between groups for different time points was assessed using JMP statistical software (SAS Institute, Inc., Cary, NC). Comparisons were made using Student’s t test for comparison of data from two groups, Dunnett’s test for comparison of multiple treatment groups with a control, and the Tukey-Kramer test for comparisons between multiple treatment groups or controls. These latter two methods make allowance for multiple comparisons. A value of P < 0.05 was used to determine statistically significant differences.
Results
OPG vs. APD in a C-26 tumor model
The normal range for total serum calcium in untreated tumor-free mice (mean ± 2 SD) was 7.77–8.89 mg/dl (1.94–2.22 mmol/liter; Fig. 1, shaded box). The sc C-26 tumor caused severe hypercalcemia (11 mg/dl or 2.75 mmol/liter) in all mice within 12–14 d of tumor inoculation, at which time treatment was initiated (designated d 0). A single iv treatment with human OPG-Fc at 0.2, 1, or 5 mg/kg caused a significant decrease in serum calcium within 12 h (P < 0.05 for each dose level vs. PBS treatment), with calcium levels reaching a nadir at 48 h after treatment. Serum calcium was in the normal range for the 1 and 5 mg/kg dose groups (Fig. 1) at the nadir. In contrast, serum calcium in PBS-treated, tumor-bearing mice was at a peak level of 14.46 mg/dl at 48 h. Mild hypercalcemia returned after the 48 h nadir in OPG-treated mice, although levels remained significantly lower than those in PBS-treated, tumor-bearing mice (P < 0.05). Treatment of tumor-bearing mice with the highest dose of APD (15 mg/kg) was associated with a transient and significant increase in serum calcium at 12 h (compared with PBS treatment), followed by a progressive reduction in serum calcium levels (Fig. 1). These animals died between the third and fourth study day, and gross examination during necropsies revealed a white precipitate in the kidneys. These lesions were presumed to be drug substance that may have precipitated as a result of the bolus iv administration of APD (41). Lower doses of APD (1.5 and 5 mg/kg) caused a gradual reduction of serum calcium in tumor-bearing mice that reached a nadir on d 4. Statistically significant reductions in serum calcium were obtained from d 2–5 after APD treatment (P < 0.05), although serum calcium levels remained above the normocalcemic range throughout the study.
FIG. 1. Serum calcium responses in C-26 tumor-bearing mice treated with OPG or APD. Hypercalcemic CDF1 mice bearing sc C-26 tumors were treated with PBS (), APD (, 1.5 mg/kg; , 5 mg/kg; , 15 mg/kg), or OPG (, 0.2 mg/kg; , 1 mg/kg; , 5 mg/kg). Nontumor-bearing mice were treated with PBS (). The shaded box represents the normal calcium range (mean ± 2 SD) obtained from 35 measurements of untreated, nontumor-bearing mice of the same age, sex, and strain. Data are expressed as means (n = 5/group). Statistically significant differences (P < 0.05) are described in Results.
C-26 tumor burden was associated with a nonsignificant reduction in cancellous bone volume in the proximal tibial metaphysis compared with tumor-free controls. Both OPG and APD caused significant gains in bone volume in tumor-bearing mice (P < 0.05; data not shown). The percent osteoclast surface to bone surface was increased by 3-fold in tumor-bearing mice compared with tumor-free mice (P < 0.05; Fig. 2A), and OPG caused a dose-dependent reduction in osteoclast surface, reaching levels significantly lower than those in normal tumor-free mice at the highest dose (P < 0.05). APD at 5 mg/kg reduced the osteoclast surface in tumor-bearing mice to levels similar to those found in tumor-free mice. Osteoblast surface was significantly increased in tumor-bearing mice, and both OPG and APD prevented this increase at all doses examined (Fig. 2B).
FIG. 2. Bone histomorphometry of the proximal tibial metaphysis in C-26 tumor-bearing mice treated with OPG or APD. Nontumor-bearing control mice were treated with PBS. Tumor-bearing mice were treated once (iv) with PBS, OPG, or APD at the indicated concentrations. Mice were killed 5 d later, and tibiae were harvested for histomorphometry. Data were not available from mice treated with APD at 15 mg/kg due to extensive mortality. A, Osteoclast surface as a percentage of total bone surface (OcS/BS); B, osteoblast surface as a percentage of total bone surface (ObS/BS). Data are expressed as the mean ± SEM (n = 5/group). a, Significant difference from PBS-treated, tumor-bearing mice; b, significant difference from PBS-treated, nontumor-bearing mice (P < 0.05, by two-way ANOVA and Dunnett’s test).
The effects of tumor burden and treatments on tibial histology are indicated in Fig. 3. Photomicrographs of representative TRAP-stained sections demonstrate the typical pharmacological effects of the highest tolerated dose of each drug (5 mg/kg).
FIG. 3. Representative photomicrographs of the proximal tibial metaphysis in C-26 tumor-bearing mice treated with OPG or APD. Sections were stained for TRAP (purple) to highlight osteoclasts (OCs; see arrow). A, Tumor-free mouse treated with PBS; B, tumor-bearing mouse treated with PBS; C, tumor-bearing mouse treated with APD (5 mg/kg); D, tumor-bearing mouse treated with OPG (5 mg/kg).
OPG vs. ZOL in a C-26 tumor model
The normal range of blood ionized calcium in this study was 1.21–1.37 mmol/liter (see shaded box, Fig. 4). Tumor-bearing mice had moderate hypercalcemia at the time of treatment (1.71 ± 0.35 mmol/liter), and this hypercalcemia progressed dramatically over the ensuing 96 h. OPG (5 mg/kg) caused significant suppression of hypercalcemia from 24–96 h after treatment (P < 0.05 vs. PBS-treated, tumor-bearing mice). Blood ionized calcium was restored to within the normal range within 24 h of OPG treatment. ZOL (5 mg/kg) caused a significant suppression of hypercalcemia from 48–96 h after treatment (P < 0.05 vs. PBS-treated, tumor-bearing mice). Antiresorptive effects were also assessed at the time the mice were killed by monitoring serum TRAP-5b, an osteoclast-specific marker of bone resorption. C-26 tumor burden was associated with a 2-fold increase in serum TRAP-5b (P < 0.05 vs. tumor-free mice; Fig 5). This increase was fully prevented by single treatments with either OPG or ZOL. OPG suppressed serum TRAP-5b to levels significantly lower than those in ZOL-treated, tumor-bearing mice or normal, tumor-free mice (P < 0.05).
FIG. 4. Blood ionized calcium in C-26 tumor-bearing mice treated with OPG or ZOL. Hypercalcemic CDF1 mice bearing sc C-26 tumors were treated once (sc) with PBS (), OPG (5 mg/kg; s), or ZOL (5 mg/kg; ). Nontumor-bearing mice were treated with PBS (). The shaded box represents the normal calcium range (mean ± 2 SD) obtained from 35 measurements of PBS-treated, nontumor-bearing mice of the same age, sex, and strain. Data are expressed as the mean ± SEM (n = 5/group). *, Significantly different from PBS-treated, tumor-bearing mice, P < 0.05. BL, Baseline.
