This work was presented in abstract form at the American Society of Bone and Mineral Research meetings, Seattle, Washington, U.S.A., 1996 and Cincinnati, Ohio, U.S.A., 1997.
Zoledronate Prevents the Development of Absolute Osteopenia Following Ovariectomy in Adult Rhesus Monkeys†
Article first published online: 1 NOV 1998
Copyright © 1998 ASBMR
Journal of Bone and Mineral Research
Volume 13, Issue 11, pages 1775–1782, November 1998
How to Cite
Binkley, N., Kimmel, D., Bruner, J., Haffa, A., Davidowitz, B., Meng, C., Schaffer, V. and Green, J. (1998), Zoledronate Prevents the Development of Absolute Osteopenia Following Ovariectomy in Adult Rhesus Monkeys. J Bone Miner Res, 13: 1775–1782. doi: 10.1359/jbmr.19188.8.131.525
- Issue published online: 4 DEC 2009
- Article first published online: 1 NOV 1998
- Manuscript Accepted: 15 JUL 1998
- Manuscript Revised: 22 JUN 1998
- Manuscript Received: 6 MAR 1998
This study assessed effects of the bisphosphonate zoledronate (ZLN) on bone density and biochemical markers of bone turnover in ovariectomized (OVX) adult female rhesus monkeys. Forty monkeys were randomly assigned to one control or four OVX groups. The control and one OVX group received saline, and the other three OVX groups received ZLN (0.5, 2.5, or 12.5 μg/kg) by a single weekly subcutaneous injection for 69 weeks. Bone mass of the total body (TB), lumbar spine (LS), distal and central radius (dual-energy X-ray absorptiometry), and skeletal turnover markers were measured at baseline and at 13, 26, 39, 52, and 69 weeks of treatment. Increased skeletal turnover and decreased bone mass (LS and TB) were demonstrable by 13 weeks post-OVX. Maximal bone loss (7–8%) at these sites occurred by 39 weeks after OVX and persisted for the study duration. Long-term ZLN treatment was well tolerated and prevented increased skeletal turnover and bone loss in a dose-dependent fashion. Progressive turnover suppression was not observed with any ZLN dose. In conclusion, after OVX, adult rhesus monkeys develop persistent increased bone turnover and absolute osteopenia of the LS and TB, making them an outstanding model of skeletal behavior in perimenopausal women. These OVX-related skeletal changes are dose-dependently blocked by ZLN.
NONHUMAN PRIMATES share many skeletal and hormonal similarities with humans including macro- and microskeletal structure, cancellous and cortical bone remodeling processes,1,2 regular menses,3,4 and natural menopause.5 Furthermore, the age of skeletal maturity is well documented,6,7 and increased skeletal turnover and bone loss following ovariectomy (OVX) have been demonstrated.8–10 As a result, nonhuman primates are currently the preferred large animal model of human skeletal physiology, particularly Haversian remodeling, not only following surgical11,12 or pharmaceutical12 estrogen depletion but also for evaluating potential osteoporosis therapies.13
The bisphosphonate class of compounds acts via inhibition of osteoclastic bone resorption.14 These agents decrease skeletal turnover and prevent bone loss following OVX in small15 and large8 animals, and are efficacious in the prevention of postmenopausal bone loss and the reduction of osteoporotic fracture in humans.16,17 It is therefore of interest to investigate whether a potent bisphosphonate,18 such as zoledronate (ZLN), would reduce skeletal turnover and preserve bone mass following OVX in animals that replicate human skeletal physiology, i.e., nonhuman primates. The purpose of this study was to establish the utility of the OVX nonhuman primate model in the profiling of pharmaceutical agents affecting skeletal metabolism by assessing the dose effectiveness of ZLN on bone mineral density (BMD) and markers of skeletal turnover.
