JOURNAL OF BONE AND MINERAL RESEARCH Volume 7. Number 8 . 1992 Mary Ann Liebert, Inc., Publishers

The Bisphosphonate, Alendronate, Prevents Bone Loss in Ovariectomized Baboons D.D. THOMPSON, J.G. SEEDOR, H. QUARTUCCIO, H.SOLOMON, C. FIORAVANTI, J. DAVIDSON, H. KLEIN, R. JACKSON, J. CLAIR, D. FRANKENFIELD, E. BROWN, H.A. SIMMONS, a n d G.A. RODAN

ABSTRACT We examined the effect of the amino bisphosphonate alendronate, administered IV every 2 weeks at 0.05 and 0.25 mg/kg for 1 year, on bone loss and parameters related to bone metabolism in ovariectomized baboons. Relative to non-OVX animals, the OVX baboons experienced increased bone turnover, reflected in biochemical and histomorphometric measurements, and bone loss assessed by dual-beam absorptiometry in the lumbar spine, which was similar to changes observed in ovariectomized women. Alendronate treatment maintained all parameters of bone turnover at control (nonovariectomized) levels and prevented the bone loss in a dose-dependent manner. We concluded that ovariectomized baboons offer a suitable model for the bone changes observed in ovariectomized women and that these changes can be prevented by sustained administration of an appropriate dose of this aminobisphosphonate.

INTRODUCTION N HUMANS, MENOPAUSE is accompanied by increased bone turnover that can result in bone loss of about 2%/ year for about 5 years and in increased susceptibility to fractures, especially in the vertebrae. ( I m Prevention of postmenopausal bone loss can be achieved by estrogen replacement therapy (ERT),whose primary action is to inhibit bone resorption. However, ERT is not always well tolerated and patient compliance is often poor; therefore, alternative therapies are being considered. Bisphosphonates are potent inhibitors of bone turnover.(” They act directly on osteoclasts to reduce bone resorption(‘) and were shown to reduce bone loss in established osteoporosis(’.*’and prevent bone loss in early menopause(” or after surgical oophorectomy. ( W Alendronate (4-amino- I -hydroxybutylidene-1-bisphosphonicacid sodium salt) is a potent new member of this class of compounds, which was shown to be effective in the treatment of hypercalcemia of malignancy(9’ and Paget’s disease(Io’in humans and in reducing immobilization-related bone Ioss(’~~ and estrogen deficiency bone loss in rats.(‘”

I

Animal models of estrogen deficiency mimic surgically induced menopause in women, in whom increased bone turnover and bone loss follow removal of the ovaries.(13-”’ In ovariectomized rats, increased boine turnover is detected within 2 weeks and bone loss is demonstrable by 8 weeks.(l””o’ In baboons, increased bone turnover and bone loss were noted by 6 months.(,’) Unlike rats, baboons have menstrual cycles similar to humans, and thus lifetime cyclic patterns of estrogen exposure should also be similar. Patterns of bone turnover and bone loss in baboons are therefore more likely to minic those in humans. The purpose of this study was to assess the effects of estrogen deficiency in ovariectomized baboons and to evaluate the ability of alendronate to reduce bone turnover and prevent bone loss due to estrogen deficiency over a 12 month treatment period.

MATERIALS AND METHODS A total of 28 adult female baboons (Pupio onubb) weighing approximately 12 kg were used in this study. Animals were fed standard laboratory food (Purina Monkey

Merck. Sharp and Dohme Research Laboratories, West Point, Pennsylvania.

95 1

THOMPSON ET AL.

952

Chow) and fruit. All animals had a history of normal, regular menstrual cycles. Each animal was evaluated by x-ray to ensure epiphyseal closure and to confirm adult skeletal status and the absence of skeletal abnormalities, such as compressed vertebrae or fractures. Baboons with incomplete epiphyseal closure or evidence of skeletal abnormalities were not admitted to the study. Animals were randomly assigned to one of four groups ( n = 7 per group).

