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Lasofoxifene (CP-336,156), a Selective Estrogen Receptor Modulator, Prevents Bone Loss Induced by Aging and Orchidectomy in the Adult Rat

Department of Cardiovascular and Metabolic Diseases, Central Research Division, Pfizer, Inc., Groton, Connecticut 06340

Address all correspondence and requests for reprints to: Dr. H. Z. Ke, Department of Cardiovascular and Metabolic Diseases, Box 8118-216, Central Research Division, Pfizer, Inc., Groton, Connecticut 06340. E-mail: huazhu_ke{at}groton.pfizer.com

	Abstract

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It has been well documented that selective estrogen receptor modulators (SERMs) can prevent bone loss in ovariectomized rats and postmenopausal women. The purposes of this study were to determine the effects of a potent and orally active SERM, lasofoxifene (CP-336,156), on bone mass, bone strength, total serum cholesterol, prostate weight, and histology in adult male orchidectomized (ORX) rats.

Sprague Dawley male rats at 10 months of age were divided into 6 groups, with 10 rats/group. The first group was necropsied on day 0 and served as basal controls. The remaining rats were either sham operated (n = 10) and treated orally with vehicle, or ORX (n = 40) and treated with either vehicle or lasofoxifene at 1, 10, or 100 µg/kg·day for 60 days. Total serum cholesterol, prostate weight and histology, distal femoral bone mineral density (DFBMD) by dual energy x-ray absorptiometry, and static and dynamic bone histomorphometry of the third lumbar vertebral body were determined. Maximal load and stiffness of the fifth lumbar vertebral body were also determined by compression tests.

Age-related decreases in DFBMD (-9%) and trabecular bone volume (TBV; -13%) of the third lumbar vertebral body were found in sham-operated rats compared with basal controls. ORX induced significant increases in total serum cholesterol (+31%), eroded surface (+48%), activation frequency of bone turnover (+103%) and significant decreases in prostate weight (-89%), DFBMD (-14%), TBV (-23%), and maximal load (-17%) compared with basal controls. Compared with sham controls, ORX induced significant increases in eroded perimeter and activation frequency. Lasofoxifene decreased body weight in all dose groups compared with both sham and ORX control values. Compared with ORX controls, ORX rats treated with lasofoxifene at 10 or 100 µg/kg·day had significantly lower percent eroded perimeter activation frequency and significantly higher DFBMD, TBV, and maximal load. Further, lasofoxifene at 10 and 100 µg/kg·day significantly decreased total serum cholesterol by 46% and 68% in ORX rats, whereas no effect was found in prostate weight and histology parameters compared with ORX control values. These data showed that lasofoxifene prevented bone loss by inhibiting bone turnover associated with aging and orchidectomy in 10-month-old male rats. Further, lasofoxifene decreased total serum cholesterol and did not affect the prostate in these rats. These results suggest that SERMs such as lasofoxifene may be useful therapeutic agents for preventing bone loss in elderly men with some degree of hypogonadism.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
OSTEOPOROTIC fractures occur more commonly in women than men after 45 yr of age (1). This difference may be attributed to the higher peak bone mass of the appendicular skeleton and the slower bone loss during aging in men than in women (2, 3). However, osteoporosis and associated fractures are a common skeletal disorder in aging men (1), to the extent that one third of hip fractures occur in men (4). It has been reported that a mutation in the estrogen receptor gene in a man caused osteoporosis and incomplete epiphyseal closure (5). More recently, results from clinical investigations indicated that bone loss in elderly men is more significantly correlated with declining estrogen levels than with declining androgen levels (6, 7, 8). Thus, it has been hypothesized that estrogen deficiency plays an important role in age-related bone loss in elderly men (9).

Selective estrogen receptor modulators (SERMs) are currently being investigated as alternatives to estrogen for the prevention and treatment of postmenopausal osteoporosis in women (10, 11, 12, 13, 14, 15, 16, 17). SERMs have been shown to maintain estrogen’s positive bone and cardiovascular effects while minimizing several of the objectionable side-effects of estrogen (11, 17). We have discovered a nonsteroidal SERM, lasofoxifene (CP-336,156). Previously we reported (18, 19) that lasofoxifene binds selectively (>100-fold selectivity against all other steroid receptors) and with high affinity to human estrogen receptor- and human estrogen receptor-ß with IC₅₀ values of 1.5 and 1.2 nM, respectively, which are similar to those seen with estradiol. No uterine hypertrophy was observed in immature (3-week-old) or aged (17-month-old) female rats treated with lasofoxifene (18). In ovariectomized (OVX) rats, lasofoxifene completely prevented OVX-induced bone loss and inhibited the increased bone turnover associated with estrogen deficiency (18).