MATERIALS AND METHODS
This protocol was approved by the University of Wisconsin Institutional Animal Care and Use Committee. Forty nonpregnant female premenopausal rhesus monkeys aged 10.1–19.9 years (mean 15.2 years), that had been bred and reared in captivity, were selected from the Wisconsin Regional Primate Research Center colony. The animals were selected for homogeneity with exclusion criteria including parturition or lactation within the last year, degenerative arthritis of the lumbar spine (LS), or abnormal (above or below mean ± 2 SD) levels of estradiol or biochemical markers of bone metabolism in the blood or urine. They were individually housed for 1 year prior to and for the duration of the study, and maintained on a 12 h light/dark cycle. Temperature and humidity were monitored continuously and maintained between 21°C and 26°C and 30–70%, respectively. Each animal was allowed ad libitum access to tap water and certified monkey chow (#5048; Purina, St. Louis, MO, U.S.A.) containing ∼1% calcium, 0.7% phosphorus, and 6.6 IU vitamin D/g. The diet was supplemented with fresh fruit (apples, bananas, and grapes) approximately every other day. The animals were randomized by total body bone mineral content (TBBMC) into five groups (n = 8/group). The monkeys of four groups were OVX at baseline via a midline ventral incision, while under halothane anesthesia administered via endotracheal tube. The fifth group served as intact, non-OVX controls. Two animals died during the study, one of respiratory compromise caused by an oral soft tissue polyp at week 66, the other of respiratory arrest due to anesthesia during dual-energy X-ray absorptiometry (DXA) scanning at week 13.
At all sampling time points (baseline and weeks 13, 26, 39, 52, and 69), blood was obtained without the use of anesthesia by femoral vein phlebotomy between 0700 and 0900 h following an overnight fast. These samples were allowed to clot for 30–45 minutes at room temperature, centrifuged at 3300–3700 rpm for at least 10 minutes, and aliquotted. These aliquots were frozen at −20°C until thawed for analysis.
Baseline urine samples were collected via bladder catheterization. Because this technique frequently yielded only small quantities of urine, specimens at subsequent time points were obtained via metabolic cage. For this, the animals were placed alone in a clean metabolic cage in a dark room with no access to food at 1500 ± 1 h. The following morning, between 0700 and 0800 h, all urine in the metabolic cage pan was removed. These samples were aliquotted within 2 hs and frozen at −20°C until analysis. Both the syringe used to collect the specimens and all aliquots were protected from light.
The following endpoints of skeletal relevance were measured:
• serum: osteocalcin, total alkaline phosphatase (ALP), skeletal alkaline phosphatase (sALP), tartrate-resistant acid phosphatase (TRAP), calcium, creatinine, intact parathyroid hormone (PTH), calcitonin, estradiol, phosphorus, and 1,25-dihydroxyvitamin D (1,25(OH)2D).
• urine: N-telopeptides of type I collagen (NTx), deoxypyridinoline (Dpd), pyridinoline (Pyd), hydroxyproline, calcium, phosphorus, and creatinine.
These assays were validated and conducted by either ClinTrials/BioResearch (Senneville, PQ, Canada) or Novartis Pharmaceuticals Laboratories (Summit, NJ, U.S.A.). The reagents/kits utilized and the intra- and interassay coefficients of variation (CVs) are reported in Table 1.
DXA scans of the total body (TB), L2–L4 LS, proximal femur, central radius (CR), and distal radius (DR) were performed at baseline and weeks 13, 26, 39, 52, and 69 utilizing ketamine/xylazine anesthesia and standardized positioning as previously described.6 All DXA scans were performed using a Lunar DPX-L densitometer (Madison, WI, U.S.A.) with pediatric and small animal software and analyzed by one operator. The radius regions of interest (ROIs) were defined as follows: DR, a 2 mm ROI placed 10–12 mm proximal to the distal tip of the radius, and CR, a 5 mm ROI placed 47.5–52.5 mm proximal to the distal tip of the radius. DXA BMD precision (% CV) for TB, LS, CR, and DR was 1.1, 1.4, 1.8, and 3.3%, respectively. Although the quality of the proximal femur scans appeared excellent on visual examination, the standard analysis routines provided unreliable results. Work is ongoing to modify and validate the software to permit reliable analysis of these femur scans.
All animals received weekly subcutaneous injections (0.1 ml/kg) of ZLN or the saline vehicle for 69 weeks. Three OVX treatment groups received ZLN at 0.5, 2.5, or 12.5 μg/kg/week. One OVX and the nonoperated group received the saline vehicle. ZLN was supplied as a lyophilized powder by Novartis Pharmaceuticals and was reconstituted with sterile saline immediately prior to each weekly administration. The study duration and dose increments were selected in accordance with the Food and Drug Administration draft guidelines for profiling osteoporosis treatment agents.19 Doses and dosing schedules were selected on the basis of prior rat studies and preliminary efficacy data from phase I clinical trials.18,20,21
All animals underwent open iliac crest and rib biopsies at baseline and 26 weeks. At necropsy, uterine weight was measured and skeletal samples for histomorphometry and biomechanical testing were obtained. Dual fluorochrome labeling with calcein, oxytetracycline, and xylenol orange was performed 15 and 5 days prior to bone sampling at baseline, 26 and 69 weeks, respectively. All histomorphometric and biomechanical data will be reported later.
