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Keywords:

  • osteoporosis;
  • 2-methylene-19-nor-(20S)-1α,25(OH)2D3;
  • bone strength;
  • bone formation;
  • biomechanics;
  • 1,25-dihydroxyvitamin D3

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

An orally active, highly potent analog of 1α,25-dihydroxyvitamin D3, 2MD, restores trabecular and cortical bone mass and strength by stimulating periosteal bone formation and decreasing trabecular bone resorption in OVX rats with established osteopenia.

Introduction: The purposes of this study were to determine the effects of long-term treatment with 2-methylene-19-nor-(20S)-1α,25(OH)2D3 (2MD) on restoring bone mass and bone strength in ovariectomized (OVX) rats with established osteopenia and 2MD effects on bone formation and bone resorption on trabecular and cortical bone surfaces.

Materials and Methods: Sprague-Dawley female rats were sham-operated (sham) or OVX at 4 months of age. Beginning at 8 weeks after OVX, OVX rats were orally dosed with 2MD at 0.5, 1, 2.5, 5, or 10 ng/kg/day for 16 weeks. Serum calcium was measured at 6, 13, and 16 weeks after treatment, and bone mass and structure, bone formation, bone resorption, and bone strength were determined at the end of the study.

Results: Serum calcium did not change significantly with 2MD at 0.5 or 1 ng/kg/day, whereas it significantly increased at 2.5, 5, or 10 ng/kg/day. 2MD significantly and dose-dependently increased total body BMD, total BMC, and stiffness of femoral shaft (FS), maximal load and stiffness of femoral neck, and toughness of the fifth lumbar vertebral body (L5) at all doses compared with OVX controls. In 2MD-treated OVX rats, there was a dose-dependent increase in total BMD and total BMC of the distal femoral metaphysis (DFM), trabecular bone volume of L3, ultimate strength and stiffness of L5, and maximal load of FS compared with OVX controls at dosages ≥1 ng/kg/day. At dosages >2.5 ng/kg/day, most of the bone mass and bone strength related parameters were significantly higher in 2MD-treated OVX rats compared with sham controls. Bone histomorphometric analysis of L3 showed dose-dependent decreases in osteoclast number and osteoclast surface on trabecular bone surface and a dose-dependent increase in periosteal bone formation associated with 2MD treatment.

Conclusions: 2MD not only restored both trabecular and cortical bone mass but also added bone to the osteopenic OVX rats beyond that of sham controls by stimulating bone formation on the periosteal surface and decreasing bone resorption on the trabecular surface. 2MD increased bone mass and strength at doses that did not induced hypercalcemia.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

OSTEOPOROSIS IS DEFINED as a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture. (1) Aging, postmenopausal estrogen deficiency, or other factors such as chronic corticosteroid usage or immobilization are among the important risk factors for osteoporosis. The reduction in BMD and bone quality (which refers to architecture, turnover, damage accumulation, and mineralization) leads to decreased bone strength and increased number of fractures, typically of the spine, wrist, and hip. In addition to the pain associated with osteoporosis, diminished quality of life, loss of independence, and mortality, the economic burden on the health care system is very high. (1)

Whereas current therapies including bisphosphonates, hormone replacement therapy (HRT), and selective estrogen receptor modulators (SERMs) are effective inhibitors of bone resorption and bone remodeling, (2–8) these agents do not stimulate bone formation and therefore do not restore bone mass and rebuild bone structure. A bone formation agent, recombinant human PTH(1-34) {rhPTH(1-34)}, has been shown to possess superior efficacy in increasing BMD and in reducing fracture risk. (9) For example, vertebral fracture was reduced 65–69% in 2 years of rhPTH(1-34) treatment(9) compared with approximately a 50% reduction in vertebral fracture with antiremodeling agents such as bisphosphonates or SERMs. (2–8) These clinical results highlight the ability of an anabolic agent to meet the unmet clinical need in osteoporosis beyond what can be achieved with antiremodeling therapies. Therefore, there is great medical need for an improved bone anabolic agent to treat patients with established osteoporosis by restoring bone mass and rebuilding bone structure to prevent further skeletal fractures.