Recent data suggest that estrogen deficiency may play an important role in age-related bone loss in elderly men. We hypothesize that SERMs prevent osteopenia in males. In this study, we examined the effects of lasofoxifene on the adult orchidectomized (ORX) male rat, which has been used as a model for osteopenia in males (20, 21, 22, 23).

	Materials and Methods

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Animals and study protocol
Sixty Sprague Dawley male rats at 10 months of age (Charles River Laboratories, Inc., Wilmington, MA), weighing approximately 630 g, were used for this study. The rats were obtained at 3 months of age and were housed singly in 20 x 32 x 20-cm cages at local vivarium conditions (24 C; 12-h light, 12-h dark cycle) for 7 months before the study. All rats were allowed free access to water and a pelleted commercial diet (Agway ProLab 3000, Agway County Food, Inc., Syracuse, NY) containing 0.97% calcium, 0.85% phosphorus, and 1.05 IU/g vitamin D₃. The experiments were conducted according to Pfizer, Inc., animal care-approved protocols, and animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Ten rats were necropsied on day 0 as basal controls. Ten rats were sham operated (sham) and treated by daily oral gavage with vehicle (10% ethanol and 90% saline, 1 ml/rat) for 60 days as sham-operated controls. The remaining rats (n = 10/group) were ORX and treated by oral gavage with either vehicle or lasofoxifene at doses of 1, 10, and 100 µg/kg·day for 60 days beginning 1 day postsurgery. All rats were given sc injections of 10 mg/kg calcein (Sigma, St. Louis, MO), a fluorochrome bone marker, at 12 and 2 days before death to determine dynamic changes in bone tissues (24).

After 60 days of treatment, body weight was determined. The rats were then necropsied, and blood was obtained by cardiac puncture. Total serum cholesterol was determined using a high performance cholesterol colorometric assay (Roche Molecular Biochemicals, Indianapolis, IN). The prostate wet weight was determined immediately at necropsy. Five-micron, paraffin-embedded, hematoxylin- and eosin-stained prostate sections were used for qualitative observation of histological changes among groups using a microscope. The third lumbar vertebral body (LV3) was removed and saved for static and dynamic bone histomorphometry. The fifth lumbar vertebral body (LV5) was removed and frozen in saline until bone strength testing.

All surgical and necropsy procedures performed in rats were under anesthesia by ip injection of a mixture of ketamine/xylazine (57 mg/0.86 mg·ml solution/kg BW).

Femoral bone mineral measurements
Each right femur was scanned ex vivo using dual energy x-ray absorptiometry (QDR-1000/W, Hologic, Inc., Waltham, MA) equipped with regional high resolution scan software. Bone area, bone mineral content (BMC), and bone mineral density (BMD) of the total femur, distal femoral metaphysis (the second 0.5 cm from the distal end of femur), and femoral shaft (mid 0.5 cm of the femur) were determined as previously described (12, 13).

LV3 cancellous bone histomorphometry
At necropsy, LV3 was removed, dissected free of soft tissue, fixed in 70% ethanol, stained in Villanueva bone stain, dehydrated in graded concentrations of ethanol, defatted in acetone, then embedded in methyl methacrylate (Eastman Organic Chemicals, Rochester, NY). Parasagittal sections of LV3 at 220 µm thickness were cut using a low speed metallurgical saw (Buehler Co., Lake Bluff, IL) and then ground to 20 µm for histomorphometric analysis (25). These 20-µm sections were used to determine indexes related to bone mass, bone structure, bone resorption, bone formation, and bone turnover using an Image Analysis System (Osteomeasure, Inc., Atlanta, GA). Histomorphometric measurements were performed in cancellous bone tissue of LV3 at distances greater than 0.5 mm from the cranial and caudal growth plates.