Ovariectorny procedure Three groups of baboons ( n = 21) underwent surgery to remove the ovaries (OVX groups). The animals were anesthetized using a mixture of ketamine and xylazine (75 mg ketamine hydrochloride and 5 mg xylazine per ml) given intramuscularly (IM) at a dose of 1 ml per 10 kg body weight and were maintained under anesthesia with intermittent intravenous IV) administration of ketamine and xylazine into the saphenous vein. Each animal was also intubated endotracheally and received glycopyrolate (Robinul, 0.05 mg/kg IM). The lower abdomen was surgically scrubbed and an incision made to expose the ovaries and uterus. The ovarian artery and vein were isolated and ligated on the proximal and distal aspects of each ovary. The ovaries were removed and the muscles and skin sutured. All animals recovered from surgery without incident. The fourth group was left with intact ovaries (nonO V X group) and was not exposed to surgery.

Transilial bone biopsy At time 0 a transilial bone biopsy from all animals was obtained at a site 3-5 mm inferior and anterior to the exposed margin of the ileum. A Michele bone trephine, 8 mm ID (inner diameter), was used to extricate the bone biopsy. Care was taken to ensure that both cortices of the biopsy and the intervening trabeculae were intact. The biopsy was fixed in 70% ethanol and refrigerated. Additional bone biopsies were obtained identically on alternate ilia at 6 and 12 months after ovariectomy. The 12 month biopsy was taken at a site 2 cm posterior to the site of the biopsy taken at time 0 to avoid the healed site.(211

Bone histomorphornetry Following fixation in 70% ethanol, the bone samples were dehydrated in successive concentrations of ethanol, cleared in xylene, and embedded in methyl methacrylate. Sections 6 pn in thickness, stained wit Masson's trichrome stain, were subjected to histomorphometric analysis with the aid of a Joyce-Loebl image analyzer (Magiscan) equipped with a Sony MP4 color camera and Bone software. (''' Histomorphometric parameters acquired included trabecular bone volume (Yo), osteoid surface and area (To), eroded (resorption) surface (Vo), and osteoclast number per mm of bone surface (number/mm). A total of 20 fields were analyzed in each biopsy for a total measured area of 4.06 mm' for each section. Two sections were analyzed in each biopsy.

Serum and urine biochemistry The urine was obtained by puncture of the bladder 12-16 h after last feeding at time 0, 3, 6, 9, and 12 months postovariectomy. At this time vertebral x-rays (anteroposterior. AP) were taken and animals were weighed. Serum and urinary calcium (mg/dl) were measured by atomic absorption spectroscopy. Serum tartrate-resistant acid phosphatase (U/dl), urinary~. phosphate (mg/dl), and urinary . . . creatinine (mg/dl) were measured with a Gemstar Analyzer (Electro-Nucleonics, Inc). Serum creatinine (mg/dl), phosphate (mg/dl), sodium (mmol), and total protein (gm/dl) were determined with a Kodak analyzer. Estradiol (pg/ml; New England Nuclear, Cambridge, MA), osteocalcin (BGP, ng/ml; Incstar, Stillwater, MN), calcitonin, and intact parathyroid hormone (pg/ml; Nichols Institute, San Juan Capistrano, CA) were determined by radioimmunoassay (RIA). 1,ZS-Dihydroxyvitamin D, was determined by RIA (pg/ml, Nichols Institute), using a modified extraction procedure (J. Haddad, personal communication).

Bone mineral content of the spine, femoral neck, and radius Bone mineral measurements were taken at time 0, 3, 6, 9, and 12 months postovariectomy on anesthetized animals. Single-photon absorptiometric measurements were taken on the radius one-third from the distal end at a site that was tatooed for future measurements, using a Lunar SP2 scanner with an l Z 5 I source (Lunar Corp.. Madison, WI). Dual-photon absorptiometric measurements of the lumbar spine (Ll-4) and femoral neck were performed with a Lunar DP3 scanner with a "'Gd source (Lunar Corp.). All bone scans were performed with the baboon in a prone position. Each baboon's radius, femur, and spine were scanned twice, repositioning between each scan. The radius was scanned with the palmar surface down. The right femur was scanned from approximately 4 inches below the pubic symphysis to approximately 4 inches below the greater trochanter. The lumbar spine was scanned from below the intervertebral space of L4-5 to above the intervertebral space of Ll-Tl2. Two operators independently processed the scan data from the three sites using software supplied with the scanners. Radial measurements were largely automated. Lumbar spine scan processing required adjustment of the baselines for adequate edge detection. For vertebrae the operators marked the intervertebral spaces, and the software was then used to calculate the bone mineral content. Similar manual processing was required for the femoral neck. A 2.86 x 0.75 cm box was superimposed on the scan image of the femoral neck, and this defined the region of interest for further processing. The software required that the computer-generated box be positioned perpendicular to the long axis of the femoral neck. A reproducibility study was conducted to assess the effect of operator variability and radionuclide source strength on the bone mineral measurements. Four normal