FIG. 5. Serum TRAP-5b in C-26 tumor-bearing mice treated with OPG or ZOL. Hypercalcemic CDF1 mice bearing sc C-26 tumors were treated once (sc) with PBS, OPG (5 mg/kg), or ZOL (5 mg/kg). Nontumor-bearing mice were treated with PBS. Serum was obtained when mice were killed (4 d after treatment) and was assayed for serum TRAP-5b, an osteoclast-specific marker of bone resorption. Data are expressed as the mean ± SEM (n = 5/group). a, Significantly different from nontumor-bearing mice; b, significantly different from PBS-treated, tumor-bearing mice (P < 0.05, by two-way ANOVA and Dunnett’s test).
Cancellous bone volume in the proximal tibial metaphysis was not significantly different among any groups (data not shown). C-26 tumor burden was associated with a 3-fold increase in osteoclast surface in the tibia (P < 0.05 vs. tumor-free controls; Fig. 6A), and OPG and ZOL both caused significant reductions in osteoclast surface in tumor-bearing mice (P < 0.05 vs. PBS-treated mice). OPG suppressed osteoclast surface to levels significantly lower than those in ZOL-treated, tumor-bearing mice or normal, tumor-free mice (P < 0.05). Osteoblast surface was not significantly influenced by tumor burden in this study. OPG caused a modest and nonsignificant reduction in osteoblast surface, whereas ZOL caused a marginally greater reduction that achieved statistical significance (Fig. 6B; P < 0.05).
FIG. 6. Bone histomorphometry of C-26 tumor-bearing mice treated with OPG or ZOL. The region of analysis consisted of 2 mm2 cancellous bone immediately distal to the proximal metaphyseal growth plate. Hypercalcemic CDF1 mice bearing sc C-26 tumors were treated once (sc) with PBS, OPG (5 mg/kg), or ZOL (5 mg/kg). Nontumor-bearing mice were treated with PBS. A, Osteoclast surface as a percentage of total bone surface (OcS/BS); B, osteoblast surface as a percentage of total bone surface (ObS/BS). Data are expressed as the mean ± SEM (n = 5/group). a, Significant difference from PBS-treated tumor-free mice, P < 0.05; b, significant difference from PBS-treated, tumor-bearing mice, P < 0.05; c, significant difference from ZOL-treated, tumor-bearing mice, P < 0.05.
OPG vs. APD in PTHrP model of hypercalcemia
The normal range of blood ionized calcium for PBS-challenged mice in the PTHrP study was 1.14–1.29 mmol/liter (Fig. 7, shaded box). Three days of PTHrP challenges resulted in significantly higher levels of blood ionized calcium compared with PBS-challenged mice (P < 0.05 vs. PBS-challenged mice). Mice then received a single iv injection of OPG or APD on d 0, and PTHrP challenges continued for an additional 4 d. OPG (5 mg/kg) restored blood ionized calcium to the normal range within 3 h, and normocalcemia was maintained for at least 24 h. Hypercalcemia returned in OPG-treated mice 2 d after OPG treatment. A single iv injection of APD (5 mg/kg) resulted in normocalcemia at 24 h, after which blood ionized calcium increased progressively. At the end of the study, PTHrP-challenged mice treated with APD had significantly higher blood ionized calcium compared with PTHrP-challenged mice treated with PBS (P < 0.05).
FIG. 7. Blood ionized calcium responses in PTHrP-challenged mice treated with OPG or APD. Hypercalcemia was induced by 3 d of twice-daily (morning and evening) sc injections of PTHrP-(1–34) (0.5 mg/kg/injection). PTHrP challenges continued during the treatment period. PTHrP-challenged mice were treated with single iv injections of PBS (), APD (5 mg/kg; ), or OPG (5 mg/kg; ). Normocalcemic (i.e. PBS-challenged mice) were treated with single iv injections of PBS (), APD (5 mg/kg; ), or OPG (5 mg/kg; ). Retroorbital blood was drawn exactly 3 h after each morning challenge with PTHrP (or PBS) for ionized calcium determinations. The shaded box represents the normal calcium range (mean ± 2 SD) obtained from 28 measurements of untreated mice of the same age, sex, and strain. Data are expressed as the mean ± SEM (n = 5/group). Statistically significant differences are indicated in Results.
Total serum TRAP was measured as a systemic marker of osteoclast activity. In PBS-challenged mice, both OPG and APD treatments caused nonsignificant reductions in serum TRAP (Fig. 8). In PTHrP-challenged mice, OPG treatment significantly reduced serum TRAP (P < 0.05 vs. PBS-treated controls), whereas APD treatment had no significant effect.
FIG. 8. Bone resorption (serum TRAP) in PTHrP-challenged mice treated with OPG or APD. Hypercalcemia was induced by 3 d of twice-daily (morning and evening) sc injections of either PBS or PTHrP (0.5 mg/kg/injection). Mice were then treated with single iv injections of PBS, OPG (5 mg/kg), or APD (5 mg/kg). Data are expressed as the mean ± SEM (n = 5/group). a, Significant difference from PBS-treated mice under similar challenge conditions (P < 0.05, by two-way ANOVA and Dunnett’s test).
Osteoclast surface in PBS-challenged mice was significantly reduced by OPG (P < 0.05), but not by APD treatment (Fig. 9A). In PTHrP-challenged mice, OPG treatment caused an 85% reduction in osteoclast surface (P < 0.05 vs. PBS-treated mice), whereas APD treatment led to a paradoxical 50% increase in osteoclast surface (P < 0.05 vs. PBS-treated mice). Eroded surface was dramatically suppressed by OPG in both PBS-challenged and PTHrP-challenged mice, whereas APD treatment failed to significantly reduce eroded surface in PBS-challenged mice (data not shown). In PTHrP-challenged mice, APD treatment caused a 2.5-fold increase in eroded surface compared with PBS-treated PTHrP-challenged mice (P < 0.05; data not shown).
FIG. 9. Bone histomorphometry of PTHrP-challenged mice treated with OPG or APD. The region of analysis consisted of 2 mm2 cancellous bone immediately distal to the proximal metaphyseal growth plate of the tibia. Mice were challenged for 3 d with twice-daily (morning and evening) sc injections of PBS or PTHrP (0.5 mg/kg/injection). Mice were then cotreated with single iv injections of PBS, OPG (5 mg/kg), or APD (5 mg/kg). A, Osteoclast surface as a percentage of total bone surface (OcS/BS); B, osteoblast surface as a percentage of total bone surface (ObS/BS); C, bone volume as a percentage of total volume (BV/TV). Data are expressed as the mean ± SEM (n = 5/group). a, Significant difference from PBS-challenged mice that were treated with PBS; b, significant difference from PTHrP-challenged mice that were treated with PBS (P < 0.05, by two-way ANOVA and Dunnett’s test).
Osteoblast surface in PBS-challenged mice was significantly reduced by both APD and OPG treatments (P < 0.05; Fig. 9B). PTHrP challenge alone led to a significant increase in osteoblast surface. APD treatment of PTHrP-challenged mice had no effect on osteoblast surface compared with PTHrP-challenged controls. OPG reduced osteoblast surface in PTHrP-challenged mice, although values remained greater than those in PBS-treated mice.