Measurements and calculations related to bone mass and structure included trabecular bone volume (TBV), thickness, number, and separation, whereas measurements and calculations related to bone resorption included eroded perimeter and percent eroded perimeter. Further, the parameters related to bone formation and turnover included percent labeling perimeter [(double labeling perimeter + 1/2 single labeling perimeter)/total trabecular perimeter x 100], percent osteoid perimeter, mineral apposition rate, bone formation rate/BV, bone formation rate/bone surface (BS), wall width (average distance between reversal line and quiescent trabecular surface), formation period, resorption period, and activation frequency. The definitions and formula for calculations of these parameters were described previously by Parfitt et al. (26) and Jee et al. (27).

Mechanical testing of LV5
Using a Material Testing System (model 810, MTS Systems Corp., Minneapolis, MN), a compression test was used to determine the mechanical properties of LV5 (28, 29). The spinous process and posterior pedicle arch were first removed from LV5 using a low speed saw. Both cranial and caudal ends of epiphyses were removed with a parallel saw set at a distance of 5.0 mm to generate two parallel surfaces (Fig. 1). The specimen was then compressed to failure at a displacement rate of 0.1 mm/sec using a 2.5-kN load cell (MTS model 661, 14A-03). The load and displacement curve were obtained from each test. The maximal load is the force that results in mechanical failure of the LV5 (Fig. 1). Stiffness was calculated from the linear portion of load and displacement curve.

View larger version (29K): [in this window] [in a new window]   Figure 1. Representation of the load cell and dimension employed in the LV5 compression test to assess maximal load and stiffness.

	Results

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Body weight
There was no significant difference in body weight at necropsy among basal controls, sham-operated controls, ORX controls, and ORX rats treated with lasofoxifene at 1 µg/kg·day (Table 1). Body weight was significantly decreased in ORX rats treated with lasofoxifene at 10 or 100 µg/kg·day compared with sham-operated controls at the end of the study (Table 1).

Total serum cholesterol
Total serum cholesterol did not significantly differ between sham-operated and ORX controls (Table 1). Total serum cholesterol significantly increased in ORX rats compared with basal controls. A dose-dependent decrease in total serum cholesterol was seen in lasofoxifene-treated ORX rats compared with vehicle-treated ORX rats. Lasofoxifene at 10 and 100 µg/kg·day significantly decreased total serum cholesterol compared with basal values, sham controls, ORX controls, or ORX rats treated with lasofoxifene at 1 µg/kg·day. There was a significant decrease in total serum cholesterol in ORX rats treated with lasofoxifene at 100 µg/kg·day compared with levels in those treated with 10 µg/kg·day (Table 1).

Femoral bone mineral measurements
Significant decreases in distal femoral metaphysis BMC (-9.6%) and BMD (-8.9%) were found in sham controls compared with basal controls (Table 2). ORX for 2 months induced a nonsignificant decrease in distal femur BMC (-4.1%) and BMD (-5.5%) compared with sham controls. Compared with basal controls, ORX induced a significant decrease in total femur BMD (-4.9%), distal femur BMC (-13.3%), and BMD (-13.9%). Lasofoxifene at a dose of 10 or 100 µg/kg·day significantly prevented the decreases in total femoral BMD, distal femoral BMC, and BMD induced by aging and ORX. All parameters listed in Table 2 did not differ between basal controls and ORX rats treated with lasofoxifene at 10 or 100 µg/kg·day. Compared with ORX rats treated with lasofoxifene at 1 µg/kg·day, ORX rats treated with lasofoxifene at 10 or 100 µg/kg·day had higher BMC and BMD in total femur, distal femoral metaphysis, and femoral shafts. Furthermore, total femoral BMD was significantly higher in ORX rats treated with lasofoxifene at 100 µg/kg·day compared with levels in rats treated with 10 µg/kg·day (Table 2).

View larger version (52K): [in this window] [in a new window]   Figure 2. TBV (A), osteoid perimeter (B), eroded perimeter (C), and activation frequency (D) of third lumbar vertebral cancellous bone in basal controls (Basal), sham-operated controls (Sham), and ORX rats treated with vehicle (ORX) or lasofoxifene at 1 µg/kg·day (1 µg), 10 µg/kg·day (10 µg), or 100 µg/kg·day (100 µg) for 2 months. a, P < 0.05 vs. Basal; b, P < 0.05 vs. Sham; c, P < 0.05 vs. ORX; d, P < 0.05 vs. ORX plus1 µg/kg·day; e, P < 0.05 vs. ORX plus 10 µg/kg·day.