Several vitamin D metabolites and analogs, including 1α,25-dihydroxyvitamin D3 {1α,25(OH)2D3}, have been studied for efficacy in increasing bone mass in animal models and osteoporotic patients. 1,25(OH)2D3 (calcitriol) and its prodrug, 1α-hydroxyvitamin D3 (alfacalcidol), have been reported to reduce vertebral and hip fractures in postmenopausal and senile osteoporosis patients, as well as in glucocorticoid-induced osteoporosis patients. (10–17) Despite its efficacy in fracture reduction, the mechanism of action of calcitriol on bone remains unclear. Sairanen et al. (18) reported that 1,25(OH)2D3 (calcitriol) treatment for 4 years increases BMD in lumbar spine and femoral neck in postmenopausal women by reducing bone turnover (decreased both serum markers of bone formation and bone resorption). However, there is increasing evidence that 1,25(OH)2D3 may directly stimulate osteoblastic bone formation. Not only do osteoblasts possess abundant vitamin D receptors, (19, 20) but calcitriol and alfacalcidol have been shown to stimulate bone formation and increase bone mass and strength in animal models of osteopenia. (21–28) Because of their hypercalcemia and hypercalciuria effects, calcitriol and alfacalcidol have a very narrow therapeutic window for the prevention and treatment of osteoporosis. (18) Extensive research has focused on the identification of structurally distinct, novel vitamin D analogs that can have positive bone effects (ideally stimulate bone formation) and improve the therapeutic index where doses can enhance bone formation without increasing urinary or serum calcium levels. (29–34) It was reported that Ro-26-9228, a vitamin D analog, increased osteoblast surface and bone mass determined by bone histomorphometry and inhibited bone resorption determined by biochemical markers in an ovariectomized (OVX) rat model at doses that did not increase serum and urine calcium. (30) Clinical studies will be needed to confirm bone anabolic activity and the therapeutic window of Ro-26-9228. ED-71 {2β-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D3}, another vitamin D analog, was reported to restore bone mass back to sham control levels by maintaining bone formation and inhibiting bone resorption at 2 and 4 weeks after OVX in rats given daily treatment for 3 months. (32, 33) Results from early clinical studies indicated that ED-71 significantly increased lumbar vertebral BMD by rapidly decreasing serum markers of bone resorption and gradually decreasing/unchanging serum markers of bone formation, whereas no overt hypercalcemia was found at these doses in osteoporotic patients. (33, 34) Longer-term and larger clinical trials are needed to confirm these beneficial effects of ED-71. Although both above-mentioned vitamin D analogs increase bone mass in animal models or osteoporotic patients, the specific mechanisms regarding their effects on bone formation and bone resorption have not yet been completely shown.

A new analog of 1α,25-dihydroxyvitamin D3, 2-methylene-19-nor-(20S)-1-α,25(OH)2D3 (2MD; Fig. 1), binds to the vitamin D receptor (VDR) with a high affinity comparable with that of the endogenous ligand, 1α,25(OH)2D3. (35) However, 2MD is a more potent agonist in a variety of cellular assays. 2MD is ∼10-50 times more potent than calcitriol in transcriptional reporter cell assays using vitamin D responsive promoters in an osteoblastic cell line, MC3T3. (36) The increased potency of 2MD relative to calcitriol in these cell systems has been correlated with the increased ability of 2MD to act at the vitamin D receptor by promoting receptor conformational change, protein/protein interactions with the receptor and co-activators SRC-1 and DRIP205, and receptor/DNA interactions at specific sites. (37) Consistent with these observations, 2MD is also more potent than calcitriol in its ability to induce differentiation in human promyelocytic HL-60 cells into monocytes, a widely studied action of vitamin D pharmacology. (38) It has also been reported that 2MD stimulates osteoblastic bone formation in vitro and increases bone mass in OVX rats with minimal effect on serum calcium. (38) However, the long-term effects on bone formation and resorption of 2MD on trabecular and cortical bone were not reported in these studies. In this study, we investigated whether long-term treatment with 2MD can restore trabecular and cortical bone mass and strength in OVX rats with established osteopenia. The bone formation and bone resorption activities were characterized by bone histomorphometry on both trabecular and cortical bone surfaces.

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Figure FIG. 1.. Chemical structure of calcitriol (1α,25-dihydroxyvitamin D3) and 2MD {2-methylene-19-nor-(20S)-1α,25(OH)2D3}.20

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MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

A total of 82 virgin Sprague-Dawley female rats (Taconic, German Town, NY, USA) were used in this study. Rats were sham-operated (n = 20) or ovariectomized (OVX, n = 62) at 4 months of age by the vendor. The rats were ∼5 months old and 4 weeks postsurgery on arrival. Rats were housed singly in 48 × 27 × 20-cm cages at local vivarium conditions (24°C; 12-h light 12-h dark cycle). Deionized water was provided to the animals ad libitum. During the study, the OVX rats were pair-fed with AIN-76A Rodent Diet (Research Diet, New Brunswick, NJ, USA) based on the average weekly food consumption of the sham control group (20 g/day). This diet contains 0.52% calcium, 0.40% phosphorus, and 1.0 IU/g vitamin D3. The experiments were conducted according to Pfizer Animal Care and Use approved protocols, and animals were maintained in accordance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals.

Eight weeks after surgery, 10 sham-operated and 8 OVX rats were necropsied and served as basal controls. The remaining 10 sham-operated (sham controls) rats were treated with vehicle (corn oil), and the remaining OVX rats were treated with 2MD at 0 (corn oil, OVX controls), 0.5, 1, 2.5, 5, and 10 ng/kg body weight/day by oral gavage at the dosing volume of 0.1 ml/rat/day for 16 weeks. There were 9–10 rats in each group.

All rats were given subcutaneous injections of calcein green (10 mg/kg body weight; Sigma, St Louis, MO, USA), a fluorochrome bone marker, 12 and 2 days before necropsy to determine dynamic changes in bone tissue. Rats were necropsied under anesthesia and killed by CO2 asphyxiation.