953

ALENDRONATE EFFECT IN OVARIECTOMIZEDBABOONS nonovariectomized baboons were studied. Each operator scanned and processed L2-4 five times on 2 different days with a new ' Y i d source and a source that was 1 year old. The results showed that the source age did not contribute to variation in bone mineral content values, nor did substantial variation exist between operators (data not shown). An additional validation study was performed in a group of nonovariectomized baboons, which were not part of this study, to assess radial, lumbar, and femoral neck scan reproducibility over time. The results showed that the mean coefficient of variation for five different scan sessions over 12 months (0,3, 6 , 9 , and 12 months) was 2.4% for the radius, 3% for the lumbar spine, and 8.8% for the femoral neck.

Treatment with alendronate The ovariectomized baboons were treated with 0, 0.05, or 0.25 mg alendronate per kg IV every 2 weeks. The baboons were anesthetized, a 21 gauge butterfly catheter was inserted in the saphenous vein, and 3 ml saline with or without drug was infused over 3 minutes. The bimonthly intravenous regimen was chosen for the following reasons: simultaneous ingestion of food reduces drug absorption and IV administration provides best assurance for full dose delivery. Previous dose-response studies in rats have indicated that the chosen dose levels may be effective and human studies on hypercalcemia of malignancy have shown 2-3 weeks efficacy after a single IV bolus, possibly corresponding to the duration of the resorption phase at a particular site. No adverse reactions to anesthesia or drug treatment over the 12 months of study were noted.

Statistical analysis Means and standard errors were computed for each variable. Analysis of variance was performed, and significance was established at p < 0.05 using Fisher's Protected Least Square Difference (PLSD) test.

RESULTS Animal weights at 3 month intervals are shown in Table 1. All four groups of animals gained weight over the 12

months of observation; the differences were significant for the non-OVX and the high-dose alendronate groups. X-rays of the spine, taken at 3 month intervals for I year, revealed no apparent crush fractures or collapsed vertebrae.

Serum biochemislry Estradiol (pg/ml) in the baboons at time 0 ranged from 2.8 to 22.8 pg/ml. At 12 months estradiol levels in the nonovariectomized baboons ranged between 2.3 and 20.7 (10.4 6.9 pg/ml SEM, standard error of the mean), and all ovariectomized baboons had undetectable levels. Serum alkaline phosphatase (U/liter) was significantly (p < 0.05) increased in vehicle-treated ovariectomized baboons at 3 months postovariectomy and remained significantly elevated at 6, 9. and 12 months (Fig. la). In ovariectomized baboons treated with either 0.05 or 0.25 mg alendronate per kg, alkaline phosphatase levels did not change relative to time 0 levels. Furthermore, no significant differences were noted at any time between the ovariectomized baboons treated with alendronate and the nonovariectomized controls. Serum tartrate-resistant acid phosphatase (U/dl) was significantly (p < 0.05) elevated at 6 and 12 months in the vehicle-treated ovariectomized group. The alendronatetreated groups also showed an increase in tartrate-resistant acid phosphatase at 12 months, albeit less than that observed in the vehicle-treated group (Fig. Ib). Osteocalcin (BGP, ng/ml) levels were significantly (p < 0.05) elevated following ovariectomy in the vehicle-treated group starting at 6 months and remained elevated at 9 and 12 months (Fig. 2a). BGP levels in alendronate-treated animals were significantly lower than in the ovariectomized vehicle-treated animals and were not significantly different from those in the nonovariectomized group. Serum PTH levels changed only in the 0.25 mg alendronate-treated group where a significant increase relative to time 0 was seen at 3 and 9 months following ovariectomy (Fig. 2b). Calcitonin (pg/ml) levels in serum were not affected by ovariectomy or treatment (Fig. 2c). Significant increases in 1,25-dihydroxyvitarnin D, (pg/ml) levels relative to time 0 were found in all groups at 6 and 12 months (Table 2). At 12 months the 1.25-dihydroxyvitamin D,