At predose baseline time points, analysis of variance F-test for the equality of group means22 was performed. At postdose time points, two-sided T-maximum trend tests based on the maximum contrast on the group means23 were employed on percentage changes from the baseline value to detect possible (monotonically increasing or decreasing) drug effects. Since these tests are sensitive to the violation of the assumption of homogeneous group variances, data were scrutinized for extreme deviation from this assumption. If heterogeneous variances were detected via a significant Bartlett's test at p < 0.001,24,25 robust versions of the analysis of variance test26 and the trend test,27 which did not require the assumption of equal group variances, were then applied. If the initial trend test was significant, the same trend test was reapplied to the remaining dose groups after excluding the highest dose group (this follow-up trend test was one-sided in the direction indicated by the initial trend test). This procedure was continued until no significant trend was detected or until all the dose groups had been exhausted. The application of follow-up trend tests in the manner described above would lead to the identification of statistical significance at lower dose levels. Individual animal BMD data were adjusted for a DXA scanner baseline shift that affected measurements collected from weeks 26–69. The adjustment was based on interpolation from a regression line, estimated from BMD phantom data collected for the DXA scanner.
Transient decreases were observed following both the baseline and 26-week surgical procedures in all groups (Fig. 1). Body weight at baseline and termination did not differ. Neither OVX nor ZLN administration was associated with weight change or clinically apparent adverse effects.
Dual-energy X-ray absorptiometry
An absolute reduction was observed in the TBBMC of the OVX-saline group (Fig. 2A). TBBMC was 5.4% lower at 13 weeks and progressed to 7.4% lower at 26 weeks; no further change was observed. The OVX-induced loss was dose dependently prevented by ZLN at both the 2.5 and 12.5 μg/kg/week doses. A 4.1% increase from baseline was observed with the 12.5 μg/kg/week ZLN dose at 69 weeks. At study termination, TBBMC in the group receiving ZLN 12.5 μg/kg/week was 10.9% higher than the OVX-saline group. A trend toward TBBMC preservation that did not reach statistical significance was observed in the group receiving ZLN 0.5 μg/kg/week.
Lumbar spine (L2–L4) BMD:
An absolute reduction was observed in the lumbar spine bone mineral density (LSBMD) of the OVX-saline group (Fig. 2B). The LSBMD was reduced from baseline by 5.1% at 13 weeks and progressed to an 8.4% reduction at 39 weeks with no subsequent change. This OVX-induced loss was prevented by ZLN at both 2.5 and 12.5 μg/kg/week. A 2.6% increase in LSBMD over baseline was observed with 12.5 μg/kg/week at 69 weeks. At study termination, LSBMD in the group receiving ZLN 12.5 μg/kg/week was 11.5% higher than the OVX-saline group. No effect on LSBMD was observed in the group receiving ZLN 0.5 μg/kg/week.
DR and CR BMD:
BMD at the radial sites decreased equally in both control and OVX-saline groups (Figs. 2C and 2D). The loss was greater at the distal (8.7% control, 10.1% OVX) than the central (4.9 control, 6.3% OVX) site. BMD decreased for the first 39 weeks, after which no further bone loss occurred. At both distal and central sites, these losses were prevented by ZLN at a dose of 12.5 μg/kg/week. No effect was observed with ZLN at 0.5 or 2.5 μg/kg/week.
Successful OVX was first confirmed by the reduction of the serum estradiol concentration to essentially undetectable levels by 13 weeks post-OVX (Table 2) which persisted for 69 weeks. Functional deficiency was confirmed by the observation of uterine atrophy at necropsy (Table 2).
Increased skeletal turnover, as measured by elevated serum osteocalcin, ALP, sALP, and TRAP, and by urinary NTx, Pyd, and Dpd, was present by 13 weeks after OVX, which persisted for the study duration. The serum osteocalcin and urinary NTx declined toward baseline levels after 39 weeks. ZLN suppressed the OVX-induced turnover increase in a dose-dependent manner (Figs. 3A, 3B, 3C, 3D).