Serum calcium and osteocalcin

After overnight fasting, blood (1.0 ml) was collected for measurements of serum calcium after 6, 13, and 16 weeks of treatment and serum osteocalcin after 6 and 16 weeks of treatment through tail bleeding while the rats were under 5% isoflurane (Baxter, Deerfield, IL, USA) anesthesia. The blood was allowed to clot at room temperature, and the serum was separated by centrifuging at 2500g for 10 minutes at ∼4°C. The serum samples were stored frozen, at approximately −70°C until analysis. Serum osteocalcin was assayed using a Rat Osteocalcin IRMA kit (Immutopics, San Clemente, CA, USA). Serum calcium was assayed using a VETACE Clinical Chemistry Analyzer (Alfa Wasserman, West Caldwell, NJ, USA).

Body weight and whole body DXA

One day before necropsy, body weight, lean and fat body mass, total body bone mineral area, BMC, and BMD were determined using DXA (Hologic QDR-4500; Hologic, Waltham, MA, USA) equipped with whole body scan software.

Ex vivo pQCT analysis

Excised femurs were scanned by a pQCT machine (Stratec XCT Research M; Norland Medical Systems, Fort Atkinson, WI, USA) with software version 5.40. A 1-mm-thick cross-section of distal femur metaphysis (DFM) was taken at 5.0 mm proximal from the distal end, and a 1-mm-thick cross-section of the femoral shaft (FS) was taken at the midpoint of the femur with a voxel size of 0.10 mm. Cortical bone was defined and analyzed using contour mode 2 and cortical mode 4. An outer threshold setting of 340 mg/cm3 was used to distinguish the cortical shell from soft tissue and an inner threshold of 529 mg/cm3 to distinguish cortical bone along the endocortical surface. Trabecular bone was determined using peel mode 4 with a threshold setting of 655 mg/cm3 to distinguish (sub)cortical from cancellous bone. An additional concentric peel of 1% of the defined cancellous bone was used to ensure (sub)cortical bone was eliminated from the analysis. Volumetric BMC, BMD, and area were determined for both trabecular and cortical bone. (39) Using the above setting, we determined that the ex vivo precision of volumetric BMC, BMD, and area of total bone, trabecular, and cortical regions ranged from 0.99% to 3.49% with repositioning.

Third lumbar vertebral cancellous bone histomorphometry

At necropsy, the third lumbar vertebral body was removed, dissected free of soft tissue, fixed in 70% ethanol, dehydrated in graded concentrations of ethanol, defatted in acetone, and embedded in methyl methacrylate (Fisher Scientific, Fair Lawn, NJ, USA). Parasagittal sections of the third lumbar vertebral body at thicknesses of 4 and 10 μm were cut. The 4-μm sections were stained with modified Masson's trichrome stain and used for measurements of bone mass, structure, and bone resorption-related indices, whereas the 10-μm sections were unstained and used for measurement of bone formation-related indices using an Image Analysis System with software version 2.2 (Osteomeasure, Atlanta, GA, USA). A tissue area within 0.5 mm from each dorsal and ventral end was selected for histomorphometric analysis.

Measurements and calculations related to trabecular bone volume and structure included trabecular bone volume, thickness, number, and separation, whereas measurements and calculations related to bone resorption included osteoclast number and osteoclast surface. Furthermore, the parameters related to bone formation and turnover included mineralizing surface ({double labeling perimeter + 1/2 single labeling perimeter}/total trabecular perimeter x 100), mineral apposition rate (MAR), bone formation rate/bone volume (BFR/BV), bone formation rate/bone surface (BFR/BS), and bone formation rate/tissue volume (BFR/TV). The definitions and formula for calculations of these parameters were described previously by Parfitt et al. (40) and Jee et al. (41)

Tibial shaft cortical bone histomorphometry

For cortical bone, undecalcified, methyl methacrylate-embedded, cross-sections of the tibial shaft at a thickness of 20 μm were prepared. Total tissue area, periosteal perimeter, cortical bone area, marrow cavity area, endocortical perimeter, and dynamic histomorphometric parameters including mineralizing surface, MAR, and bone formation rate were determined on periosteal and endocortical surfaces. (40, 41)

Biomechanical testing of L5, femoral neck (FN), and FS

Biomechanical testing of L5, femoral neck (FN), and FS was performed using an Instron 5500 servo-electric testing machine and associated Merlin II software (version 4.05; Instron, Canton, MA, USA). A compression test was used to determine the ultimate strength, stiffness, and toughness for L5. A cantilever bending test was used to determine the maximal load to failure and stiffness for the FN, whereas a three-point bending test was used to determine the maximal load to failure and stiffness for the FS. A detailed description of these tests has been previously reported. (42)

Statistics

Data are expressed as mean ± SE. Statistics were calculated using StatView 4.0 packages (Abacus Concepts, Berkeley, CA, USA). The ANOVA test was used for all groups necropsied at the end of the study, and Fisher's protected least significant difference (PLSD) was used to compare differences between each group. The difference between the groups at 16 weeks and basal controls was determined by Student's t-test. p < 0.05 was considered a significant difference.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Serum calcium and serum osteocalcin