TABLE1. BODYWEIGHT^ Alendronate (mglkg) Time (months)

Non-OVX

0 3 6 9 12

14.42 (0.37) 15.36 (0.41) 16.10 (0.44) 16.41 (0.67) 17.20 (0.97)b

OVX- Vehicle

0.05

0.25

14.09 (0.42) 13.94 (0.47) 14.02 (0.58) 14.50 (0.67) 14.79 (0.66)

13.84 (0.48) 13.96 (0.38) 14.10 (0.37) 14.61 (0.47) 14.90 (0.52)

13.90 (0.39) 14.21 (0.61) 14.33 (0.60) 15.00 (0.64) 15.78 (0.45)b

aMean body weights (kg) of each of the baboon groups at each time point. bp < 0.05 versus time 0.

THOMPSON ET AL.

954

300

f

250

200

1100

5 0

3

0 6 Time (Months)

N 9

I

1.511 0

12

I

3

6

9

1 12

Time (Monthm)

FIG. 1. (a) Serum alkaline phosphatase (ALP) levels in ovariectomized (OVX) baboons treated with alendronate for 12 months. ALP was measured as described in Materials and Methods. (b) Serum tartrate-resistant acid phosphatase (ALP) levels in ovariectomized baboons treated with alendronate for 12 months. ALP was measured as described in Materials and Methods.

TABLE 2. 1,25-DIHYDROXYVITAMIN DJ (PG/ML)a Alendronate (mg/kg) Time (months)