Effects of lasofoxifene. There was a significant decrease in eroded perimeter (-48%), but no significant difference was found in other parameters listed in Table 3 and Fig. 2 in ORX rats treated with lasofoxifene at 1 µg/kg·day compared with ORX controls. Compared with ORX controls, ORX rats treated with lasofoxifene at 10 µg/kg·day had significantly higher trabecular thickness (+16%) and significantly lower percent labeling perimeter, percent osteoid perimeter, percent eroded perimeter, bone formation rate/BV, bone formation rate/BS, and formation period. ORX rats treated with lasofoxifene at 100 µg/kg·day had significantly greater TBV, trabecular thickness, wall width, formation period, and resorption period and significantly lower labeling perimeter, osteoid perimeter, eroded perimeter, mineral apposition rate, bone formation rate/BV, bone formation rate/BS, and activation frequency compared with ORX controls. Compared with sham controls, there was no significant difference in all other parameters listed in Table 3 and Fig. 2 in ORX rats treated with lasofoxifene at 10 or 100 µg/kg·day, with the exception of an increase in mineral apposition rate with 10 µg/kg·day and an increase in trabecular thickness with 100 µg/kg·day.

Compared with ORX rats treated with lasofoxifene at 1 µg/kg·day, ORX rats treated with lasofoxifene at 10 µg/kg·day had significantly higher trabecular bone thickness and mineral apposition rate and significantly lower labeling perimeter, osteoid perimeter, and formation period. ORX rats treated with lasofoxifene at 100 µg/kg·day had significantly higher TBV, trabecular thickness, trabecular number, wall width, formation period, and resorption period compared with those treated with lasofoxifene at 1 µg/kg·day (Table 3 and Fig. 2). Trabecular separation, labeling perimeter, osteoid perimeter, mineral apposition rate, bone formation rate/BV, bone formation rate/BS, and activation frequency were significantly lower in ORX rats treated with lasofoxifene at 100 µg/kg·day than in rats treated with lasofoxifene at 1 µg/kg·day. Further, ORX rats treated with lasofoxifene at 100 µg/kg·day had significantly higher wall width, formation period, and resorption period and significantly lower mineral apposition rate, bone formation rate/BV, bone formation rate/BS, and activation frequency compared with ORX rats treated with lasofoxifene at 10 µg/kg·day (Table 3 and Fig. 2).

Mechanical testing of LV5
Maximal load did not differ between basal and sham controls (Fig. 3). A nonsignificant decrease (-11%) in maximal load was found in ORX controls compared with sham controls. ORX significantly decreased maximal load (-17%) compared with that in basal controls. No significant difference was found in ORX rats treated with 1 µg/kg·day lasofoxifene compared with basal, sham, or ORX controls. ORX rats treated with lasofoxifene at 10 or 100 µg/kg·day had significantly higher maximal load (both +30%) compared with ORX controls (Fig. 3). No statistically difference was observed in ORX rats treated with lasofoxifene at 10 or 100 µg/kg·day compared with those treated with lasofoxifene at 1 µg/kg·day (Fig. 3).

View larger version (31K): [in this window] [in a new window]   Figure 3. Maximal load (A) and stiffness of LV5 in basal controls (Basal), sham-operated controls (Sham), and ORX rats treated with vehicle (ORX) or lasofoxifene at 1 µg/kg·day (1 µg), 10 µg/kg·day (10 µg), or 100 µg/kg·day (100 µg) for 2 months. a, P < 0.05 vs. Basal; b, P < 0.05 vs. Sham; c, P < 0.05 vs. ORX; d, P < 0.05 vs. ORX plus 1 µg/kg·day; e, P < 0.05 vs. ORX plus 10 µg/kg·day.

	Discussion

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The mechanism for body weight loss in lasofoxifene-treated ORX rats is currently unknown. Food intake was not determined in this study. However, more recent studies suggest that lasofoxifene decreased food intake by approximately 15% in aged male rats (Ke, H. Z., et al., in preparation). Thus, we hypothesize that a decrease in food intake in lasofoxifene-treated ORX rats may in part contribute to a loss in body weight. Studies are currently underway to determine which component of body composition changes in response to lasofoxifene treatment in aged male rats.