In PBS-treated mice, OPG caused a significant increase in cancellous bone volume at the proximal tibial metaphysis (P < 0.05 vs. PBS-treated controls; Fig. 9C). APD treatment caused an appreciable, but statistically insignificant, increase in bone volume. Twice-daily PTHrP challenges also caused a significant increase in bone volume, which was an expected pharmacological effect of intermittent PTHrP injections. In these PTHrP-challenged mice, treatment with APD caused significant blunting of the anabolic effect of PTHrP on bone volume (P < 0.05 vs. PBS-treated mice), although values remained higher than those in PBS-treated mice. OPG caused no blunting of PTHrP-mediated increases in bone volume (Fig. 9C).
Discussion
HHM is one of the most prevalent and clinically significant paraneoplastic syndromes. The typical etiology for HHM is the tumor production of factors such as PTHrP that systemically activate osteoclasts. PTHrP also stimulates renal calcium reabsorption, which can exacerbate hypercalcemia. In the absence of specific inhibitors of renal calcium reabsorption, therapy for HHM has focused on antiresorptives, such as bisphosphonates. Most HHM patients respond favorably to bisphosphonates when the drugs are preceded by saline hydration (3, 9, 42, 43, 44), but a significant number of patients remain hypercalcemic after these treatments (10, 45, 46, 47, 48). Resistance to APD in some HHM patients was highly correlated with pretreatment nephrogenous cAMP levels, which is consistent with a PTHrP-mediated renal response (49). In other studies, resistance to bisphosphonates was attributed to inadequate inhibition of bone resorption (1, 2, 15). Greater doses of bisphosphonates might provide better control of hypercalcemia, but treatment with iv bisphosphonates such as APD (50) or ZOL is limited by concerns about renal toxicity (50, 51). Because of these limitations, other classes of antiresorptives, such as RANKL inhibitors, may be warranted for patients who are resistant to bisphosphonate therapy.
RANKL inhibitors might be particularly well suited for inhibiting osteoclast-mediated hypercalcemia, particularly when PTHrP is involved. It is clear that functional RANK/RANKL interactions are essential for PTHrP-mediated bone resorption, because PTHrP fails to cause hypercalcemia or induce the appearance of osteoclasts in RANK–/– mice (35). Recombinant OPG, the native RANKL inhibitor, blocks the hypercalcemic response of mice injected with either PTHrP (52) or recombinant RANKL (30). OPG was previously shown to reverse hypercalcemia within 2 h in mice and rats, an effect that was unrelated to changes in renal calcium reabsorption (39) or urinary calcium excretion (39, 40). OPG induces osteoclast apoptosis in mice within 6 h (33). These rapid effects on bone resorption are probably related to the unique mechanism of action for RANKL inhibitors. RANKL inhibitors work without binding to a bone matrix substrate and can therefore cause a nearly immediate arrest of osteoclast formation, activation, and survival. In contrast, bisphosphonates must bind to bone matrix and then be liberated or consumed by osteoclasts to suppress bone resorption. Whether these fundamental mechanistic differences result in meaningful differences in therapeutic responses was previously unknown, due in part to the lack of direct comparisons.
The current studies provide the first direct comparisons of the effects of a RANKL inhibitor vs. bisphosphonates on hypercalcemia and bone histomorphometry. In two different models of HHM, the RANKL inhibitor OPG caused a more rapid reversal of established hypercalcemia compared with APD or ZOL, which represent the current standard of care for the treatment of hypercalcemia. OPG treatment also resulted in consistently greater reductions in osteoclast surface and bone resorption markers, and these responses were associated with the rapid and sustained reversal of hypercalcemia. In the PTHrP challenge study, OPG caused a dramatic reduction in osteoclast surface that was associated with consistent suppression of hypercalcemia, whereas APD caused a paradoxical increase in osteoclast surface that was associated with the exacerbation of hypercalcemia. OPG also caused greater reductions in osteoclast surface and serum TRAP-5b compared with ZOL in the C-26 tumor model. These data consistently demonstrate that greater suppression of bone resorption is associated with better control of hypercalcemia.
In the PTHrP study, both OPG and APD caused significant and similar increases in cancellous bone volume in PBS-challenged mice. Twice-daily PTHrP challenges resulted in even greater gains in cancellous bone volume, which is consistent with the known anabolic effects of intermittent PTHrP (53). Although APD treatment significantly blunted the ability of PTHrP to increase cancellous bone volume, OPG had no effect on this anabolic response. The blunting of PTH anabolism could have been related to the APD-mediated exacerbation of hypercalcemia and bone resorption (eroded surface) that occurred near the end of the treatment period. It is also possible that APD blunting of anabolism could be related to a poorly understood mechanism that has been previously described in postmenopausal women (54), elderly men (55), and aged sheep (56) treated with PTH plus bisphosphonate. In contrast, OPG has been shown to add to the anabolic effects of intermittent PTH in arthritic mice (57) and in aged osteopenic rats (58). OPG may cooperate with PTH cotherapy by effectively blocking the catabolic effects of PTH or by fully permitting the anabolic effects of PTH.
The present studies have important limitations that may have influenced the results. For OPG treatment, the sc route of administration and the doses employed were similar to those of regimens that were deemed safe and efficacious in clinical trials (59). All studies used a human OPG-Fc construct that is strongly immunogenic in normal mice and rats. It is therefore possible that the duration of OPG pharmacology in these studies was negatively impacted by host immune responses to this foreign protein. The doses of bisphosphonates used in these studies were not clinically relevant. We used a very high dose of bisphosphonate (5 mg/kg) to determine the maximum benefit that could be expected of this therapeutic class. The bisphosphonates were administered either by iv bolus injection (APD) or by sc injection (ZOL), which contrasts with the slower iv infusion recommended for the clinical use of APD and ZOL. The iv bolus infusion of APD in the C-26 study was associated with significant renal toxicity at the highest dose (15 mg/kg), and these mice died 3 d after treatment. Necropsies showed gross evidence of nephrotoxicity, a finding consistent with reports from other rodent studies in which APD was given at similar doses (60, 61). The iv bolus delivery of 5 mg/kg APD (and OPG) appeared to be well tolerated based on the lack of changes in uric acid, blood urea nitrogen, and creatinine measured at death (data not shown). It remains possible that subclinical renal deterioration occurred in mice treated with iv APD at 5 mg/kg, which would potentially influence renal handling of calcium. We elected to deliver ZOL by sc injection to avoid concerns about renal toxicity, which has been observed in rats with an iv dose of 3 mg/kg (62). Subcutaneous injections of ZOL may have reduced bioavailability, a concern we attempted to minimize by using very high doses (50- to 100-fold greater than clinically relevant doses). It is possible that slower iv infusion of ZOL would have resulted in more rapid suppression of hypercalcemia compared with the sc route.
It is important to note that in both models, hypercalcemia eventually returned in treated mice regardless of the antiresorptive used. This phenomenon has been described previously in bisphosphonate studies, but it was unclear whether the return of hypercalcemia was related to renal effects or to the inadequate control of osteoclasts (20, 37). In the present PTHrP study, hypercalcemia returned in APD-treated mice to levels even greater than those in untreated mice. Serum TRAP was also significantly elevated in these mice, suggesting that inadequate control of bone resorption was at least partly responsible for the return of hypercalcemia. However, hypercalcemia also returned to a lesser degree in OPG-treated mice despite clear evidence of significantly suppressed bone resorption. In these mice, it is likely that renal PTHrP responses were largely responsible for the return of hypercalcemia. The unfortunate conclusion from these data is that even the total suppression of bone resorption, if theoretically possible, might still be inadequate for effectively managing HHM when circulating PTHrP levels are persistently elevated.
