Full text of DOI:10.1359/jbmr.2001.16.2.348

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 16, Number 2, 2001
© 2001 American Society for Bone and Mineral Research
The Effect of a Single Dose of Osteoprotegerin in
Postmenopausal Women*
PIROW J. BEKKER,1 DONNA HOLLOWAY,1 ARLINE NAKANISHI,1 MICHAEL ARRIGHI,1
PHILIP T. LEESE,2 and COLIN R. DUNSTAN1
ABSTRACT
Osteoprotegerin (OPG), a tumor necrosis factor (TNF) receptor family member, is a critical regulator of bone
resorption. It is an important inhibitor of the terminal differentiation and activation of osteoclasts. This
randomized, double-blind, placebo-controlled, sequential dose escalation study was conducted in postmeno-
pausal women to determine the effect of a single subcutaneous (sc) dose of OPG on bone resorption as
indicated by the biochemical markers, urinary N-telopeptide (NTX) and deoxypyridinoline (DPD), which are
stable collagen degradation products. NTX levels decreased within 12 h after OPG administration. At the
highest dose administered (3.0 mg/kg), a mean percent decrease in NTX of approximately 80% was observed
4 days after dosing. Six weeks after dosing a mean decrease of 14% in NTX was observed. The levels of
bone-specific alkaline phosphatase (BSAP), a marker of bone formation, did not change for approximately 3
weeks after dosing. Thereafter, a modest decrease, reaching approximately 30% at 6 weeks, was observed in
the 3.0-mg/kg dose group. The rapid decrease from baseline in NTX and delayed decrease in BSAP indicated
that OPG acted primarily on osteoclasts to decrease bone resorption. OPG injections are well tolerated. This
study, for the first time, indicates that a single sc injection of OPG is effective in rapidly and profoundly
reducing bone turnover for a sustained period and that OPG therefore may be effective in treatment of bone
diseases characterized by increased bone resorption such as osteoporosis. (J Bone Miner Res 2001;16:
348–360)
Key words: osteoprotegerin, osteoporosis, bone resorption, bone turnover, osteoclast
recently discovered ligand of the TNF family, receptor
activator of NF-␬B ligand (RANKL),(3) also known as
TNF-related activation-induced cytokine (TRANCE),(4)
OPG ligand (OPGL),(5) or osteoclast differentiation fac-
tor (ODF).(6) RANKL is expressed on osteoblastic and
stromal cells(5,6) and has been found to be an essential
requirement for osteoclast differentiation(5,6) and activa-
tion in mice.(7) RANKL binds to a membrane bound
receptor in the TNF receptor family on osteoclast pre-
cursors and osteoclasts to induce differentiation and ac-
tivation, respectively. This receptor originally was iso-
lated in dendritic cells and is known as RANK.(3)
Absence of RANKL or RANK results in total absence of
INTRODUCTION
STEOPROTEGERIN (OPG) is a recently discovered se-
creted soluble member of the tumor necrosis factor
O
(TNF) receptor family,(1,2) which in rodents has been
found to be a potent inhibitor of osteoclast differentiation
and function.(1) OPG acts by binding and inactivating a
*Presented in part at a plenary session at the 1999 meeting of the
American Society of Bone and Mineral Research in St. Louis, MO,
USA (J Bone Miner Res 1999;14:1192).
P. Bekker, D. Holloway, A. Nakanishi, M. Arrighi, and C. Dunstan
are employees of Amgen, Inc., which sponsored this study.
1Amgen, Inc., Thousand Oaks, California, USA.
2Quintiles, Lenexa, Kansas, USA.
348

OSTEOPROTEGERIN IN POSTMENOPAUSAL WOMEN
349
osteoclasts, as was shown in the RANKL and RANK tration of the following medications was not allowed: hor-
knockout mice.(8,9)
mone replacement therapy, raloxifene, calcitonin, parathy-
Transgenic mice overexpressing OPG develop osteope- roid hormone (PTH), Ͼ1.5 g daily elemental calcium
trosis,(1) and, conversely, OPG knockout mice develop se- supplementation, Ͼ1000 IU daily vitamin D supplementa-
vere osteoporosis.(10) Mice that are heterozygous for the tion, glucocorticosteroids, anabolic steroids, or calcitriol.
null mutation of the OPG gene have osteoporosis of inter- Administration of the following medications within 12
mediate severity, indicating that adequate OPG expression months before enrollment was not allowed: bisphospho-
is an essential requirement for normal bone mass in mice. nates or fluoride for osteoporosis. Subjects also were
Administration of OPG to ovariectomized rats decreases ineligible if they had any of the following conditions: hy-
osteoclastic activity and restores bone mass to normal lev- perparathyroidism, hyperthyroidism, hypothyroidism, os-
els.(1)
teomalacia, rheumatoid arthritis, acute osteoarthritis/gout,
OPG is detectable in serum of adult humans.(11) RANKL malignancy (except basal cell carcinoma) within the last 6
has been shown to promote the formation of osteoclasts months, renal disease (serum creatinine concentration Ͼ 2
from precursors in human peripheral blood and to increase mg/dl), Paget’s disease of bone, any bone disease that might
bone resorption by these cells in vitro.(12,13) In vitro studies interfere with interpretation of the findings (e.g., osteope-
of human-derived osteoblast cell lines have shown that the trosis or osteogenesis imperfecta), recent fracture (within 6
expression of both OPG and RANKL is regulated by osteo- months), weight greater than 100 kg (to limit the total
tropic hormones and cytokines.(14) Together, these results volume of study drug injections), or malabsorption syn-
indicate that OPG may have an important role in human drome. Furthermore, subjects who had participated in an-
bone metabolism.