Serum calcium did not differ significantly among sham controls, OVX controls, and OVX rats treated with 2MD at 0.5 and 1 ng/kg/day at 6, 13, and 16 weeks after treatment (Fig. 2A). Compared with both sham and OVX controls, serum calcium increased significantly and dose-dependently in OVX rats treated with 2MD at 2.5, 5, and 10 ng/kg/day at 6 weeks, and the elevated serum calcium was maintained at the elevated levels at 13 and 16 weeks of treatment (Fig. 2A). Serum osteocalcin increased nonsignificantly in OVX controls compared with sham controls at 6 and 16 weeks of treatment (+14% and +36%, respectively; Fig. 2B). Serum osteocalcin did not differ significantly among sham controls, OVX controls, and OVX rats treated with 2 MD at 0.5 and 1 ng/kg/day at both 6 and 16 weeks of treatment. There was a significant increase in serum osteocalcin by 34%, 76%, and 111% in OVX rats treated with 2 MD at 2.5, 5, and 10 ng/kg/day, respectively, compared with OVX controls at week 6. There was a continuous increase in osteocalcin in OVX rats treated with 2 MD at 2.5, 5, and 10 ng/kg/day compared with OVX controls at week 16 (+72%, 100%, and 151%, respectively; Fig. 2B).

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Figure FIG. 2.. Changes in (A) serum calcium and (B) serum osteocalcin in vehicle-treated sham controls (Sham), vehicle-treated OVX controls (OVX), and OVX rats treated with 2MD at 0.5, 1, 2.5, 5, or 10 ng/kg/day at 6, 13, or 16 weeks after treatment. Error bars represent SE.ap < 0.05 vs. Sham;bp < 0.05 vs. OVX.20

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Whole body DXA analysis

At the end of the study, there was a significant increase of 29% in body weight in OVX controls compared with sham controls (Fig. 3A). Body weight did not change significantly in OVX rats treated with 2MD at 0.5, 1, and 2.5 ng/kg/day, whereas body weight decreased significantly in OVX rats treated with 2MD at 5 and 10 ng/kg/day compared with OVX controls. The decrease in body weight was caused by a significant decrease in fat body mass (Fig. 3B) and unchanged lean body mass (data not shown) in OVX rats treated with 2MD at 5 and 10 ng/kg/day compared with OVX controls.

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Figure FIG. 3.. Changes in (A) body weight, (B) fat body mass, and (C) total body BMD in vehicle-treated sham controls (Sham), vehicle-treated OVX controls (OVX), and OVX rats treated with 2MD at 0.5, 1, 2.5, 5, or 10 ng/kg/day for 16 weeks. Error bars represent SE.ap < 0.05 vs. Sham, bp < 0.05 vs. OVX.20

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Total body BMD decreased significantly by 6% in OVX controls compared with sham controls at the end of the study (Fig. 3C). Compared with OVX controls, total body BMD increased significantly by 4%, 7%, 14%, 23%, and 31% in OVX rats treated with 2MD at 0.5, 1, 2.5, 5, and 10 ng/kg/day, respectively. At doses ≥2.5 ng/kg/day, 2MD not only restored total body BMD back to the sham control level but also increased BMD significantly above sham controls (Fig. 3C).

Ex vivo pQCT analysis of DFM

Images obtained from the DFM showed a significant bone loss in baseline OVX and OVX vehicle groups compared with their age-matched sham controls (Fig. 4A). 2MD dose-dependently increased both cortical and trabecular bone between doses of 0.5 and 10 ng/kg/day with a dramatic deposition of a large amount of bone in periosteal surface, endocortical surface, and marrow cavity of DFM at the two highest dose groups (Fig. 4A).

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Figure FIG. 4.. Images obtained from (A) the distal femoral metaphysis and (B) the femoral shaft by pQCT analysis show a dose-dependent increase in bone mass on periosteal, endocortical, and trabecular surfaces by treatment with 2MD in established osteopenia OVX rats. Massive bone formation was found in the higher-dose groups. 2MD induced new trabecular bone formation in the marrow cavity of long bone shaft where there was no or minimal trabecular bone in sham or OVX rats.20

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In DFM, there was a significant decrease in total BMD by 16%, trabecular BMD by 18%, and cortical thickness by 25%, whereas there was a significant increase in total tissue area by 10%, marrow cavity area by 53%, periosteal circumference by 5%, and endocortical circumference by 43% in OVX baseline controls compared with sham baseline controls (Table 1). These results indicated that OVX caused a significant loss of trabecular and endocortical bone and a significant increase of periosteal bone 8 weeks after surgery. A consistent decrease in trabecular BMD was found in OVX vehicle compared with sham vehicle at the end of the study. Except for a slight but significant increase in cortical BMD, 2MD at 0.5 ng/kg/day showed no significant difference compared with OVX controls in other pQCT parameters listed in Table 1. There was a significant increase in total BMC, total BMD, trabecular BMD, and cortical BMC in OVX rats treated with 2MD at 1 ng/kg/day compared with OVX controls. However, trabecular BMD at 1 ng/kg/day of 2MD-treated OVX rats was still significantly lower than those of sham controls. At doses ≥2.5 ng, 2MD completely restored all the parameters listed in Table 1 back to or above sham level. For instance, total BMC increased significantly in the 2.5-, 5-, and 10-ng groups compared with both OVX rats treated with vehicle (+30%, +63%, and +85%, respectively) and sham controls (+17%, +46%, and +66%, respectively). These data indicate that 2MD at doses ≥2.5 ng/kg/day not only restored the lost bone but also added extra bone to the established osteopenic OVX rats.