Non-OVX

OVX- Vehicle

0.05

0.25 ~~

0 6 12

77.6 (18.4) 147.5 (8.l)b 133.9 (14.2)b

92.3 (17.9) 145.0 (21.8)b 200.7 (15.6)b

125.0 (19.1) 163.9 (9.7)b 176.8 (21.2)b

~~~~

97.2 (8.1) 197.7 (21.8)b 194.7 (13.8)b

aMean serum 1,25-dihydroxyvitamin D, levels at 0, 6. and 12 months for each group of baboons. bp < 0.05 relative to time 0 value.

levels in the ovariectomized groups were significantly higher than in the nonovariectomized group. Serum calcium levels varied between 7.6 and 9.7 mg/dl but were higher at all time points in the untreated ovariectomized baboons than in the nonovariectomized and treated groups (Table 3). Serum phosphate values ranged between 3.95 and 5.9 mg/dl during the study period. The ovariectomized, vehicle-treated animals had higher blood phosphate levels at all time points examined, but statistical differences were noted only at 3 and 9 months. Only in one group of alendronate-treated animals at one time point (3 months) was there a statistically significant decrease in phosphate levels relative to the vehicle-treated animals. Creatinine (mg/dl). sodium (mmol/liter), and total protein (gm/dl) showed no significant differences between groups over time (Table 3).

Urinary biochemistry No significant differences as a function of time or between groups were noted for urinary calcium or creatinine or phosphorus values (data not shown).

Iliac bone histomorphometry Transilial bone biopsy samples from each baboon were quantitated at time 0, 6, and 12 months postovariectomy.

Trabecular bone volume (Vo)at time 0 was similar in all groups, about 37% (Fig. 3a). The trabecular bone volume in the vehicle-treated group declined to 27% 6 months postovariectomy and remained at this level at 12 months. No reduction in trabecular bone volume was detected in biopsies from the alendronate-treated groups at 6 or 12 months. It thus appeared that 0.05 and 0.25 mg alendronate/kg given IV twice per month prevented the decrease in trabecular bone volume. Osteoid surface (OS/BS) and volume (OV/BV) were significantly (p < 0.05) increased in the ovariectomized vehicle-treated group at 6 and 12 months postovariectomy. No significant differences in osteoid volume or surface between the nonovariectomized and the ovariectomized group treated with 0.05 mg alendronate per kg were noted over the 12 months of observation. In the animals treated with 0.25 mg alendronate, these parameters were significantly lower at 6 but not at 12 months (Figs. 3b and c). Osteoclast number per mm trabecular bone surface was significantly (p < 0.05) elevated in the vehicle-treated group at 6 and 12 months compared to time 0, and the group treated with 0.25 mg alendronate per kg showed a significant decrease at 6 months (Fig. 4a). No change in osteoclast number was noted with time or between the nonovariectomized and ovariectomized group treated with 0.05 mg alendronate per kg. Eroded surface (To) was significantly (p < 0.05) increased in the vehicle-treated group

955

ALENDRONATE EFFECT IN OVARIECTOMIZED BABOONS 0 Non-Ovr

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FIG. 2. (a) Serum osteocalcin (BGP) levels in ovariectomized baboons treated with alendronate for 12 months. BGP was measured by radioimmunoassay as described in Materials and Methods. (b) Serum intact parathyroid hormone (PGH) levels in ovariectomized baboons treated with alendronate for 12 months. PTH was measured by radioimmunoassay as described in Materials and Methods. (c) Serum calcitonin levels in ovariectomized baboons treated with alendronate for 12 months. Calcitonin was measured by radioimmunoassay as described in Materials and Methods.

FIG. 3. (a)Trabecular bone volume (To) measured in transilial bone biopsies in ovariectomized baboons treated with alendronate for 12 months. The biopsy procedure, sample preparation, and histomorphometric analysis are described in Materials and Methods. (b) OS/BS surface (To) measured in transilial bone biopsies in ovariectomized baboons treated with alendronate for 12 months and (c) osteoid volume (To). The biopsy procedure. sample preparation, and histomorphometric analysis are described in Materials and Methods.