other clinical trial within the previous 30 days or a study
Consequently, it was hypothesized that administration of involving physical exercise within the previous 3 months
OPG to humans would result in a decrease in bone resorp- were ineligible. Subjects who substantially changed their
tion. This study was conducted to determine the antiresorp- levels of physical exercise within 6 months before study
tive activity and safety of a single dose of OPG in post- entry or participated in intense physical activity (such as
menopausal women.
marathon training) also were excluded. Alcohol intake of
three or more drinks per day was not allowed for study
participation. Last, subjects with any organic or psychiatric
disorder or abnormal electrocardiogram or laboratory pa-
rameters, which in the opinion of the study investigator or
sponsor could compromise the study outcome, were not
allowed to participate.
MATERIALS AND METHODS
Study design
This was a randomized, double-blind, placebo-controlled,
single-dose, dose escalation study to determine the safety
and tolerability, as well as the bone antiresorptive effect, of
OPG. Eligible subjects were randomized within each dose
level in a 10:3 ratio to receive a single subcutaneous (sc)
dose in the abdominal region of either OPG or placebo,
respectively. Four dose levels of OPG were studied: 0.1,
0.3, 1.0, and 3.0 mg/kg. OPG used in this study consisted of
a genetically engineered Fc-OPG fusion molecule (Fc-
fragment of immunoglobulin G1 [IgG1]) that retained its
binding affinity for RANKL and had similar in vitro activity
in inhibiting osteoclast differentiation. The concentration of
the solution was either 10 mg/ml (for the 0.1-mg/kg and
0.3-mg/kg cohorts) or 30 mg/ml (for the 1.0-mg/kg and
3.0-mg/kg cohorts). Higher concentrations of OPG were not
available for this study. For the higher dose cohorts, doses
were split and administered as two or more injections.
Placebo consisted of only vehicle and was identical in
appearance to the OPG solution.
Study procedures
On the day of dosing, subjects received a single dose of
study drug in the morning and were then followed for 84
days. The following procedures were performed during the
study: medical and medication history (prestudy), physical
examinations, vital signs, hematology, serum chemistry,
coagulation parameters, immunoglobulins, urinalysis, T cell
enumeration (CD3, CD4, and CD8), neutralizing antibodies
to OPG, and electrocardiogram (ECG) (prestudy and peri-
odically during the study). To assess the effect of OPG
administration on bone metabolism, urinary N-telopeptide
(NTX)/creatinine (Osteomark, Seattle, WA, USA), urinary
deoxypyridinoline (DPD)/creatinine (DPD by high-
performance liquid chromatography), serum bone-specific
alkaline phosphatase (BSAP; Tandem-R Ostase; Hybritech,
Inc., San Diego, CA, USA), serum osteocalcin (ELSA OS-
TEO, CIS Biointernational, Gifs/Yvette, France), serum in-
tact PTH (iPTH; Nichols assay), serum albumin-adjusted
calcium, serum phosphorus, urinary calcium/creatinine, and
Study subjects
Fifty-two healthy postmenopausal women (no menses for urinary phosphorus/creatinine were assessed prestudy and
at least 12 months) were enrolled at a single study center in periodically after dosing. The urinary parameters were as-
Lenexa, KS, USA. Study subjects were between the ages of sessed on a second morning void. Duplicate predose sam-
40 and 70 years old, were ambulatory, and provided written ples were assessed for NTX, DPD, BSAP, and osteocalcin
informed consent before enrollment. For any hysterecto- and the average of these two assays was used as the baseline
mized woman Յ60 years of age, serum estradiol had to be value.
Յ20 ng/ml or serum follicle-stimulating hormone (FSH)
Adverse events and concomitant medications were as-
Ն50 IU/liter. Within 6 months before enrollment, adminis- sessed at all study visits after dosing. This study was con-

350
BEKKER ET AL.