Table Table 1.. pQCT Analysis of Distal Femoral Metaphysis
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Ex vivo pQCT analysis of the FS

Figure 4B shows the representative images obtained from pQCT analysis of FSs. These images show that OVX increased marrow cavity area in both baseline and vehicle-treated groups compared with their age-matched sham controls. 2MD dose-dependently reduced the marrow cavity area and increased the cross-sectional area compared with both sham and OVX controls. Interestingly, 2MD induced new trabecular bone formation in the marrow cavity of femoral shafts where there was no or minimal trabecular bone in sham or OVX rats, suggesting that the pre-existing bone surface might not be required for 2MD-induced bone formation. More definitive studies on more severe osteopenic rat or mouse models will be needed to confirm this observation.

In the FS, the OVX baseline group had significantly higher total tissue area and periosteal and endocortical circumferences and significantly lower cortical BMD compared with sham baseline (Table 2). Similar differences were found between vehicle-treated OVX and sham controls. Compared with OVX vehicle controls, 2MD treatment at all doses significantly and dose-dependently increased total BMC (6-33%), cortical BMC (7-33%), cortical bone area (6-30%), and cortical thickness (4-40%). Compared with sham vehicle controls, 2MD-treated OVX rats at doses of 2.5, 5, and 10 ng/kg/day significantly and dose-dependently increased total BMC (17%, 24%, and 30%, respectively), cortical BMC, cortical bone area, and cortical thickness (Table 2). Furthermore, a significant increase in total BMD, total tissue area, and periosteal circumference and a significant decrease in endocortical circumference was found in higher doses of 2MD-treated OVX rats compared with sham and OVX controls.

Table Table 2.. pQCT Analysis of Femoral Shafts
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Third lumbar vertebral cancellous bone histomorphometry

Compared with the sham baseline group, the OVX baseline group had significantly lower trabecular number and significantly higher trabecular separation and indices of bone formation and bone resorption (Table 3). Similarly, higher bone turnover was still observed between OVX vehicle compared with sham vehicle at the end of the study. In addition, there was a significant decrease (−35%) in trabecular bone volume (BV/TV) in OVX vehicle compared with sham vehicle (Table 3).

Table Table 3.. Selective Trabecular Bone Histomorphometric Parameters of LV3
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Compared with OVX vehicle, 2MD treatment at doses ≥1 ng/kg/day significantly increased BV/TV by 40–185% (Table 3). BV/TV in OVX rats treated with 2MD at 5 and 10 ng/kg/day increased significantly compared with the sham vehicle group, indicating that 2MD added extra cancellous bone to OVX rats. The higher BV/TV in 2MD-treated OVX rats was achieved by increased trabecular thickness and number. However, trabecular thickness increased more dramatically than did trabecular number, as shown in Table 3 and Fig. 5. Bone resorption indices, osteoclast surface and osteoclast number, decreased significantly in 2MD-treated OVX at doses ≥2.5 ng/kg/day compared with OVX vehicle, indicating that 2MD decreased trabecular bone resorption. Mineralizing surface (MS/BS) decreased significantly in 2MD-treated OVX rats at doses ≥1 ng/kg/day compared with OVX vehicle. However, MS/BS did not differ significantly between all doses of 2MD-treated OVX and sham vehicle. Bone formation rate/bone volume (BFR/BV) decreased significantly in 2MD-treated OVX rats at doses ≥1 ng/kg/day compared with OVX vehicle. Furthermore, BFR/BV decreased significantly in 2MD-treated OVX rats at doses of 5 and 10 ng/kg/day compared with sham vehicle. MAR, BFR/BS, and BFR/TV did not differ significantly between all doses of 2MD-treated OVX rats and OVX vehicle (Table 3).

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Figure FIG. 5.. Dose-dependent increase in trabecular bone volume, trabecular thickness, and trabecular number in the third lumbar vertebral body was observed with 2MD treatment for 16 weeks in established osteopenia OVX rats. Much thicker trabeculae were observed in 2MD-treated rats compared with sham and OVX controls at the end of the study. Masson's trichrome-stained, 10-μm-thick sections.20

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Tibial shaft cortical bone histomorphometry

As reported previously by other investigators, (43–45) OVX did not induce osteopenia or significant bone loss in rat long bone shafts. However, we still can use this skeletal site to study the effects of 2MD on periosteal and endocortical surfaces. In this study, OVX increased total tissue area and marrow cavity area in baseline and at the end of the study compared with age-matched sham controls (Table 4). At baseline, OVX rats had higher periosteal mineralizing surface (P-MS/BS) and bone formation rate (P-BFR/BS) than sham controls. Periosteal bone formation decreased dramatically with age in both sham and OVX rats between baseline groups and vehicle-treated groups. By the end of the study, there was no significant difference in periosteal bone formation between OVX vehicle and sham vehicle. Endocortical bone formation did not differ significantly between OVX and sham at baseline but increased significantly in the OVX vehicle group compared with the sham vehicle group (Table 4).