at 6 and 12 months, but the other groups were unchanged tomy or alendronate treatment did not change bone minfrom time 0 (Fig. 4b). eral content in the radius over the 12 month period. Bone mineral content of the femoral neck did not change in the untreated ovariectomized group but was significantly increased (p < 0.05) in the groups treated with 0.05 and 0.25 Bone mineral content of the radius, femoral mg alendronate per kg at 9 and 12 months compared to neck, and spine time 0. In the nonovariectomized group bone mineral conTable 4 contains data for each of the four groups at tent was also significantly increased at 12 months relative each time point for the radius and femoral neck, Ovariec- to time 0.

THOMPSON ET AL.

956 TABLE3. MEANSERUM VALUES FOR BIOCHEMICAL PARAMETERSa

Alendronate (mg/kg) Time (months)

Non-0 VX

0VX- Vehicle

0.25

0.05

Ca (mgldl)

0 3 6 9 12

8.16 (0.20) 9.14 (0.10) 8.I4 (0.1I) 8.46 (0.22) 8.60 (0.22)

7.61 (0.24) 9.94 (0.14)b 8.38 (0.20) 9.67 (0.08)b 8.90(0.12)

7.85 (0.15) 9.51 (0.18) 8.06 (0.23) 9.05 (0.17)c 8.76 (0.16)

7.93 (0.19) 9.59 (0.20) 7.75 (0.19)c 9.14 (0.24) 8.48 (0.20)

4.67 (0.36) 4.37 (0.32) 4.83 (0.43) 3.95 (0.25) 4.46 (0.36)

5.23 (0.17) 5.47 (0.35)b 5.21 (0.30) 5.90 (0.39)b 4.74(0.16)

5.54 (0.34) 4.30 (0.21)c 5.04 (0.42) 4.89 (0.32)b 5.66 (0.43)c

5.54 (0.20) 4.87 (0.27) 5.43 (0.20) 4.91 (0.29)b 4.99 (0.30)

153.43 (1.17) 137.57 (4.39) 136.00 (3.70) 133.57 (5.39) 142.86 (2.14)

153.33 (1.20) 136.43 (4.08) 142.43 (7.94) 138.29 (4.08) 142.86 (0.88)

149.50 (1.86) 149.33 (3.90) 134.00 (5.25) 140.86 (5.88) 146.29 (1.30)

153.00 (1.65) 145.86 (9.00) 141.29 (4.79) 137.29 (1.48) 143.29 (1.36)

0.89 (0.06) 0.93 (0.08) 1.06 (0.22) 0.67 (0.04) 0.73 (0.05)

0.81 (0.07) 0.90(0.15) 0.84(0.06) 0.87 (0.14) 0.84 (0.07)

0.80 (0.04) 0.84 (0.16) 0.74(0.09) 0.86 (0.05) 0.90 (0.06)

0.83 (0.08) 1.03 (0.11) 0.91 (0.06) 1.04 (0.08) 0.91 (0.06)

6.86 (0.26) 6.85 (0.21) 6.49 (0.22) 6.66 (0.17) 6.50 (2.18)

7.11 (0.11) 7.28 (0.14) 7.09 (0.23) 6.83 (0.14) 6.46 (0.21)

6.87 (0.24) 7.39 (0.09) 6.87 (0.13) 6.64 (0.21) 6.34 (0.19)

6.70 (0.18) 7.84(0.33) 7.07 (0.24) 7.31 (0.27) 6.46 (0.12)

PO, (mg/dl)

0 3 6 9 12 Na (mmol/liter) 0 3 6 9 12 Creatinine (mg/dl)

0 3 6 9 12 Total protein (g/dl)

0 3 6 9 12

~

~

~

aEstirnated as described in Materials and Methods for each of the baboon groups at each time point. bp < 0.05 relative to non-OVX. cp < relative to OVX-vehicle.

Lumbar spine (Ll-4)bone mineral content was significantly (p < 0.05) reduced at 6 months in the vehicletreated ovariectomized group compared to time 0 but then increased at 9 and 12 months; however, the increase was not statistically significant (Fig. 5). No change in spinal bone mineral content was noted in the group treated with 0.05 mg alendronate per kg over 12 months of study. The lumbar bone mineral content of the group treated with 0.25 mg alendronate per kg was significantly elevated at 9 and 12 months, and the nonovariectomized animals showed a mean increase in spinal bone mineral content that was not statistically significant.

DISCUSSION Various animal models that mimic estrogen deficiency bone loss in humans have been explored. Rats lose a considerable amount of cancellous bone following ovariectomy.(La-20’ and bone loss can be prevented by estrogen re-

placement. There are many rat skeletal features, however, such as longitudinal growth during adulthood and the absence of Haversian systems in cortical bone, that differ from those in humans. In addition, it was reported that in rats estrogen treatment decreased the amount of cortical bone,(131whereas in women it is estrogen deficiency that The rat estrus causes a reduction in cortical bone. cycle is about 4 days versus 28 in humans, which could translate into a different estrogen exposure pattern and possibly average exposure level of the rat skeleton. These differences do not invalidate the rat as a model for studying certain aspects of estrogen deficiency bone IOSS(~’-~~’ but may limit its applicability for evaluating the effect of long-term therapy on bone preservation in the human disease.(261Dogs, which have an estrus cycle of 260 days but possess larger bones with Haversian canals, were reported to lose bone following ovariectomy in some studies but not in Ewes also lose bone following ovariectomy and may provide a suitable model for estrogen deficiency bone Postovariectomy bone loss was also re-

957

ALENDRONATE EFFECT IN OVARIECTOMIZEDBABOONS 0 Non-Ova

I

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3

0

6 Time (Months)

9

12

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0

3

6 Time (Months)