TABLE 1. DEMOGRAPHICS AND BASELINE CHARACTERISTICS OF THE STUDY POPULATION, MEAN (SEM)
OPG groups
Placebo
0.1 mg/kg
0.3 mg/kg
1.0 mg/kg
3.0 mg/kg
(n ϭ 12)
(n ϭ 10)
(n ϭ 10)
(n ϭ 10)
(n ϭ 10)
Age (years)
57.0 (2.5)
8.4 (2.6)*
1.64 (0.02)
73.3 (3.4)
51.1 (4.5)
56.8 (1.8)
10.2 (3.3)†
1.63 (0.01)
71.8 (3.6)
36.4 (4.3)
60.6 (2.2)
11.2 (2.9)‡
1.61 (0.02)*
71.5 (5.2)
54.3 (10.3)
57.2 (3.0)
9.5 (3.5)*
1.65 (0.01)
70.0 (2.7)
35.7 (3.3)
57.7 (2.2)
10.6 (2.8)
1.61 (0.02)
70.1 (5.0)
50.4 (6.8)
Years post menopausea
Height (m)
Weight (kg)
Urinary NTX/creatinine (nmol
BCE/mmol)
Serum BSAP (␮g/liter)
Serum iPTH (pg/ml)
Serum albumin-adjusted calcium
(mg/dl)
15.4 (1.5)
36.8 (4.0)
9.1 (0.10)
12.5 (1.3)
34.1 (4.4)
9.0 (0.09)
10.9 (1.1)
40.4 (3.4)
9.2 (0.12)
12.7 (0.4)
42.7 (2.8)
8.8 (0.06)
15.4 (1.0)
43.3 (3.6)
8.8 (0.06)
Urine calcium/creatinine (mg/g)§
142.3 (35.9)
115.2 (16.5)
138.2 (18.8)
85.1 (18.2)
126.0 (25.5)
Reference ranges: N-telopeptide/creatinine, 12–180 nmol BCE/mmol; bone-specific alkaline phosphatase, 6.4–24.4 ␮g/liter; iPTH,
10–65 pg/ml; serum albumin-adjusted calcium, 8.5–10.5 mg/dl; urine calcium/creatinine, 20–280 mg/g.
SI conversion factors: serum albumin-adjusted calcium, 1 mg/dl ϭ 4 mmol/l; serum iPTH, 1 pg/ml ϭ 0.106 pmol/l; urine
calcium/creatinine, 1 mg/g ϭ 0.0028 mmol/mmol.
BCE, bone collagen equivalent.
a Excludes subjects with hysterectomy and an unknown date of menopause.
* n ϭ 9; †n ϭ 7; ‡n ϭ 8; §p Ͻ 0.05, ANOVA.
ducted in accordance with the Helsinki Declaration and was ble at baseline, with the exception of a significant difference
approved by the local institutional review board.
(ANOVA, p Ͻ 0.05) in urine calcium/creatinine. All but 3
subjects were white. There were 2 black subjects (one each
in the placebo and 0.1-mg/kg OPG groups) and one His-
panic subject (0.3-mg/kg OPG group). The mean height and
weight data indicate that the study population was relatively
obese (mean body mass index [BMI] Ͼ 25 kg/m2 across all
dose groups). There were 3, 3, 2, 1, and 0 hysterectomized
subjects in the placebo, 0.1-, 0.3-, 1.0-, and 3.0-mg/kg OPG
groups, respectively. The ages of hysterectomized women
ranged from 52 to 70 years (compared with 41–70 years for
the rest of the study population). The baseline mean urinary
NTX/creatinine values were somewhat variable across treat-
ment groups and were in the lower part of the reference
range. However, these values are comparable with pub-
lished values for postmenopausal women.(15,16) Bone den-
sitometry was not a required study procedure. Therefore, it
is unknown how many of the study subjects had osteopo-
rosis.
Data analysis
Descriptive statistics (n, mean, and SEM) were calculated
for demographics, baseline characteristics, and bone turn-
over markers. As prospectively specified per protocol, data
from the 3 subjects randomized to placebo within each of
the 4 cohorts were combined to provide a single pooled
placebo cohort of 12 subjects for comparison with the OPG
treatment groups. Before pooling, baseline data for the
placebo subjects were assessed for the ability to pool the
placebo data. The treatment groups were examined for
comparability at baseline.
To compare treatment groups, bone turnover marker
changes were computed as changes at a particular time point
from the baseline value and expressed as a percentage
relative to the baseline value. Serum albumin-adjusted cal-
cium (mg/dl) was calculated as serum calcium (mg/dl) ϩ
0.8 ϫ (4 Ϫ albumin [g/dl]). Within each time point, percent
change from baseline in NTX, DPD, and BSAP for each
OPG treatment group was compared against the placebo
group using analysis of variance (ANOVA) with a Dun-
nett’s adjustment for multiple comparisons (␣ ϭ 0.05).
Adjustments for multiple comparisons across the repeated
measures were not made.
Bone metabolism
OPG treatment produced a dose-dependent decrease in
urinary NTX (Table 2 and Fig. 1). Table 2 presents the
mean and SEM of the percent change from baseline at each
time point and for each treatment group. The table also
presents the p values for the comparison between each OPG
group and the placebo group and the corresponding 95%
CIs for the difference in the means, adjusted for multiple
comparisons using Dunnett’s.