The current studies provide the first direct comparisons of the effects of a RANKL inhibitor and bisphosphonates on hypercalcemia and bone histomorphometry. OPG caused more rapid and more complete inhibition of hypercalcemia and bone resorption markers compared with high doses of APD or ZOL in two models of HHM. These responses to OPG were accompanied by greater reductions in osteoclast numbers relative to those produced by either bisphosphonate. Hypercalcemia eventually returned in both models, but to a lesser extent after OPG treatment. These observations suggest that RANKL inhibitors may be particularly effective at controlling HHM, particularly when PTHrP is involved.
Acknowledgments
Holly B. Zoog assisted in the preparation of the manuscript.
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Address all correspondence and requests for reprints to: Dr. Paul J. Kostenuik, Metabolic Disorders Research, M/S 15-2-B, Amgen, Inc., 1 Amgen Center Drive, Thousand Oaks, California 91320-1789. E-mail: paulk@amgen.com.
Abstract
Humoral hypercalcemia of malignancy (HHM) is mediated primarily by skeletal and renal responses to tumor-derived PTHrP. PTHrP mobilizes calcium from bone by inducing the expression of receptor activator for nuclear factor-B ligand (RANKL), a protein that is essential for osteoclast formation, activation, and survival. RANKL does not influence renal calcium reabsorption, so RANKL inhibition is a rational approach to selectively block, and thereby reveal, the relative contribution of bone calcium to HHM. We used the RANKL inhibitor osteoprotegerin (OPG) to evaluate the role of osteoclast-mediated hypercalcemia in two murine models of HHM. Hypercalcemia was induced either by sc inoculation of syngeneic colon (C-26) adenocarcinoma cells or by sc injection of high-dose recombinant PTHrP (0.5 mg/kg, sc, twice per day). In both models, OPG (0.2–5 mg/kg) caused rapid reversal of established hypercalcemia, and the speed and duration of hypercalcemia suppression were significantly greater with OPG (5 mg/kg) than with high-dose bisphosphonates (pamidronate or zoledronic acid, 5 mg/kg). OPG also caused greater reductions in osteoclast surface and biochemical markers of bone resorption compared with either bisphosphonate. In both models, hypercalcemia gradually returned despite clear evidence of ongoing suppression of bone resorption by OPG. These data demonstrate that osteoclasts and RANKL are important mediators of HHM, particularly in the early stages of the condition. Aggressive antiresorptive therapy with a RANKL inhibitor therefore might be a rational approach to controlling HHM.
Introduction
HYPERCALCEMIA IS A significant complication of certain human malignancies, including squamous cell, renal cell, and breast carcinomas. Humoral hypercalcemia of malignancy (HHM) results primarily from the systemic activation of osteoclasts by tumor-derived factors (1, 2, 3). Tumor cell secretion of PTHrP is the principal mechanism of HHM (4, 5). PTHrP acts through PTH/PTHrP receptors in bone and kidney to stimulate osteoclastic bone resorption and calcium reabsorption, respectively (3, 6). The relative calcemic contributions of bone and kidney are difficult to quantify in HHM, although tumors that produce PTHrP appear to have a greater renal contribution to hypercalcemia than tumors that do not produce PTHrP (1, 2, 7). In the absence of effective inhibitors of renal calcium reabsorption, therapy for HHM has focused on antiresorptives (4). Bisphosphonates are effective in many cases of HHM, suggesting that osteoclasts play an important role in mediating hypercalcemia. Bisphosphonates are usually effective at reducing hypercalcemia when preceded by saline hydration (reviewed in Ref. 8). However, an informal meta-analysis of published clinical studies indicates that as many as one quarter of HHM patients fail to achieve normocalcemia after hydration and bisphosphonate therapy (7, 9, 10, 11, 12, 13, 14, 15, 16, 17). Bisphosphonate resistance has been observed in hypercalcemic patients treated with pamidronate (APD) (18) or zoledronic acid (ZOL) (19). Bisphosphonate resistance has been attributed to renal calcium reabsorption (5, 15) and, in some cases, to inadequate inhibition of bone resorption (1, 2, 15).
Animal studies have underscored the difficulty of controlling PTHrP-mediated hypercalcemia with antiresorptives. In rat studies, the infusion of PTHrP caused hypercalcemia that was only partially and transiently inhibited by treatment with a high dose (3 mg/kg) of APD or alendronate (20). It is unclear whether the persistence of hypercalcemia in these bisphosphonate-treated rats was related to residual bone resorption, renal calcemic effects, or both. It is difficult to determine the extent of residual bone resorption after bisphosphonate treatment, because these agents often fail to reduce osteoclast numbers in vivo (21, 22, 23). In some animal and human studies, bisphosphonate treatment has led to paradoxical increases in osteoclast numbers (10, 24, 25, 26, 27). Thus, osteoclast counts are not a reliable surrogate for the determination of bone resorption after bisphosphonate treatment (28), which has confounded attempts to assess the relative contributions of bone vs. kidney in HHM.
Osteoprotegerin (OPG) is a novel antiresorptive agent with a different mechanism of action than bisphosphonates. OPG is a soluble secreted protein of the TNF receptor family (29) that functions as a decoy receptor for receptor activator of NF-B ligand (RANKL) (30). RANKL is a TNF family member that binds to RANK on osteoclasts and their precursors to promote osteoclast differentiation, activation, and survival (30, 31, 32, 33, 34). The very existence of osteoclasts appears to require functional RANK-RANKL interactions, because mice that lack the genes for either RANKL (33) or RANK (35) are virtually devoid of osteoclasts. In rats, a single treatment with human recombinant OPG resulted in a greater than 95% reduction in osteoclast surface (36). Time-course analysis showed that both the suppression and the eventual recovery of osteoclast surface were temporally coordinated with changes in serum levels of recombinant OPG, biochemical markers of bone turnover, bone volume, and bone density (36). Thus, osteoclast surface appears to be a robust and sensitive measure of bone resorption after OPG treatment. This unique relationship could assist in distinguishing the relative contributions of bone and kidney to humoral hypercalcemia.
We have previously demonstrated that OPG delays the onset of hypercalcemia and reverses established hypercalcemia in a syngeneic mouse model of HHM. The sc C-26 colon adenocarcinoma model employed in those studies caused significant elevations in serum PTHrP in association with increased osteoclast surface (37). OPG treatment significantly delayed the onset of hypercalcemia in tumor-bearing mice, and this response was accompanied by a 99% reduction in osteoclast surface. Interestingly, hypercalcemia gradually returned in OPG-treated animals despite this profound reduction in osteoclast surface. Similar phenomena were observed in another murine model of HHM using a different RANKL inhibitor. RANK-Fc, a soluble extracellular fragment of RANK, significantly delayed the onset of hypercalcemia in nude mice bearing a PTHrP-secreting human lung tumor xenograft. Hypercalcemia eventually developed in these mice despite a 95% reduction in osteoclast numbers (38). These observations are consistent with an increasing contribution of renal calcium reabsorption with time in response to tumor-derived PTHrP. The control of hypercalcemia with OPG was previously shown to occur without changes in renal calcium reabsorption (39) or urinary calcium excretion (39, 40). OPG is therefore a suitable molecule to isolate the role of osteoclasts in the pathogenesis of HHM. Tumors may produce other proresorptive cytokines besides PTHrP, so we tested whether direct PTHrP challenge of mice would recapitulate the gradual return of hypercalcemia associated with the C-26 model despite continued osteoclast suppression by OPG. The effects of high-dose bisphosphonates were also examined to explore potential mechanisms of therapeutic resistance to bisphosphonate treatment.