In addition to the above findings, we found age-related changes between 10 (basal controls) and 12 (sham controls) months of age in male rats. There were significant decreases in prostate weight (-44%), distal femoral BMC and BMD, vertebral TBV, thickness, and mineral apposition rate at 12 months of age compared with 10 months of age in these male rats. These age-related changes might be due in part to the decrease in testosterone levels at 12 vs. 10 months of age. Our previous finding (31) that serum testosterone levels decreased approximately 30% at 12 months (82.6 ± 13.9 ng/dl) compared with 10 months (118.5 ± 21.5 ng/dl) of ages in male rats support this hypothesis. These results were in agreement with our earlier findings (33), which indicated that male rats achieve their peak cancellous bone mass at or before 10 months of age, and an age-related cancellous bone loss occurs thereafter. Thus, the skeletal metabolism status of 10-month-old male rats might be similar to that of 50-yr-old men, as Agnusdei et al. (32) reported that bone loss in men accelerates after 50 yr of age. Thus, adult male rats at or older than 10 months may be a very useful model to study age-related bone loss in men.

The decreased bone mass in ORX rats in the current study was mainly accounted for by age-related bone loss. As discussed above, an age-related decrease in bone mass was observed in sham controls compared with basal controls. ORX for 2 months induced only nonsignificant decreases in distal femoral BMC and BMD, lumbar vertebral TBV, and maximal load compared with sham controls. Longer than 2 months may be needed to induce significantly lower bone mass in adult ORX rats compared with sham controls. Vanderschueren et al. (22) reported that ORX in 13-month-old male rats induced a significant decrease in bone mass at 4 months but not at 1 month postsurgery compared with sham controls. Nevertheless, bone mass and bone strength decreased significantly in ORX controls compared with basal controls in the current study. These data also demonstrated the importance of basal (time zero) controls in such studies.

Androgen deficiency may express its effects on skeletal tissues via the androgen receptor and/or the estrogen receptor, as previously reviewed by Vanderschueren et al. (33). It has been shown that either androgen (21) or estrogen (22) replacement can prevent bone changes induced by androgen deficiency in ORX rats. Similar to the effects of estrogen (22), our data showed that treatment with lasofoxifene, which selectively binds to the estrogen receptors (>100-fold selectivity against all other steroid receptors), prevented bone loss and bone turnover associated with aging and androgen deficiency in adult male rats. At an oral dose as low as 1 µg/kg·day, lasofoxifene partially inhibited the increases in bone resorption. At this dose level, however, bone mass and strength did not differ between vehicle- and lasofoxifene-treated ORX rats. The decreases in bone mass and strength and the increases in bone resorption and turnover were prevented by lasofoxifene at doses of 10 and 100 µg/kg·day in ORX rats. These bone-protective effects of lasofoxifene in ORX male rats were similar to those previously reported in female OVX rats (18). In the OVX female rats, we found that lasofoxifene at doses equal to or greater than 10 µg/kg·day completely prevented bone loss and inhibited bone turnover associated with estrogen deficiency.

It has been well documented that SERMs decrease total serum cholesterol in OVX female rats (11, 12, 13, 14, 15, 16) and decrease total serum cholesterol and low density lipoprotein in postmenopausal women (10, 17). It has also been reported that oral estrogen treatment improves serum lipid levels in elderly men (34). Although the rat is not an ideal model of human lipid modulation, we found that lasofoxifene decreased total serum cholesterol in ORX male rats in this study, indicating that lasofoxifene acts as an estrogen agonist for serum lipoproteins in male rats, similar to that in OVX female rats. The mechanism of the lipid-lowering effects of lasofoxifene in ORX male rats is not clear. The decreases in food intake and body weight in these rats induced by lasofoxifene treatment may result in the decreased total serum cholesterol.

In summary, we found that lasofoxifene acted as an estrogen agonist on bone and serum cholesterol and had no effect on prostate weight in the adult ORX rat model. These results suggested that SERMs such as lasofoxifene might be useful agents for the prevention of osteoporosis not only in postmenopausal women, but also in elderly men with some degree of hypogonadism.

	Acknowledgments

 
The authors thank Dr. Webster Jee of University of Utah for help with the histomorphometric measurements, Dr. Victor Shen of Helen Hayes Hospital for help with the mechanical tests of bones, and Drs. Mei Li and David B. MacLean of Pfizer Central Research for helpful discussions and suggestions.

Received August 26, 1999.

	References

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