Table Table 4.. Selective Cortical Bone Histomorphometric Parameters of Tibial Shafts
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Treatment of OVX rats with 2MD dose-dependently increased total tissue area, cortical bone area, periosteal perimeter, P-MS/BS, P-MAR, P-BFR/BS, E-MS/BS, and E-BFR/BS, and dose-dependently decreased marrow cavity area and endocortical perimeter compared with sham vehicle and OVX vehicle (Table 4). There was no significant difference in cortical bone mass or periosteal and endocortical bone formation between OVX rats treated with vehicle and 1 ng/kg/day of 2MD. At 1 ng/kg/day, 2MD treatment significantly increased cortical bone area by 5.4% and significantly decreased marrow cavity area by 17.1% compared with OVX vehicle. OVX rats treated with 2MD at 2.5 ng/kg/day had significantly higher cortical bone area (+13.3%), P-MS/BS (+942%), P-MAR (+253%), and P-BFR/BS (+2488%) and significantly lower marrow cavity area (−28%) and endocortical perimeter (−14%) compared with vehicle-treated OVX rats. Similarly, at the higher doses (5 and 10 ng/kg/day), 2MD further increased cortical bone mass and periosteal bone formation, and further decreased marrow cavity area (Table 4).

Biomechanical testing of L5, FN, and FS

Ultimate strength of L5 decreased significantly in OVX baseline and vehicle-treated OVX rats compared with their respective sham controls (−32% and -36%, respectively; Fig. 6A). In OVX rats, 2MD at 1 and 2.5 ng/kg/day completely restored the ultimate strength back to the sham level. 2MD at 5 and 10 ng/kg/day significantly increased ultimate strength compared with both OVX vehicle (+147% and +184%, respectively) and sham vehicle (+57% and +81%, respectively). There was no significant difference in stiffness of L5 among sham baseline, OVX baseline, sham vehicle, OVX vehicle, and OVX rats treated with 2MD at 0.5 ng/kg/day (Fig. 6B). 2MD treatment at 1 and 2.5 ng/kg/day significantly increased stiffness of L5 compared with OVX vehicle, while significantly increasing stiffness of L5 compared with both sham and OVX vehicle at 5 and 10 ng/kg/day (Fig. 6B). Similarly, toughness of L5 was significantly increased at 0.5, 1, and 2.5 ng/kg/day of 2MD-treated OVX rats compared with OVX vehicle. At 5 and 10 ng/kg/day, 2MD significantly increased toughness of L5 compared with both sham and OVX vehicle (Fig. 6C).

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Figure FIG. 6.. Changes in (A) ultimate strength, (B) stiffness, and (C) toughness of the fifth lumbar vertebral body (LV5) in sham and OVX baseline controls, vehicle-treated sham controls, vehicle-treated OVX controls, and OVX rats treated with 2MD at 0.5, 1, 2.5, 5, or 10 ng/kg/day for 16 weeks. Error bars represent SE.ap < 0.05 vs. Sham, bp < 0.05 vs. OVX.20

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Maximal load in the FN did not differ significantly between sham and OVX at baseline and at end of vehicle treatment (Fig. 7A). However, 2MD treatment significantly increased maximal load of FN at all doses administered compared with vehicle in OVX rats (+23%, +22%, +29%, +34%, and +32% for 0.5, 1, 2.5, 5, and 10 ng/kg/day, respectively). Furthermore, OVX rats treated with 2MD at 5 and 10 ng/kg/day had significantly higher maximal load of FN compared with sham vehicle (Fig. 7B). Stiffness of FN in OVX rats treated with vehicle was significantly decreased compared with sham treated with vehicle (Fig. 7B). Treatment of OVX rats with 2MD at all doses completely restored the stiffness back to the sham level.