Materials and Methods
Animals
All animal studies were conducted in accordance with federal animal care guidelines and in compliance with the Amgen institutional animal care and use committee. The C-26 studies used male CDF1 mice (10–12 wk old), and the PTHrP study used male BDF1 mice (4 wk old; Charles River Laboratories, Wilmington, MA). All mice were fed standard laboratory rodent chow (Harlan Teklad, Madison, WI) throughout the experiments. Each experimental group consisted of five mice.
C-26 tumor
C-26 cells were obtained from the Tumor Repository of the National Cancer Institute (Bethesda, MD). C-26 tumor cells were passaged sc, and dissected tumors were cut into fragments and cultured at 37 C in 5–6% CO2. Adherent cells were cultured in DMEM (Invitrogen Life Technologies, Inc., Grand Island, NY), 1x penicillin-streptomycin/glutamine (Invitrogen Life Technologies, Inc.), and 1x nonessential amino acids (Invitrogen Life Technologies, Inc.). Cell cultures were harvested by trypsinization (Invitrogen Life Technologies, Inc.) and resuspended in unsupplemented DMEM at a concentration of 2.5 x 106 cells/ml. Mice were injected sc in a shaved area of the right flank with 0.5 x 106 cells in a 200-μl cell suspension.
OPG vs. APD in a C-26 tumor model
Total serum calcium levels and body weights were determined daily starting 12 d after C-26 tumor inoculation. Mice were bled retroorbitally, and total serum calcium was determined with a Hitachi 717 serum chemistry analyzer (Roche, Indianapolis, IN). Normal serum calcium ranges were determined from 35 measurements of PBS-treated control mice. Normocalcemia was defined as the mean of these measurements [8.33 mg/dl (2.02 mmol/liter)] ± 2 SD [0.56 mg/dl (0.14 mmol/liter)]. Treatments were performed when an individual tumor-bearing mouse achieved a serum calcium level of 11 mg/dl or greater (2.75 mmol/liter; designated d 0). APD (Novartis, East Hanover, NJ) was suspended in PBS and injected once into the tail vein at a dose of 1.5, 5, or 15 mg/kg. The recombinant OPG used in these studies was a human OPG construct fused to the Fc portion of human IgG1 (29). OPG was suspended in PBS and injected once into the tail vein at doses of 0.2, 1, and 5 mg/kg. Blood was drawn from the retroorbital sinus daily for the next 5 d to monitor total serum calcium. Mice were then killed by isofluorane inhalation. The left tibia was harvested for histomorphometry.
OPG vs. ZOL in a C-26 tumor model
C-26 tumor cells were injected sc, and blood ionized calcium levels were monitored using a calcium/pH analyzer (model 634, Chiron Diagnostics, Norwood, MA). The normocalcemic range for this study, as determined by 35 measurements of PBS-treated control mice, was 1.29 ± 0.08 mmol/liter (mean ± 2 SD). Ten days after tumor inoculation, C-26 tumor-bearing mice were significantly hypercalcemic (mean ± 1 SD, 1.71 ± 0.35 mmol/liter). OPG (5 mg/kg) or ZOL (5 mg/kg) was injected once (sc) into tumor-bearing mice; tumor-free control mice were injected with PBS. Blood ionized calcium was monitored regularly for the next 96 h. Terminal serum was collected by cardiac puncture for analysis of tartrate-resistant acid phosphatase-5b (TRAP-5b), and the left tibia was harvested for histomorphometry. Serum TRAP-5b (Immunodiagnostic Systems, Inc., Fountain Hills, AZ) was measured in duplicate in accordance with the manufacturer’s instructions.
OPG vs. APD in PTHrP model of hypercalcemia
Mice were challenged twice daily (morning and evening, by sc injection), with 0.5 mg/kg PTHrP-(1–34) (Bachem, Torrance, CA). Control mice received PBS injections twice daily. Retroorbital blood was drawn exactly 3 h after each morning challenge to monitor changes in blood ionized calcium. Blood ionized calcium levels were determined using a calcium/pH analyzer (model 634, Chiron Diagnostics). The normocalcemic range for this study, as determined from 35 measurements of PBS-treated control mice, was 1.21 ± 0.08 mmol/liter (mean ± 2 SD). After 3 d of challenges, PTHrP-treated mice were significantly hypercalcemic (1.43 ± 0.01 mol/liter). At this time (d 0), mice were given a single iv injection of OPG (5 mg/kg), APD (5 mg/kg), or PBS. Mice continued to be challenged in the morning and evening with PTHrP or PBS. Retroorbital blood samples were obtained exactly 3 h after each morning or evening challenge to monitor blood ionized calcium. Terminal blood samples were drawn when the mice were killed (96 h after treatment) for clinical chemistry analysis. The left tibia was harvested for histomorphometry.
Tissue processing and histomorphometry
Tibiae were fixed in phosphate-buffered zinc formalin, decalcified in formic acid, embedded in paraffin, and sectioned as previously described (29). Sections were stained for TRAP activity (leukocyte acid phosphatase kit, Sigma-Aldrich Corp., St. Louis, MO) and counterstained with hematoxylin. The area of analysis consisted of 2 mm2 cancellous bone immediately distal to the proximal growth plate. Histomorphometry was performed by tracing the cancellous bone surface with the aid of a digital video camera attached to a microscope. Analysis was performed using Osteomeasure bone analysis software (Osteometrics, Inc., Decatur, GA). Osteoclasts were scored based on contact with the bone surface and TRAP-positive staining. Osteoblasts were scored based on contact with bone surface and plump cuboidal morphology.
Statistical analyses
The statistical significance of differences between groups for different time points was assessed using JMP statistical software (SAS Institute, Inc., Cary, NC). Comparisons were made using Student’s t test for comparison of data from two groups, Dunnett’s test for comparison of multiple treatment groups with a control, and the Tukey-Kramer test for comparisons between multiple treatment groups or controls. These latter two methods make allowance for multiple comparisons. A value of P < 0.05 was used to determine statistically significant differences.