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FIG. 4. (a) Number of osteoclasts per mm bone surface measured in transilial bone biopsies from ovariectomized baboons treated with alendronate for 12 months. The biopsy procedure, sample preparation, and histomorphometric analysis are described in Materials and Methods. (b) Eroded surface (To) measured in transilial bone biopsies in ovariectomized baboons treated with alendronate for 12 months. The biopsy procedure, sample preparation, and histomorphometric analysis are described in Materials and Methods.

TABLE 4. BONEMINERAL CONTENT IN RADIUS AND FEMORAL NECK OF WITH ALENDRONATE TREATED

Radius Non-OVX OVX vehicle OVX 0.05 OVX 0.25 Femoral neck Non-OVX OVX vehicle OVX 0.05 OVX 0.25 ap

ovx BABOONS

Time 0

3 Months

6 Months

9 Months

12 Months

0.50 (0.015) 0.53 (0.015) 0.51 (0.018) 0.52 (0.022)

0.50 (0.018) 0.51 (0.013) 0.52 (0.016) 0.52 (0.020)

0.50 (0.018) 0.52 (0.022) 0.51 (0.010) 0.51 (0.023)

0.51 (0.015) 0.52 (0.020) 0.52 (0.016) 0.52 (0.022)

0.51 (0.018)

0.57 (0.012) 0.63 (0.034) 0.58 (0.015) 0.63 (0.026)

0.60 (0.024) 0.60 (0.023) 0.59 (0.013) 0.69 (0.024)

0.61 (0.029) 0.63 (0.029) 0.59 (0.026) 0.69 (0.026)

0.63 (0.024) 0.64 (0.036) 0.64a (0.023) 0.73a (0.026)

0.69a (0.032) 0.67 (0.025) 0 . 6 9 (0.032) 0.74a (0.017)

0.52 (0.015) 0.53 (0.021) 0.53 (0.024)

< 0.05 relative to time 0.

ported in primate species. including baboon^,'^^-^^^^^^ which have a menstrual cycle similar to that of humans. The findings reported here further document the similarity in estrogen deficiency bone loss in baboons and humans. As in ovariectomized and in pharmacologically ovariectomized primates, 15-17.Ja1 significant increases in the osteoblast activity markers alkaline phosphatase and osteocalcin were observed in vehicle-treated baboons 3 and 6 months postovariectomy. respectively. The level of these osteoblast activity markers remained elevated over the 12 months of observation. Similarly, tartrate-resistant acid phosphatase, a marker of osteoclastic activity, was also elevated at 6 and 12 months. The serum calcium and phosphate levels were slightly higher in OVX animals (3-9% for calcium and 6 4 9 % for phosphate), and the differences were statistically significant at 3 and 9 months. No differences in sodium, creatinine, or total protein were observed. The changes in serum parathyroid hormone reflected the calcium changes, showing a significant reduc-

tion in the OVX animals at 3 and 9 months compared to controls. These findings suggest that the changes in parathyroid hormone level are secondary to increases in bone resorption. As previously reported for women, there were no significant changes in serum calcitonin levels throughout the study. The mean calcium and phosphate concentrations in urine samples and the calcium to creatinine ratios were higher in the ovariectomized animals at all time points except the last. but there was a relatively large error in these measurements and the differences were not statistically significant (data not shown). The increase in bone turnover indicated by the biochemical markers was accompanied by histomorphometric changes in the iliac crest bone biopsies. The ovariectomized animals showed a significant increase in osteoclast number and eroded surfaces at 6 and 12 months relative to controls, as well as an increase in osteoid area and surface, reflective of increased bone turnover. These changes were associated with a decrease in trabecular bone volume,

958

THOMPSON ET AL.

had closed epiphyses, the exact age of these animals is not known. Extensive calibration of the instruments indicated that this was not due to experimental error. Alendronate, which has been shown to be a potent in6 hibitor of osteoclastic bone resorption in v i t ~ o ' ~ and ' ] in viva, 14.1 I . l 1 . 3 1 1 effectively prevented the increase in bone turnover and the bone loss caused by estrogen deficiency. Treatment of ovariectomized baboons with either 0.05 or 0.25 mg alendronate per kg every 2 weeks IV prevented the increases in serum alkaline phosphatase, osteocalcin, and tartrate-resistant acid phosphatase, markers of bone turnover. Bone turnover estimated by histomorphometric measurements, such as osteoid area, also showed a reduction -6' to control levels in both ovariectomized groups treated 0 3 6 9 12 Months with alendronate. The resorption surface extent and osteoclast number were also similar to that of nonovariecto+ =p< 05 lrom tlme 0 mized animals. Similarly, the trabecular bone volume was =p< 05 lrom OVX 0 25 treated group FIG. 5. Bone