RESULTS
Demographics and baseline characteristics
There was a small reduction in NTX at day 1.5 (12 h after
A summary of the subject characteristics at baseline is dosing) in the placebo group (mean Ϯ SEM of Ϫ23.7 Ϯ
presented in Table 1. The treatment groups were compara- 5.9% change from baseline) that probably is related to the

OSTEOPROTEGERIN IN POSTMENOPAUSAL WOMEN
351
TABLE 2. PERCENT CHANGE FROM BASELINE IN URINARY NTX/CREATININE (%)
OPG groups
1.0 mg/kg
Percent change from
baseline in urinary
NTX/creatinine (%)
Placebo
(n ϭ 12)
0.1 mg/kg
(n ϭ 10)
0.3 mg/kg
(n ϭ 10)
3.0 mg/kg
(n ϭ 10)
(n ϭ 10)
Day 1.5
mean (SEM)
95% CI*
p†
Ϫ23.7 (5.9)
Ϫ42.1 (4.5)
(Ϫ37.7, 1.01)
0.068
Ϫ45.4 (6.4)
(Ϫ41.0, Ϫ2.3)
0.024
Ϫ59.8 (4.9)
(Ϫ55.5, Ϫ16.8)
Ͻ0.001
Ϫ66.4 (5.5)
(Ϫ62.0, Ϫ23.3)
Ͻ0.001
—
—
Day 2
mean (SEM)
95% CI*
p†
Ϫ6.3 (5.0)
Ϫ2.8 (7.5)
(Ϫ18.4, 25.3)
0.985
Ϫ44.8 (5.6)
(Ϫ60.4, Ϫ16.6)
Ͻ0.001
Ϫ54.0 (8.8)
(Ϫ69.6, Ϫ25.9)
Ͻ0.001
Ϫ65.2 (3.1)
(Ϫ80.8, Ϫ37.1)
Ͻ0.001
—
—
Day 3
mean (SEM)
95% CI*
p†
3.1 (5.5)
—
Ϫ11.4 (6.4)
(Ϫ35.0, 5.9)
0.234
Ϫ48.2 (6.7)
(Ϫ71.8, Ϫ30.9)
Ͻ0.001
Ϫ66.4 (6.9)
(Ϫ90.1, Ϫ49.1)
Ͻ0.001
Ϫ74.6 (2.7)
(Ϫ98.2, Ϫ57.3)
Ͻ0.001
—
Day 4
mean (SEM)
95% CI*
p†
6.6 (6.1)
—
Ϫ12.1 (6.4)
(Ϫ38.9, 1.4)
0.074
Ϫ37.6 (7.6)
(Ϫ64.4, Ϫ24.1)
Ͻ0.001
Ϫ72.1 (4.3)
(Ϫ98.9, Ϫ58.6)
Ͻ0.001
Ϫ77.7 (2.5)
(Ϫ104.5, Ϫ64.2)
Ͻ0.001
—
Day 5
mean (SEM)
95% CI*
p†
Ϫ1.9 (6.2)
Ϫ14.9 (5.2)
(Ϫ32.3, 6.4)
0.284
Ϫ42.9 (7.1)
(Ϫ60.4, Ϫ21.6)
Ͻ0.001
Ϫ70.2 (4.9)
(Ϫ87.7, Ϫ49.0)
Ͻ0.001
Ϫ79.8 (2.9)
(Ϫ97.2, Ϫ58.5)
Ͻ0.001
—
—
Day 8
mean (SEM)
95% CI*
p†
19.7 (6.8)
Ϫ8.1 (5.7)
(Ϫ47.8, Ϫ7.9)
0.003
Ϫ36.9 (5.9)
(Ϫ76.6, Ϫ36.7)
Ͻ0.001
Ϫ67.1 (5.4)
(Ϫ107.4, Ϫ66.3)
Ͻ0.001
Ϫ75.2 (3.6)
(Ϫ114.9, Ϫ75.0)
Ͻ0.001
—
—
Day 11
mean (SEM)
95% CI*
p†
0.6 (8.0)
—
Ϫ5.1 (4.5)
(Ϫ28.7, 17.3)
0.929
Ϫ18.4 (8.6)
(Ϫ42.0, 4.0)
0.133
Ϫ55.4 (4.0)
(Ϫ79.1, Ϫ33.1)
Ͻ0.001
Ϫ69.5 (5.5)
(Ϫ93.8, Ϫ46.5)
Ͻ0.001
—
Day 15
mean (SEM)
95% CI*
p†
Ϫ5.2 (5.6)
Ϫ3.6 (7.2)
(Ϫ18.6, 21.9)
0.999
Ϫ20.0 (6.0)
(Ϫ35.0, 5.5)
0.214
Ϫ36.2 (3.9)
(Ϫ51.8, Ϫ10.1)
0.002
Ϫ56.6 (5.6)
(Ϫ71.6, Ϫ31.1)
Ͻ0.001
—
—
Day 22
mean (SEM)
95% CI*
p†
1.6 (5.9)
—
11.3 (9.5)
(Ϫ14.1, 33.6)
0.695
Ϫ26.1 (6.2)
(Ϫ51.5, Ϫ3.8)
0.018
Ϫ20.9 (7.0)
(Ϫ46.3, 1.4)
0.071
Ϫ43.9 (4.1)
(Ϫ69.3, Ϫ21.6)
Ͻ0.001
—
Day 29
mean (SEM)
95% CI*
p†
9.4 (10.5)
0.9 (6.3)
(Ϫ44.6, 27.6)
0.937
Ϫ24.0 (6.7)
(Ϫ69.5, 2.7)
0.077
Ϫ10.6 (8.0)
(Ϫ56.1, 16.1)
0.443
Ϫ10.7 (16.1)
(Ϫ56.2, 16.1)
0.441
—
—
Day 43
mean (SEM)
95% CI*
p†
10.6 (9.7)
13.5 (5.4)
(Ϫ26.8, 32.5)
0.998
1.6 (10.3)
(Ϫ38.6, 20.6)
0.862
7.3 (8.6)
(Ϫ33.8, 27.1)
0.996
Ϫ13.6 (7.0)