Results
OPG vs. APD in a C-26 tumor model
The normal range for total serum calcium in untreated tumor-free mice (mean ± 2 SD) was 7.77–8.89 mg/dl (1.94–2.22 mmol/liter; Fig. 1, shaded box). The sc C-26 tumor caused severe hypercalcemia (11 mg/dl or 2.75 mmol/liter) in all mice within 12–14 d of tumor inoculation, at which time treatment was initiated (designated d 0). A single iv treatment with human OPG-Fc at 0.2, 1, or 5 mg/kg caused a significant decrease in serum calcium within 12 h (P < 0.05 for each dose level vs. PBS treatment), with calcium levels reaching a nadir at 48 h after treatment. Serum calcium was in the normal range for the 1 and 5 mg/kg dose groups (Fig. 1) at the nadir. In contrast, serum calcium in PBS-treated, tumor-bearing mice was at a peak level of 14.46 mg/dl at 48 h. Mild hypercalcemia returned after the 48 h nadir in OPG-treated mice, although levels remained significantly lower than those in PBS-treated, tumor-bearing mice (P < 0.05). Treatment of tumor-bearing mice with the highest dose of APD (15 mg/kg) was associated with a transient and significant increase in serum calcium at 12 h (compared with PBS treatment), followed by a progressive reduction in serum calcium levels (Fig. 1). These animals died between the third and fourth study day, and gross examination during necropsies revealed a white precipitate in the kidneys. These lesions were presumed to be drug substance that may have precipitated as a result of the bolus iv administration of APD (41). Lower doses of APD (1.5 and 5 mg/kg) caused a gradual reduction of serum calcium in tumor-bearing mice that reached a nadir on d 4. Statistically significant reductions in serum calcium were obtained from d 2–5 after APD treatment (P < 0.05), although serum calcium levels remained above the normocalcemic range throughout the study.
FIG. 1. Serum calcium responses in C-26 tumor-bearing mice treated with OPG or APD. Hypercalcemic CDF1 mice bearing sc C-26 tumors were treated with PBS (), APD (, 1.5 mg/kg; , 5 mg/kg; , 15 mg/kg), or OPG (, 0.2 mg/kg; , 1 mg/kg; , 5 mg/kg). Nontumor-bearing mice were treated with PBS (). The shaded box represents the normal calcium range (mean ± 2 SD) obtained from 35 measurements of untreated, nontumor-bearing mice of the same age, sex, and strain. Data are expressed as means (n = 5/group). Statistically significant differences (P < 0.05) are described in Results.
C-26 tumor burden was associated with a nonsignificant reduction in cancellous bone volume in the proximal tibial metaphysis compared with tumor-free controls. Both OPG and APD caused significant gains in bone volume in tumor-bearing mice (P < 0.05; data not shown). The percent osteoclast surface to bone surface was increased by 3-fold in tumor-bearing mice compared with tumor-free mice (P < 0.05; Fig. 2A), and OPG caused a dose-dependent reduction in osteoclast surface, reaching levels significantly lower than those in normal tumor-free mice at the highest dose (P < 0.05). APD at 5 mg/kg reduced the osteoclast surface in tumor-bearing mice to levels similar to those found in tumor-free mice. Osteoblast surface was significantly increased in tumor-bearing mice, and both OPG and APD prevented this increase at all doses examined (Fig. 2B).
FIG. 2. Bone histomorphometry of the proximal tibial metaphysis in C-26 tumor-bearing mice treated with OPG or APD. Nontumor-bearing control mice were treated with PBS. Tumor-bearing mice were treated once (iv) with PBS, OPG, or APD at the indicated concentrations. Mice were killed 5 d later, and tibiae were harvested for histomorphometry. Data were not available from mice treated with APD at 15 mg/kg due to extensive mortality. A, Osteoclast surface as a percentage of total bone surface (OcS/BS); B, osteoblast surface as a percentage of total bone surface (ObS/BS). Data are expressed as the mean ± SEM (n = 5/group). a, Significant difference from PBS-treated, tumor-bearing mice; b, significant difference from PBS-treated, nontumor-bearing mice (P < 0.05, by two-way ANOVA and Dunnett’s test).
The effects of tumor burden and treatments on tibial histology are indicated in Fig. 3. Photomicrographs of representative TRAP-stained sections demonstrate the typical pharmacological effects of the highest tolerated dose of each drug (5 mg/kg).
FIG. 3. Representative photomicrographs of the proximal tibial metaphysis in C-26 tumor-bearing mice treated with OPG or APD. Sections were stained for TRAP (purple) to highlight osteoclasts (OCs; see arrow). A, Tumor-free mouse treated with PBS; B, tumor-bearing mouse treated with PBS; C, tumor-bearing mouse treated with APD (5 mg/kg); D, tumor-bearing mouse treated with OPG (5 mg/kg).
OPG vs. ZOL in a C-26 tumor model
The normal range of blood ionized calcium in this study was 1.21–1.37 mmol/liter (see shaded box, Fig. 4). Tumor-bearing mice had moderate hypercalcemia at the time of treatment (1.71 ± 0.35 mmol/liter), and this hypercalcemia progressed dramatically over the ensuing 96 h. OPG (5 mg/kg) caused significant suppression of hypercalcemia from 24–96 h after treatment (P < 0.05 vs. PBS-treated, tumor-bearing mice). Blood ionized calcium was restored to within the normal range within 24 h of OPG treatment. ZOL (5 mg/kg) caused a significant suppression of hypercalcemia from 48–96 h after treatment (P < 0.05 vs. PBS-treated, tumor-bearing mice). Antiresorptive effects were also assessed at the time the mice were killed by monitoring serum TRAP-5b, an osteoclast-specific marker of bone resorption. C-26 tumor burden was associated with a 2-fold increase in serum TRAP-5b (P < 0.05 vs. tumor-free mice; Fig 5). This increase was fully prevented by single treatments with either OPG or ZOL. OPG suppressed serum TRAP-5b to levels significantly lower than those in ZOL-treated, tumor-bearing mice or normal, tumor-free mice (P < 0.05).
FIG. 4. Blood ionized calcium in C-26 tumor-bearing mice treated with OPG or ZOL. Hypercalcemic CDF1 mice bearing sc C-26 tumors were treated once (sc) with PBS (), OPG (5 mg/kg; s), or ZOL (5 mg/kg; ). Nontumor-bearing mice were treated with PBS (). The shaded box represents the normal calcium range (mean ± 2 SD) obtained from 35 measurements of PBS-treated, nontumor-bearing mice of the same age, sex, and strain. Data are expressed as the mean ± SEM (n = 5/group). *, Significantly different from PBS-treated, tumor-bearing mice, P < 0.05. BL, Baseline.
FIG. 5. Serum TRAP-5b in C-26 tumor-bearing mice treated with OPG or ZOL. Hypercalcemic CDF1 mice bearing sc C-26 tumors were treated once (sc) with PBS, OPG (5 mg/kg), or ZOL (5 mg/kg). Nontumor-bearing mice were treated with PBS. Serum was obtained when mice were killed (4 d after treatment) and was assayed for serum TRAP-5b, an osteoclast-specific marker of bone resorption. Data are expressed as the mean ± SEM (n = 5/group). a, Significantly different from nontumor-bearing mice; b, significantly different from PBS-treated, tumor-bearing mice (P < 0.05, by two-way ANOVA and Dunnett’s test).
Cancellous bone volume in the proximal tibial metaphysis was not significantly different among any groups (data not shown). C-26 tumor burden was associated with a 3-fold increase in osteoclast surface in the tibia (P < 0.05 vs. tumor-free controls; Fig. 6A), and OPG and ZOL both caused significant reductions in osteoclast surface in tumor-bearing mice (P < 0.05 vs. PBS-treated mice). OPG suppressed osteoclast surface to levels significantly lower than those in ZOL-treated, tumor-bearing mice or normal, tumor-free mice (P < 0.05). Osteoblast surface was not significantly influenced by tumor burden in this study. OPG caused a modest and nonsignificant reduction in osteoblast surface, whereas ZOL caused a marginally greater reduction that achieved statistical significance (Fig. 6B; P < 0.05).