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Figure FIG. 7.. Changes in (A) maximal load and (B) stiffness of the femoral neck (FN), and (C) maximal load and (D) stiffness of femoral shaft (FS) in sham and OVX baseline controls, vehicle-treated sham controls, vehicle-treated OVX controls, and OVX rats treated with 2MD at 0.5, 1, 2.5, 5, or 10 ng/kg/day for 16 weeks. Error bars represent SE.ap < 0.05 vs. Sham, bp < 0.05 vs. OVX.20

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As expected, maximal load and stiffness of FS did not differ between sham and OVX at baseline and at the end of vehicle treatment (Fig. 7C). 2MD treatment at doses ≥1 ng/kg/day significantly increased maximal load compared with OVX vehicle (+9%, +30%, +45%, and +47% for 1, 2.5, 5, and 10 ng/kg/day; Fig. 7C). Similarly, OVX rats treated with 2MD at 0.5, 2.5, 5, and 10 ng/kg/day had significantly higher stiffness than OVX vehicle (Fig. 7D). At doses ≥2.5 ng/kg/day, 2MD-treated OVX rats significantly increased maximal load and stiffness of FS compared with sham vehicle (Figs. 7C and 7D).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The results from this study showed that 2MD is an orally active and highly potent agent for restoring bone mass in established osteopenic OVX rats. At doses as low as 0.5 ng/kg/day, 2MD significantly restored total body BMD, toughness of lumbar vertebral body, and maximal load and stiffness of FN in the OVX rat model. At 1 ng/kg/day, 2MD completely restored most parameters related to bone mass, bone structure, and bone strength back to sham control levels in OVX rats. At these doses (0.5 and 1 ng/kg/day), no significant change in serum calcium was found associated with 2MD treatment. These results show the possibility of identifying doses of 2MD that have therapeutic efficacy in bone without causing sustained hypercalcemia.

At a dose of 2.5 ng/kg/day, 2MD not only restored bone mass, structural parameters, and bone strength back to the sham control levels, but also added extra bone to the OVX rats. For instance, at this dose, 2MD-treated OVX rats had higher total body BMD, higher total BMC in the distal femur and FS, higher cortical bone area in the tibial shaft, and higher maximal load in the FN and FS compared with sham controls. A slight increase in serum calcium was observed in OVX rats treated with 2MD at 2.5 ng/kg/day. Thus, clinical studies are needed to determine whether mild hypercalcemia would occur in humans at the similar exposure dose. At a dose of 5 or 10 ng/kg/day, 2MD in OVX rats induced significant increases in trabecular and cortical bone mass above the levels of sham controls. However, the accompanied hypercalcemia may limit such exposure in humans. In this study with an OVX rat model, the bone efficacy of 2MD was observed at doses ≥0.5 ng/kg/day, whereas the hypercalcemic response was observed at doses ≥2.5 ng/kg/day. These results suggest that 2MD may have a significant, although narrow, therapeutic window in term of bone efficacy versus hypercalcemia in humans, if the OVX rats serve well as a model for osteoporotic patients regarding hypercalcemia. Furthermore, urine calcium, which is a more sensitive parameter for changes in calcium homeostasis, and kidney function/nephrotoxicity will need to be assessed in future studies to determine the therapeutic window for 2MD in animal models or in humans.

In this study, we clearly showed by bone histomorphometry of cortical bone that 2MD dose-dependently increased periosteal mineralizing surface, MAR, and bone formation rate. The increased periosteal bone formation led to an increase in total tissue area and contributed to the increase in cortical bone area. Because bone remodeling is at a very minimal level on periosteal surface in rats at this age (∼10 months old), the ability of 2MD to stimulate bone formation on this surface revealed that 2MD can stimulate formation mode of bone modeling. This observation has not been reported for other vitamin D analogs such as ED71 and Ro-26-9228. (30, 32) This is an important observation because adding new bone to periosteal surface would likely improve bone strength in long bone shafts and the FN as was found in this study. FN fractures commonly occur in osteoporotic patients. If a safe therapeutic dose can be identified in humans, 2MD may offer beneficial effects in protecting against fractures in the FN. In addition to the contribution from new periosteal bone, increased bone strength in the FS and FN may also stem from a dose-dependent decrease in marrow cavity area and increased endocortical bone formation induced by 2MD treatment.

On the trabecular surface of L3, after 16 weeks of daily treatment, decreased osteoclast number and surface were observed in 2MD-treated OVX rats at doses ≥2.5 ng/kg/day compared with OVX controls, indicating that 2MD dose-dependently decreases bone resorption on trabecular surface. A slight and significant decrease in mineralizing surface and bone formation rate/bone volume referent, and no significant change in MAR, bone formation rate/bone surface referent, and bone formation rate/tissue volume referent was observed with higher doses of 2MD. These results revealed that long-term treatment with 2MD inhibited trabecular bone turnover (remodeling). However, trabecular bone resorption was inhibited to a much greater extent than was bone formation. For example, osteoclast surface decreased significantly by 82%, whereas mineralizing surface decreased only by 32% in the highest dose of 2MD-treated OVX rats compared with OVX controls (Table 3). The differential effects of 2MD on trabecular bone resorption and formation lead to a positive bone balance. The dramatic increase in trabecular thickness (Fig. 5; Table 3) induced by 2MD treatment resulted from the imbalance in bone remodeling in favor of bone formation. A similar finding was reported for ED71, another vitamin D analog. ED71 inhibited bone resorption and maintained bone formation in the trabecular bone of OVX rats. (32) Although the positive trabecular bone gain induced by long-term 2MD treatment can be explained partially by the observation of imbalanced bone remodeling in favor of bone formation, an earlier stimulation of trabecular bone formation is still a likely possibility, because such a dramatic increase in trabecular bone volume and thickness at the higher doses was unlikely induced only by imbalanced bone remodeling. Another vitamin D analog, Ro-26-9228, has been reported to increase osteoblast number and the expression of osteopontin and osteocalcin on trabecular bone after 3 weeks of treatment. (31) A short-term study or a time-course study will be needed to determine the short-term effects of 2MD on trabecular bone formation in OVX rats.