(Ϫ54.7, 6.2)
0.155
—
—
Day 57
mean (SEM)
95% CI*
p†
13.9 (10.1)
18.9 (7.4)
(Ϫ25.0, 35.1)
0.981
Ϫ8.2 (8.5)
(Ϫ52.1, 8.0)
0.210
10.7 (7.8)
(Ϫ33.3, 26.8)
0.997
9.2 (7.7)
(Ϫ34.7, 25.4)
0.986
—
—
Day 71
mean (SEM)
95% CI*
p†
8.6 (7.5)
—
19.6 (8.8)
(Ϫ18.3, 40.2)
0.760
4.1 (9.6)
(Ϫ33.8, 24.8)
0.986
7.1 (8.7)
(Ϫ32.7, 29.7)
Ͼ0.999
12.8 (7.9)
(Ϫ25.1, 33.5)
0.990
—
Day 85
mean (SEM)
95% CI*
p†
10.7 (9.4)
19.9 (5.8)
(Ϫ25.2, 43.6)
0.907
5.3 (11.6)
(Ϫ39.8, 29.0)
0.985
11.8 (8.3)
(Ϫ34.3, 36.5)
Ͼ0.999
12.8 (12.2)
(Ϫ32.4, 36.5)
Ͼ0.999
—
—
* Ninety-five percent Dunnett’s adjusted CI for the difference of means relative to placebo; †compared against placebo using Dunnett’s
adjustment for multiple comparison.

352
BEKKER ET AL.
FIG. 1. The effect of OPG treat-
ment on a second morning void
urinary NTX/creatinine, expressed
as percent change from baseline
over time. Data are presented as
mean and SEM. Dosing occurred
on day 1.
FIG. 2. Individual actual uri-
nary NTX/creatinine data over
the course of the study for the 6
placebo subjects (dotted lines) in
the two highest-dose cohorts (1.0
mg/kg and 3.0 mg/kg) as well as
all subjects who received 1.0
mg/kg (dashed lines) or 3.0
mg/kg OPG sc (solid lines). The
timescale is logarithmic to visual-
ize the early changes more clearly.
Dosing occurred on day 1.
previously reported diurnal variation in this marker.(17) subjects), day 2 (4 subjects), day 3 (1 subject), day 4 (2
However, in all the OPG groups except the 0.1-mg/kg subjects), day 5 (3 subjects), day 8 (3 subjects), and day 11
group, a statistically significantly (p Ͻ 0.05) greater reduc- (1 subject) after dosing. This finding indicates that the
tion in NTX compared with placebo was observed at 12 h, suppression of bone resorption is profound.
indicating that OPG decreased bone resorption as early as
12 h after administration.
Statistically significant (p Ͻ 0.025) decreases in the per-
cent change from baseline in NTX were observed at the
Six subjects who received 3.0 mg/kg OPG had NTX 0.3-mg/kg dose on days 1.5, 2, 3, 4, 5, 8, and 22 relative to
levels below the assay’s limit of quantification at day 1.5 (4 placebo. Statistically significant (p Յ 0.002) decreases also

OSTEOPROTEGERIN IN POSTMENOPAUSAL WOMEN
353
TABLE 3. PERCENT CHANGE FROM BASELINE IN DPD/CREATININE (%)
OPG groups
Percent change from
baseline in DPD/
creatinine (%)
Placebo
(n ϭ 12)
0.1 mg/kg
(n ϭ 10)
0.3 mg/kg
1.0 mg/kg
3.0 mg/kg
(n ϭ 10)
(n ϭ 10)
(n ϭ 10)
Day 1.5
Mean (SEM)
95% CI*
p†
Ϫ34.0 (3.6)
Ϫ39.8 (4.2)