FIG. 6. Bone histomorphometry of C-26 tumor-bearing mice treated with OPG or ZOL. The region of analysis consisted of 2 mm2 cancellous bone immediately distal to the proximal metaphyseal growth plate. Hypercalcemic CDF1 mice bearing sc C-26 tumors were treated once (sc) with PBS, OPG (5 mg/kg), or ZOL (5 mg/kg). Nontumor-bearing mice were treated with PBS. A, Osteoclast surface as a percentage of total bone surface (OcS/BS); B, osteoblast surface as a percentage of total bone surface (ObS/BS). Data are expressed as the mean ± SEM (n = 5/group). a, Significant difference from PBS-treated tumor-free mice, P < 0.05; b, significant difference from PBS-treated, tumor-bearing mice, P < 0.05; c, significant difference from ZOL-treated, tumor-bearing mice, P < 0.05.
OPG vs. APD in PTHrP model of hypercalcemia
The normal range of blood ionized calcium for PBS-challenged mice in the PTHrP study was 1.14–1.29 mmol/liter (Fig. 7, shaded box). Three days of PTHrP challenges resulted in significantly higher levels of blood ionized calcium compared with PBS-challenged mice (P < 0.05 vs. PBS-challenged mice). Mice then received a single iv injection of OPG or APD on d 0, and PTHrP challenges continued for an additional 4 d. OPG (5 mg/kg) restored blood ionized calcium to the normal range within 3 h, and normocalcemia was maintained for at least 24 h. Hypercalcemia returned in OPG-treated mice 2 d after OPG treatment. A single iv injection of APD (5 mg/kg) resulted in normocalcemia at 24 h, after which blood ionized calcium increased progressively. At the end of the study, PTHrP-challenged mice treated with APD had significantly higher blood ionized calcium compared with PTHrP-challenged mice treated with PBS (P < 0.05).
FIG. 7. Blood ionized calcium responses in PTHrP-challenged mice treated with OPG or APD. Hypercalcemia was induced by 3 d of twice-daily (morning and evening) sc injections of PTHrP-(1–34) (0.5 mg/kg/injection). PTHrP challenges continued during the treatment period. PTHrP-challenged mice were treated with single iv injections of PBS (), APD (5 mg/kg; ), or OPG (5 mg/kg; ). Normocalcemic (i.e. PBS-challenged mice) were treated with single iv injections of PBS (), APD (5 mg/kg; ), or OPG (5 mg/kg; ). Retroorbital blood was drawn exactly 3 h after each morning challenge with PTHrP (or PBS) for ionized calcium determinations. The shaded box represents the normal calcium range (mean ± 2 SD) obtained from 28 measurements of untreated mice of the same age, sex, and strain. Data are expressed as the mean ± SEM (n = 5/group). Statistically significant differences are indicated in Results.
Total serum TRAP was measured as a systemic marker of osteoclast activity. In PBS-challenged mice, both OPG and APD treatments caused nonsignificant reductions in serum TRAP (Fig. 8). In PTHrP-challenged mice, OPG treatment significantly reduced serum TRAP (P < 0.05 vs. PBS-treated controls), whereas APD treatment had no significant effect.
FIG. 8. Bone resorption (serum TRAP) in PTHrP-challenged mice treated with OPG or APD. Hypercalcemia was induced by 3 d of twice-daily (morning and evening) sc injections of either PBS or PTHrP (0.5 mg/kg/injection). Mice were then treated with single iv injections of PBS, OPG (5 mg/kg), or APD (5 mg/kg). Data are expressed as the mean ± SEM (n = 5/group). a, Significant difference from PBS-treated mice under similar challenge conditions (P < 0.05, by two-way ANOVA and Dunnett’s test).
Osteoclast surface in PBS-challenged mice was significantly reduced by OPG (P < 0.05), but not by APD treatment (Fig. 9A). In PTHrP-challenged mice, OPG treatment caused an 85% reduction in osteoclast surface (P < 0.05 vs. PBS-treated mice), whereas APD treatment led to a paradoxical 50% increase in osteoclast surface (P < 0.05 vs. PBS-treated mice). Eroded surface was dramatically suppressed by OPG in both PBS-challenged and PTHrP-challenged mice, whereas APD treatment failed to significantly reduce eroded surface in PBS-challenged mice (data not shown). In PTHrP-challenged mice, APD treatment caused a 2.5-fold increase in eroded surface compared with PBS-treated PTHrP-challenged mice (P < 0.05; data not shown).
FIG. 9. Bone histomorphometry of PTHrP-challenged mice treated with OPG or APD. The region of analysis consisted of 2 mm2 cancellous bone immediately distal to the proximal metaphyseal growth plate of the tibia. Mice were challenged for 3 d with twice-daily (morning and evening) sc injections of PBS or PTHrP (0.5 mg/kg/injection). Mice were then cotreated with single iv injections of PBS, OPG (5 mg/kg), or APD (5 mg/kg). A, Osteoclast surface as a percentage of total bone surface (OcS/BS); B, osteoblast surface as a percentage of total bone surface (ObS/BS); C, bone volume as a percentage of total volume (BV/TV). Data are expressed as the mean ± SEM (n = 5/group). a, Significant difference from PBS-challenged mice that were treated with PBS; b, significant difference from PTHrP-challenged mice that were treated with PBS (P < 0.05, by two-way ANOVA and Dunnett’s test).
Osteoblast surface in PBS-challenged mice was significantly reduced by both APD and OPG treatments (P < 0.05; Fig. 9B). PTHrP challenge alone led to a significant increase in osteoblast surface. APD treatment of PTHrP-challenged mice had no effect on osteoblast surface compared with PTHrP-challenged controls. OPG reduced osteoblast surface in PTHrP-challenged mice, although values remained greater than those in PBS-treated mice.
In PBS-treated mice, OPG caused a significant increase in cancellous bone volume at the proximal tibial metaphysis (P < 0.05 vs. PBS-treated controls; Fig. 9C). APD treatment caused an appreciable, but statistically insignificant, increase in bone volume. Twice-daily PTHrP challenges also caused a significant increase in bone volume, which was an expected pharmacological effect of intermittent PTHrP injections. In these PTHrP-challenged mice, treatment with APD caused significant blunting of the anabolic effect of PTHrP on bone volume (P < 0.05 vs. PBS-treated mice), although values remained higher than those in PBS-treated mice. OPG caused no blunting of PTHrP-mediated increases in bone volume (Fig. 9C).