The serum concentration of PTH, calcium, phosphorus, and calcitonin was not altered by OVX. Serum calcium was reduced at week 26, and PTH was elevated at week 52 in the 12.5 μg/kg/week ZLN treatment group (data not shown).
No OVX- or ZLN-induced changes were observed in serum creatinine. However, a transient increase in creatinine concentration of ∼10% was observed in all groups at 26 weeks; this returned to baseline at 39 weeks (data not shown.)
The primary objective of this study was to evaluate the effect of three doses of ZLN upon OVX-induced changes in bone turnover and loss in skeletally mature female rhesus monkeys. In addition, we clearly defined the skeletal effects of OVX in adult rhesus monkeys and demonstrated the utility of this primate model for profiling response to osteotropic compounds. OVX in these animals induced skeletal changes that closely mimic those observed at menopause in humans. However, bone mass stabilizes by 9 months post-OVX in these animals, but not for about 5 years after menopause in humans.28,29 Thus, this model provides a 6-fold timeframe compression for studying estrogen depletion-induced bone loss in a species with cortical and cancellous bone physiology that closely matches humans. Both increases in serum and urine markers of bone turnover and an ∼5% reduction in LS and TB bone mineral were demonstrable by 13 weeks post-OVX. Comparable amounts of estrogen depletion-induced bone loss in humans are often not observed for 2 years, again suggesting considerable timeframe compression in observing estrogen depletion bone loss in the adult rhesus monkey. Maximal elevations in turnover markers persisted until 39 weeks post-OVX and were associated with continued bone loss. Subsequently, some of the turnover markers (serum osteocalcin, urinary NTx) declined toward baseline, and further bone loss was not observed. Thus, maximal bone loss appears to occur by 39 weeks post-OVX in these adult animals and persists at that level for at least another 6 months.
Nonhuman primates are increasingly utilized as a model to test pharmaceutical efficacy in bone loss prevention following estrogen depletion. Preliminary data suggest that estradiol replacement is efficacious, but the published experience remains limited.8,13,30,31 This study clearly defines the time course of changes in skeletal turnover and bone loss following estrogen depletion as well as the dose-dependent response to antiresorptive therapy in this important animal model. Specifically, these data indicate that the effect of agents that prevent estrogen depletion-induced bone loss in adult nonhuman primates can be observed within 13 weeks. This suggests that pharmaceutical efficacy in the prevention of OVX-induced bone loss, as measured by BMD and bone turnover markers, can be demonstrated in studies of only 13 weeks duration. Furthermore, the degree of OVX-induced bone loss observed by 13 weeks indicates that evaluation of rhesus monkeys shortly after OVX should be performed to determine if bone loss and turnover increases are demonstrable at earlier time points.
Estrogen depletion in nonhuman primates is associated with skeletal changes similar to those observed in women at menopause.1,9,11,32–35 Some prior studies in which OVX was performed in young, growing animals report osteopenia relative to a control group, i.e., without persistent absolute bone loss from the pre-OVX state. The occurrence of persistent bone loss is desirable in an animal model of menopausal bone loss because bone density declines permanently in postmenopausal women. Persistent bone loss has been documented in few prior reports with nonhuman primates.9,10,30 It has been suggested that dietary manipulation (reduction of calcium intake) may be required to demonstrate absolute osteopenia in nonhuman primates.9 Our data document that reduced calcium intake is not necessary to demonstrate absolute osteopenia. Rather than dietary manipulation, use of animals that have attained stable peak skeletal mass seems more relevant, because dietary manipulation at menopause is not necessary to evoke bone loss. The age of peak skeletal mass has been established at ∼11 and ∼9 years in rhesus and cynomolgus monkeys, respectively.6,7 We suggest that use of mature animals not only avoids the confounding effect of growth on bone mass, but also permits demonstration of absolute, persistent estrogen depletion osteopenia. Furthermore, the use of purpose-bred laboratory monkeys is preferable since wild caught animals increase bone mass when brought to captivity.9
Bisphosphonates are potent antiresorptive agents with established efficacy in the treatment and prevention of osteoporosis.16,36 In both in vitro and in vivo screening models, the bisphosphonate ZLN has been established as a highly potent inhibitor of osteoclastic bone resorption, which dose dependently increases bone density in intact rats and prevents bone loss in OVX rats.18,37 In this study, increases in skeletal turnover and bone loss induced by OVX were entirely prevented by ZLN at 2.5 μg/kg/week and 12.5 μg/kg/week. Low-dose ZLN (0.5 μg/kg/week) prevented increases in bone turnover markers but not OVX-induced bone loss. The dissociation of bone turnover marker values from bone mass changes with 0.5 μg/kg/week ZLN shows that these markers are reliable indicators of bisphosphonate actions on cells of the bone remodeling system, but not necessarily on the more critical bone mass endpoints that are clearly related to fracture risk in humans. Progressive suppression of skeletal turnover markers was not observed. Consistent with this, interim histomorphometry reports on these animals demonstrate significant amounts of cancellous and cortical bone turnover at 26 weeks with all doses of ZLN.38,39 The observed decline in serum calcium and the concomitant increase in serum PTH have previously been reported with bisphosphonate treatment.40 ZLN treatment produced dose-dependent skeletal changes as may have been anticipated with a potent bisphosphonate, suggesting that it is likely to prove beneficial in osteoporosis prevention/treatment in humans.