mineral content (BMC) percentage change also maintained at control levels, approximately 37%. Alendronate treatment also prevented the bone loss defrom time 0 averaged for lumbar vertebrae L1-4. BMC measurements on anesthetized baboon were carried out tected by absorptiometry. The changes in lumbar BMC in every 3 months as described in Materials and Methods. animals receiving 0.25 mg/kg per 2 weeks 1V followed closely those of nonovariectomized controls at all times exSymbols as in other figures. amined, showing a net gain of 4% at 12 months. The animals receiving 0.05 mg/kg had intermediate values bewhich was observed at 6 months and persisted at 12 tween those receiving the higher dose and vehicle-treated months. These changes are consistent with similar observa- animals, showing a net gain of about 2% at 12 months. The reduction in bone turnover produced by alendrotion made in estrogen-deficient women. 1 1 3 , 1 ' , 3 1 1 The bone loss caused by estrogen deficiency is associated nate and reflected in biochemical, histologic, and radiowith increased bone turnover and is due to increased bone logic measurements was not accompanied by findings that resorption, which is not compensated by an adequate in- suggest adverse effects in these animals. The small changes crease in bone formation. I J 3 ) The molecular and cellular in biochemical parameters could all be attributed to the mechanisms for these estrogen effects are not well under- pharmacologic effect of this agent on bone turnover. The stood. The presence of estrogen receptors in bone normal histologic apperance of the bone from alendrocell^'^^.'^' suggests that estrogen may act directly on nate-treated animals and the normal bone turnover rate, which was similar to that measured in control animals, bone.[3o1 The bone loss caused by ovariectomy was also detected suggest that the bone accrued by alendronate treatment by bone mineral content (BMC) measurements using ab- should function normally. There is extensive epidemiologic sorptiometry. A significant reduction in lumbar BMC (6% and experimental evidence showing a positive relationship below time 0) was observed in vehicle-treated animals 6 between bone mass or bone mineral content and bone It is thus expected that the increase in bone months postovariectomy. During that time the control animineral content produced by alendronate in the vertebrae mals gained about 4% in BMC. and a difference of 5-8% between the two groups was observed throughout the and in the femoral neck will translate into increased bone study. The lack of alendronate effects on BMC at the dis- strength at these sites. In conclusion, ovariectomized baboons, similar to ovarital radius is probably a result of the lower bone turnover at these sites. The ovariectomized animals also showed some ectomized women, experienced increased bone turnover gain in lumbar BMC between 6 and 12 months, albeit not and bone loss. Alendronate, administered IV every 2 weeks statistically significant. At 12 months the drop in BMC rel- at 0.05 and 0.25 mg/kg, prevented the estrogen deficiencyative to time 0 was only 2%. This could reflect, at least in related increase in bone turnover and the related bone loss part, the filling in of the remodeling spaces generated by over 12 months of observation. Thus, alendronate could the increased activation caused by estrogen deficiency. If prove effective in preventing bone loss in postmenopausal the trabecular bone volume in the iliac crest biopsy, which women. was 25% lower than at time 0 at both 6 and 12 months postovariectomy, is representative of vertebral cancellous bone, the increase in lumbar BMC may also reflect periosteal changes. The natural history of bone mineral content changes in baboons has not been recorded thus far. If ACKNOWLEDGMENTS it is similar t o that in humans, the rise in BMC in control animals by 4% may suggest that they had not reached peak We thank Dr. R. Balena for her critical review and Ms. bone mass at the start of the study. Except that all animals D. McDonald for the preparation of this manuscript. 'O

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ALENDRONATE EFFECT IN OVARIECTOMIZED BABOONS

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Address reprint requests to: Cideon A . Rodan. M.D.. Ph.D. Merck. Sharp and Dohme Research Laboratories West Point, PA 19486 Received for publication October 15, 1991; in revised form January 2, 1992; accepted January 3. 1992.