(Ϫ18.9, 7.1)
0.624
Ϫ38.8 (3.6)
(Ϫ17.8, 8.2)
0.763
Ϫ52.9 (3.6)
(Ϫ32.0, Ϫ6.0)
0.002
Ϫ47.5 (3.5)
(Ϫ26.5, Ϫ0.5)
0.039
—
—
Day 2
Mean (SEM)
95% CI*
p†
0.0 (4.5)
—
5.0 (9.9)
(Ϫ17.5, 27.5)
0.95
Ϫ22.5 (7.2)
(Ϫ44.9, 0.1)
0.051
Ϫ26.6 (4.5)
(Ϫ49.1, Ϫ4.1)
0.016
Ϫ22.3 (4.9)
(Ϫ44.7, 0.3)
0.054
—
Day 3
Mean (SEM)
95% CI*
p†
14.0 (8.1)
3.6 (6.8)
(Ϫ33.7, 13)
0.638
Ϫ15.8 (8.3)
(Ϫ53.2, Ϫ6.5)
0.008
Ϫ31.2 (4.4)
(Ϫ68.6, Ϫ21.8)
Ͻ0.001
Ϫ29.2 (3.3)
(Ϫ66.6, Ϫ19.9)
Ͻ0.001
—
—
Day 4
Mean (SEM)
95% CI*
p†
15.5 (3.6)
1.0 (6.2)
(Ϫ31.7, 2.7)
0.121
Ϫ14.4 (6.3)
(Ϫ47.1, Ϫ12.7)
Ͻ0.001
Ϫ36.7 (4.5)
(Ϫ69.5, Ϫ35.1)
Ͻ0.001
Ϫ39.8 (3.5)
(Ϫ72.5, Ϫ38.2)
Ͻ0.001
—
—
Day 5
Mean (SEM)
95% CI*
p†
7.7 (3.6)
—
Ϫ9.8 (4.9)
(Ϫ34.6, Ϫ0.4)
0.044
Ϫ16.1 (7.0)
(Ϫ40.9, Ϫ6.7)
0.004
Ϫ40.8 (3.9)
(Ϫ65.6, Ϫ31.3)
Ͻ0.001
Ϫ42.2 (4.7)
(Ϫ67.0, Ϫ32.8)
Ͻ0.001
—
Day 8
Mean (SEM)
95% CI*
p†
9.2 (4.2)
—
Ϫ2.6 (4.7)
(Ϫ28.9, 5.3)
0.257
Ϫ21.7 (6.3)
(Ϫ48.0, Ϫ13.8)
Ͻ0.001
Ϫ42.6 (5.4)
(Ϫ69.4, Ϫ34.2)
Ͻ0.001
Ϫ40.6 (3.8)
(Ϫ67.0, Ϫ32.7)
Ͻ0.001
—
Day 11
Mean (SEM)
95% CI*
p†
9.2 (4.5)
—
Ϫ1.2 (6.4)
(Ϫ28.8, 7.9)
0.427
Ϫ10.7 (6.6)
(Ϫ38.3, Ϫ1.6)
0.029
Ϫ31.3 (2.6)
(Ϫ58.9, Ϫ22.2)
Ͻ0.001
Ϫ40.7 (5.5)
(Ϫ68.9, Ϫ31.0)
Ͻ0.001
—
Day 15
Mean (SEM)
95% CI*
p†
Ϫ0.7 (5.8)
Ϫ7.4 (4.6)
(Ϫ23.2, 9.8)
0.703
Ϫ18.0 (3.3)
(Ϫ33.8, Ϫ0.8)
0.037
Ϫ21.4 (3.1)
(Ϫ37.7, Ϫ3.8)
0.012
Ϫ33.2 (5.2)
(Ϫ49.0, Ϫ16.0)
Ͻ0.001
—
—
Day 22
Mean (SEM)
95% CI*
p†
3.4 (4.1)
—
3.2 (6.1)
(Ϫ16.7, 16.2)
Ͼ0.999
Ϫ24.2 (4.7)
(Ϫ44.1, Ϫ11.1)
Ͻ0.001
Ϫ20.5 (4.6)
(Ϫ40.4, Ϫ7.5)
0.002
Ϫ30.0 (3.4)
(Ϫ49.9, Ϫ17)
Ͻ0.001
—
Day 29
Mean (SEM)
95% CI*
p†
7.9 (5.3)
—
Ϫ5.0 (5.1)
(Ϫ31.1, 5.3)
0.234
Ϫ7.4 (5.5)
(Ϫ33.5, 2.8)
0.121
Ϫ16.0 (5.3)
(Ϫ42.1, Ϫ5.8)
0.006
Ϫ20.6 (4.3)
(Ϫ46.7, Ϫ10.4)
0.001
—
Day 43
Mean (SEM)
95% CI*
p†
Ϫ0.1 (5.8)
Ϫ7.2 (4.4)
(Ϫ28.3, 14.2)
0.822
Ϫ24.1 (4.9)
(Ϫ45.2, Ϫ2.8)
0.022
Ϫ6.6 (9.7)
(Ϫ28.3, 15.4)
0.872
Ϫ1.3 (4.7)
(Ϫ23.0, 20.7)
Ͼ0.999
—
—
Day 57
Mean (SEM)
95% CI*
p†
1 (6.3)
—
—
4.5 (7.6)
(Ϫ23.7, 30.8)
0.993
Ϫ19.6 (9.6)
(Ϫ47.9, 6.7)
0.190
Ϫ7.8 (5.3)
(Ϫ36.0, 18.5)
0.840
14.3 (9.6)
(Ϫ13.9, 40.6)
0.558
Day 71
Mean (SEM)
95% CI*
p†
Ϫ7.8 (5.5)
Ϫ17.3 (6.3)
(Ϫ35.9, 16.9)
0.784
Ϫ22.8 (8.1)
(Ϫ41.4, 11.3)
0.423
Ϫ1.7 (7.0)
(Ϫ22.0, 34.2)
0.954
17.3 (10.6)
(Ϫ1.2, 51.5)
0.066
—
—
Day 85
Mean (SEM)
95% CI*
p†
Ϫ5.9 (4.8)
Ϫ11.7 (6.6)
(Ϫ28.3, 16.6)
0.913
Ϫ19.8 (7.7)
(Ϫ36.4, 8.5)
0.343
Ϫ1.5 (5.6)
(Ϫ18.7, 27.4)
0.971
17.4 (7.0)
(0.8, 45.7)
0.041
—
—
* Ninety-five percent Dunnett’s adjusted CI for the difference of means relative to placebo; †compared against placebo using Dunnett’s
adjustment for multiple comparison.