The inhibitory effects of 2MD on lumber vertebral trabecular bone resorption observed in this study were similar to those reported for 1α,25-dihydroxyvitamin D3 {1,25(OH)2D3} and alfacalcidol. (21, 27, 28) However, there was some discrepancy regarding the effects on trabecular bone formation parameters among 2MD, 1,25(OH)2D3, and alfacalcidol. In this study, we found the mineralizing surface (MS/BS) and bone formation rate/bone volume referents were significantly decreased in 2MD-treated OVX rats compared with OVX vehicle controls. Erben et al. (21) reported that daily treatment with 1,25(OH)2D3 for 3 months in established osteopenic OVX rats did not significantly affect trabecular bone formation parameters in the first lumbar vertebral body. Li et al. (27) reported that alfacalcidol significantly increased MS/BS and bone formation rate/bone surface referents when given to established osteopenic OVX rats for 4 weeks. However, when given to rats immediately after OVX, alfacalcidol significantly decreased the bone formation rate/bone surface referent compared with OVX controls. (28) The discrepancy regarding the effects on trabecular bone formation between 2MD and 1,25(OH)2D3 may be, at least in part, caused by the duration of the treatment and the status of bone turnover at the time of administration or because of mechanistic differences between these distinct pharmacological agents.

Although slightly decreased bone formation and bone turnover was observed on the trabecular surface after long-term 2MD administration, serum osteocalcin, a marker of bone formation and bone turnover, was still significantly increased in higher doses of 2MD-treated OVX rats compared with OVX controls in this study. This may indicate that the increased bone formation activity on the periosteal surface is to a greater extent than the slightly decreased bone formation on the trabecular surface; thus, the overall bone formation activity in the whole body is increased by higher doses of 2MD. Similar results were reported for alfacalcidol(28) and another vitamin D analog, ED-71. (32) At the higher dose, ED-71 was found to increase serum osteocalcin but decrease bone resorption and have no effect on trabecular bone formation, although no data were reported regarding its effect on cortical bone. (32) These results show the importance of studying the effects of vitamin D analogs on all bone surfaces because they may have different effects on different bone surfaces.

The detailed mechanisms of increased periosteal bone formation and decreased trabecular bone resorption and formation induced by long-term 2MD treatment are not completely understood. 2MD may directly stimulate bone formation on the periosteal surface through activation of VDR, because it has been shown that 2MD directly stimulated osteoblastic mineralization in culture. (38) Furthermore, osteocalcin and osteopontin in rats and human are upregulated by 1,25(OH)2D3 through the VDR. (46–48) The effects of 2MD on inhibiting trabecular bone turnover may be a direct effect through activation of VDR, an indirect effect through suppression of endogenous PTH, or a combination of both. Decreased serum PTH level has been reported for alfacalcidol in rats. (27) In this study, we did not determine serum PTH level. However, in another separate study, we found that 2MD at doses ≥5 ng/kg/day significantly decreased serum PTH in rats.

As stated in the NIH Consensus Statement, (1) osteoporosis is defined as a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture. Bone strength reflects the integration of BMD and bone quality, and bone quality refers to architecture, turnover, damage accumulation, and mineralization. Thus, the increased bone strength induced by 2MD treatment in this study could stem from a combination of any two or all of these features. In this study, we showed that 2MD significantly increased BMD in both trabecular and cortical bone, improved trabecular and cortical bone structure (increased trabecular thickness and number and increased cross-sectional area), and decreased trabecular bone turnover after long-term therapy in OVX rats. As reported by other investigators, (49) the decrease in bone resorption and bone turnover in patients taking antiresorptive therapy accounts for a large proportion of the reduction in fracture risk. Thus, the decreased bone resorption and bone turnover induced by long-term 2MD treatment may contribute positively to the antifracture efficacy of this agent in humans. The effects of 2MD on damage accumulation and mineralization was not measured in this study and will require further study. However, it is reasonable to hypothesize that 2MD may slightly increase trabecular mineralization because of its effects on inhibiting trabecular bone resorption. Nevertheless, we found that a significant increase in bone strength in L5, the FS, and the FN in the lower doses of 2MD, in which serum calcium did not change significantly, in established osteopenic OVX rats. This was one of the most important observations in this study.

In summary, an orally active, highly potent analog of 1,25(OH)2D3, 2MD, restores trabecular and cortical bone mass and strength by stimulating periosteal bone formation and decreasing trabecular bone resorption in OVX rats with established osteopenia. These results indicate that 2MD may have therapeutic potential in human skeletal disorders such as osteoporosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The authors thank Dr Victor Shen of Skeletech, Inc. for help in biomechanical testing of bones and all members of the Pfizer Early Candidate Management Team for support for this project.

References

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  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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