Discussion
HHM is one of the most prevalent and clinically significant paraneoplastic syndromes. The typical etiology for HHM is the tumor production of factors such as PTHrP that systemically activate osteoclasts. PTHrP also stimulates renal calcium reabsorption, which can exacerbate hypercalcemia. In the absence of specific inhibitors of renal calcium reabsorption, therapy for HHM has focused on antiresorptives, such as bisphosphonates. Most HHM patients respond favorably to bisphosphonates when the drugs are preceded by saline hydration (3, 9, 42, 43, 44), but a significant number of patients remain hypercalcemic after these treatments (10, 45, 46, 47, 48). Resistance to APD in some HHM patients was highly correlated with pretreatment nephrogenous cAMP levels, which is consistent with a PTHrP-mediated renal response (49). In other studies, resistance to bisphosphonates was attributed to inadequate inhibition of bone resorption (1, 2, 15). Greater doses of bisphosphonates might provide better control of hypercalcemia, but treatment with iv bisphosphonates such as APD (50) or ZOL is limited by concerns about renal toxicity (50, 51). Because of these limitations, other classes of antiresorptives, such as RANKL inhibitors, may be warranted for patients who are resistant to bisphosphonate therapy.
RANKL inhibitors might be particularly well suited for inhibiting osteoclast-mediated hypercalcemia, particularly when PTHrP is involved. It is clear that functional RANK/RANKL interactions are essential for PTHrP-mediated bone resorption, because PTHrP fails to cause hypercalcemia or induce the appearance of osteoclasts in RANK–/– mice (35). Recombinant OPG, the native RANKL inhibitor, blocks the hypercalcemic response of mice injected with either PTHrP (52) or recombinant RANKL (30). OPG was previously shown to reverse hypercalcemia within 2 h in mice and rats, an effect that was unrelated to changes in renal calcium reabsorption (39) or urinary calcium excretion (39, 40). OPG induces osteoclast apoptosis in mice within 6 h (33). These rapid effects on bone resorption are probably related to the unique mechanism of action for RANKL inhibitors. RANKL inhibitors work without binding to a bone matrix substrate and can therefore cause a nearly immediate arrest of osteoclast formation, activation, and survival. In contrast, bisphosphonates must bind to bone matrix and then be liberated or consumed by osteoclasts to suppress bone resorption. Whether these fundamental mechanistic differences result in meaningful differences in therapeutic responses was previously unknown, due in part to the lack of direct comparisons.
The current studies provide the first direct comparisons of the effects of a RANKL inhibitor vs. bisphosphonates on hypercalcemia and bone histomorphometry. In two different models of HHM, the RANKL inhibitor OPG caused a more rapid reversal of established hypercalcemia compared with APD or ZOL, which represent the current standard of care for the treatment of hypercalcemia. OPG treatment also resulted in consistently greater reductions in osteoclast surface and bone resorption markers, and these responses were associated with the rapid and sustained reversal of hypercalcemia. In the PTHrP challenge study, OPG caused a dramatic reduction in osteoclast surface that was associated with consistent suppression of hypercalcemia, whereas APD caused a paradoxical increase in osteoclast surface that was associated with the exacerbation of hypercalcemia. OPG also caused greater reductions in osteoclast surface and serum TRAP-5b compared with ZOL in the C-26 tumor model. These data consistently demonstrate that greater suppression of bone resorption is associated with better control of hypercalcemia.
In the PTHrP study, both OPG and APD caused significant and similar increases in cancellous bone volume in PBS-challenged mice. Twice-daily PTHrP challenges resulted in even greater gains in cancellous bone volume, which is consistent with the known anabolic effects of intermittent PTHrP (53). Although APD treatment significantly blunted the ability of PTHrP to increase cancellous bone volume, OPG had no effect on this anabolic response. The blunting of PTH anabolism could have been related to the APD-mediated exacerbation of hypercalcemia and bone resorption (eroded surface) that occurred near the end of the treatment period. It is also possible that APD blunting of anabolism could be related to a poorly understood mechanism that has been previously described in postmenopausal women (54), elderly men (55), and aged sheep (56) treated with PTH plus bisphosphonate. In contrast, OPG has been shown to add to the anabolic effects of intermittent PTH in arthritic mice (57) and in aged osteopenic rats (58). OPG may cooperate with PTH cotherapy by effectively blocking the catabolic effects of PTH or by fully permitting the anabolic effects of PTH.
The present studies have important limitations that may have influenced the results. For OPG treatment, the sc route of administration and the doses employed were similar to those of regimens that were deemed safe and efficacious in clinical trials (59). All studies used a human OPG-Fc construct that is strongly immunogenic in normal mice and rats. It is therefore possible that the duration of OPG pharmacology in these studies was negatively impacted by host immune responses to this foreign protein. The doses of bisphosphonates used in these studies were not clinically relevant. We used a very high dose of bisphosphonate (5 mg/kg) to determine the maximum benefit that could be expected of this therapeutic class. The bisphosphonates were administered either by iv bolus injection (APD) or by sc injection (ZOL), which contrasts with the slower iv infusion recommended for the clinical use of APD and ZOL. The iv bolus infusion of APD in the C-26 study was associated with significant renal toxicity at the highest dose (15 mg/kg), and these mice died 3 d after treatment. Necropsies showed gross evidence of nephrotoxicity, a finding consistent with reports from other rodent studies in which APD was given at similar doses (60, 61). The iv bolus delivery of 5 mg/kg APD (and OPG) appeared to be well tolerated based on the lack of changes in uric acid, blood urea nitrogen, and creatinine measured at death (data not shown). It remains possible that subclinical renal deterioration occurred in mice treated with iv APD at 5 mg/kg, which would potentially influence renal handling of calcium. We elected to deliver ZOL by sc injection to avoid concerns about renal toxicity, which has been observed in rats with an iv dose of 3 mg/kg (62). Subcutaneous injections of ZOL may have reduced bioavailability, a concern we attempted to minimize by using very high doses (50- to 100-fold greater than clinically relevant doses). It is possible that slower iv infusion of ZOL would have resulted in more rapid suppression of hypercalcemia compared with the sc route.
It is important to note that in both models, hypercalcemia eventually returned in treated mice regardless of the antiresorptive used. This phenomenon has been described previously in bisphosphonate studies, but it was unclear whether the return of hypercalcemia was related to renal effects or to the inadequate control of osteoclasts (20, 37). In the present PTHrP study, hypercalcemia returned in APD-treated mice to levels even greater than those in untreated mice. Serum TRAP was also significantly elevated in these mice, suggesting that inadequate control of bone resorption was at least partly responsible for the return of hypercalcemia. However, hypercalcemia also returned to a lesser degree in OPG-treated mice despite clear evidence of significantly suppressed bone resorption. In these mice, it is likely that renal PTHrP responses were largely responsible for the return of hypercalcemia. The unfortunate conclusion from these data is that even the total suppression of bone resorption, if theoretically possible, might still be inadequate for effectively managing HHM when circulating PTHrP levels are persistently elevated.
The current studies provide the first direct comparisons of the effects of a RANKL inhibitor and bisphosphonates on hypercalcemia and bone histomorphometry. OPG caused more rapid and more complete inhibition of hypercalcemia and bone resorption markers compared with high doses of APD or ZOL in two models of HHM. These responses to OPG were accompanied by greater reductions in osteoclast numbers relative to those produced by either bisphosphonate. Hypercalcemia eventually returned in both models, but to a lesser extent after OPG treatment. These observations suggest that RANKL inhibitors may be particularly effective at controlling HHM, particularly when PTHrP is involved.
Acknowledgments
Holly B. Zoog assisted in the preparation of the manuscript.
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