354
BEKKER ET AL.
FIG. 3. The effect of OPG
treatment on a second morning
void urinary DPD/creatinine, ex-
pressed as percent change from
baseline over time. Data are pre-
sented as mean and SEM. Dosing
occurred on day 1.
were observed at the 1.0-mg/kg dose level on days 1.5, 2, 3,
In the 1.0-mg/kg and 3.0-mg/kg dose groups, BSAP de-
4, 5, 8, 11, and 15 relative to placebo. The mean percent creased statistically significantly compared with placebo by
changes from baseline in NTX are statistically significantly day 43 (Table 4 and Fig. 4). In addition, the mean percent
(p Ͻ 0.001) different at the 3.0-mg/kg dose compared with change in the 1.0-mg/kg OPG group was statistically sig-
placebo on days 1.5, 2, 3, 4, 5, 8, 11, 15, and 22. On day 43, nificantly different compared with placebo on day 71 and in
the mean Ϯ SEM percent decrease from baseline in NTX is the 3.0-mg/kg OPG group on days 57–85. There was a
still 13.6 Ϯ 7.0% in the 3.0-mg/kg cohort (not statistically statistically significant mean increase in BSAP compared
significantly different compared with placebo).
with placebo from day 22 onward in the 0.3-mg/kg OPG
group. A similar increase is not observed in serum osteo-
calcin levels. There are no consistent changes in serum
osteocalcin in the OPG groups compared with placebo (data
not shown).
The albumin-adjusted serum calcium levels in the OPG-
treated subjects show early but modest decreases from base-
line (Fig. 5). Similar changes are observed in the serum
phosphorus levels (data not shown). There also are increases
in iPTH levels in subjects who received 1.0 mg/kg and 3.0
mg/kg of OPG (Fig. 6), which return to baseline levels by
approximately day 29. Transient decreases are observed in
urinary calcium/creatinine levels in subjects receiving 0.3–
3.0 mg/kg of OPG (Fig. 7). Urinary phosphorus/creatinine
levels do not change relative to the placebo control group
(data not shown).
The maximum NTX reduction was observed at day 5 in
each of the dose levels (mean Ϯ SEM of Ϫ14.9 Ϯ 5.2% in
the 0.1-mg/kg dose level, Ϫ42.9 Ϯ 7.1% in the 0.3-mg/kg
dose level, Ϫ70.2 Ϯ 4.9% in the 1.0-mg/kg dose level, and
Ϫ79.8 Ϯ 2.9% in the 3.0-mg/kg dose level.
In Fig. 2 the individual actual NTX levels over time for
the placebo subjects in the 1.0-mg/kg and 3.0-mg/kg dose
cohorts, as well as for all subjects who received 1.0 mg/kg
or 3.0 mg/kg OPG, are shown. The timescale is logarithmic
to emphasize the early changes in the marker levels. The
data indicate, as expected, that there was great variability
among baseline values. Placebo subjects had a slight diurnal
decrease at the day 1.5 point but no consistent changes over
time. However, the 1.0-mg/kg and 3.0-mg/kg subjects
showed consistent decreases reaching a nadir at approxi-
mately day 5.
Changes in urinary DPD, although not as marked as
NTX, confirm the dose-dependent rapid suppression of
bone turnover (Table 3 and Fig. 3). The mean percent
Safety and tolerability
No serious adverse events or dropouts due to adverse
changes from baseline in the 0.3-, 1.0-, and 3.0-mg/kg OPG events were reported. The most common adverse event,
groups are statistically significantly different compared with which was considered possibly or probably related to study
placebo on days 3–22. In addition, the mean percent change drug by the investigator, was mild injection site reaction.
in the 0.3-mg/kg OPG group is statistically significantly One case was reported in each dose group. No clinically
different compared with placebo on day 43; in the 1.0- significant changes in any other laboratory variables were
mg/kg OPG group on day 1.5 and day 29; and in the noted. All subjects had postexposure samples assayed for
3.0-mg/kg OPG group on days 1.5, 29, and 85.
neutralizing antibodies to OPG, and all tests were negative.