Treatment of osteoporotic women with bisphosphonates increases LSBMD progressively for 3 years.16 Our data demonstrate an increase in LSBMD and TBBMC of over 2% by week 39, but subsequent progressive elevation was not observed. This outcome concurs with prior studies in which bisphosphonate administration to animals with normal bone mass was not associated with an increase in bone density.41,42 This absence of progressive bone density elevation is consistent with mechanical regulation of bone mass as initially proposed by Frost43 and recently reviewed by Rodan in the context of bisphosphonate action.44 It should be noted that the studies showing progressive BMD gains in humans were done in persons whose baseline BMD was ∼2 SD below young adult normal. The current study was done in nonhuman primates with normal baseline BMD. Commencing ZLN treatment in nonhuman primates with established osteopenia might yield a much different outcome.
The BMD decline of the distal and CR in both the OVX and control groups treated with saline was unexpected. It did not appear to be the result of technical factors. Furthermore, the housing environment for all animals was stable, during and for 1 year preceding this study, and no behavioral or activity changes were noted. This observation remains unexplained and may be a previously unappreciated aspect of this animal model. Prevention of this decline by the highest dose of ZLN is interesting and might provide insight into the mechanism of bisphosphonate action. However, at this time, the mechanism(s) of this observation remain unclear. Further insight may come from ongoing histomorphometric studies of these animals.
The elevation of serum creatinine in all groups at the 26-week time point was unexpected. It was not related to surgery because the serum samples were obtained 5 days prior to the 26-week bone biopsies. However, all animals did receive oxytetracycline (40 mg/kg by intravenous injection over 2 minutes) 10 days prior to obtaining the 26-week samples. Intravenous tetracycline administration has been reported to produce nephrotoxicity in rats, dogs,45 and, rarely, humans.46 The serum creatinine returned to baseline values at the subsequent sampling time point, and no long-term sequelae were appreciated.
In summary, OVX skeletally mature female rhesus monkeys are excellent models of estrogen-depletion bone loss, in that OVX leads to an increase in markers of bone turnover and absolute osteopenia. These findings were demonstrable at 13 weeks following OVX. Maximal elevations in skeletal turnover persisted for 39 weeks and were associated with progressive bone loss at the TB and LS. Subsequently, turnover returned toward baseline in OVX animals (though ZLN-treated animals persisted with turnover at ∼50% of OVX-saline-treated animals), and further bone loss was not demonstrable. ZLN treatment at doses from 2.5–12.5 μg/kg/week was well tolerated and prevented the OVX-induced increases in skeletal turnover and bone loss. Consistent with mechanical regulation of bone mass, progressive elevation of BMD was not observed in animals whose ZLN treatment was begun at or near their peak skeletal mass. Progressive skeletal turnover marker suppression with ZLN treatment was not observed. These results suggest that ZLN is likely to prove beneficial in human osteoporosis prevention/treatment.
We thank Kirk Boehm and Les Sander for technical advice and support. This work was supported by Novartis Pharmaceuticals. This is Wisconsin Regional Primate Research Center publication #38-004.
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