OSTEOPROTEGERIN IN POSTMENOPAUSAL WOMEN
355
TABLE 4. PERCENT CHANGE FROM BASELINE IN BSAP (%)
OPG groups
1.0 mg/kg
Percent change from
baseline in BSAP
(%)
Placebo
(n ϭ 12)
0.1 mg/kg
(n ϭ 10)
0.3 mg/kg
(n ϭ 10)
3.0 mg/kg
(n ϭ 10)
(n ϭ 10)
Day 2
Mean (SEM)
95% CI*
p†
Ϫ0.8 (2.6)
Ϫ4.5 (3.1)
(Ϫ13.8, 6.3)
0.757
0.6 (3.5)
(Ϫ8.7, 11.5)
0.991
Ϫ1.2 (2.7)
(Ϫ10.5, 9.6)
Ͼ0.999
Ϫ7.4 (2.4)
(Ϫ16.7, 3.4)
0.292
—
—
Day 3
Mean (SEM)
95% CI*
p†
3.8 (3.5)
—
Ϫ0.8 (2.3)
(Ϫ16.1, 6.8)
0.707
4.1 (3.8)
(Ϫ11.2, 11.7)
Ͼ0.999
4.7 (4.0)
(Ϫ10.6, 12.3)
0.999
Ϫ3.5 (2.1)
(Ϫ18.7, 4.2)
0.326
—
Day 4
Mean (SEM)
95% CI*
p†
Ϫ4.6 (2.3)
Ϫ6.1 (2.0)
(Ϫ12.5, 9.6)
0.992
4.6 (4.8)
(Ϫ1.9, 20.3)
0.129
Ϫ2.5 (3.0)
(Ϫ8.9, 13.2)
0.969
Ϫ5.5 (3.1)
(Ϫ12, 10.1)
0.999
—
—
Day 5
Mean (SEM)
95% CI*
p†
0.0 (2.0)
—
Ϫ1.2 (2.9)
(Ϫ11.5, 9.1)
0.995
8.9 (3.9)
(Ϫ1.4, 19.2)
0.109
Ϫ0.6 (3.4)
(Ϫ10.9, 9.7)
Ͼ0.999
Ϫ1.6 (2.2)
(Ϫ11.9, 8.7)
0.986
—
Day 8
Mean (SEM)
95% CI*
p†
0.9 (3.5)
—
Ϫ4.1 (3.7)
(Ϫ19.2, 9.2)
0.797
6.0 (5.1)
(Ϫ9.1, 19.4)
0.782
Ϫ0.1 (4.4)
(Ϫ15.6, 13.7)
Ͼ0.999
1.3 (3.9)
(Ϫ13.8, 14.7)
Ͼ0.999
—
Day 11
Mean (SEM)
95% CI*
p†
0.9 (3.5)
—
Ϫ7.6 (2.9)
(Ϫ19.7, 2.7)
0.188
1.7 (4.0)
(Ϫ10.4, 12)
0.999
0.3 (2.8)
(Ϫ11.7, 10.7)
Ͼ0.999
Ϫ1.1 (2.4)
(Ϫ13.2, 9.2)
0.976
—
Day 15
Mean (SEM)
95% CI*
p†
Ϫ4.0 (5.6)
Ϫ5.6 (3.1)
(Ϫ17.2, 14.1)
0.997
5.6 (4.3)
(Ϫ6.1, 25.2)
0.362
5.2 (5.0)
(Ϫ6.5, 24.8)
0.401
2.0 (3.2)
(Ϫ9.7, 21.7)
0.746
—
—
Day 22
Mean (SEM)
95% CI*
p†
Ϫ8.2 (3.0)
Ϫ11.7 (2.3)
(Ϫ18.5, 11.5)
0.942
18.4 (6.6)
(11.6, 41.6)
Ͻ0.001
Ϫ13.1 (3.8)
(Ϫ20.0, 10.0)
0.827
Ϫ15.3 (4.8)
(Ϫ22.1, 7.9)
0.586
—
—
Day 29
Mean (SEM)
95% CI*
p†
Ϫ5.6 (3.5)
Ϫ6.3 (3.9)
(Ϫ15.4, 14.0)
Ͼ0.999
19.9 (5.8)
(10.8, 40.2)
Ͻ0.001
Ϫ13.2 (4.0)
(Ϫ22.4, 7.0)
0.497
Ϫ19.9 (3.2)
(Ϫ29.0, 0.4)
0.058
—
—
Day 43
Mean (SEM)
95% CI*
p†
Ϫ10.0 (2.7)
Ϫ5.2 (3.2)
(Ϫ8.9, 18.6)
0.789
7.2 (6.5)
(3.5, 31.0)
0.010
Ϫ25.7 (2.8)
(Ϫ29.8, Ϫ1.5)
0.026
Ϫ29.7 (2.9)
(Ϫ33.8, Ϫ5.6)
0.004
—
—
Day 57
Mean (SEM)
95% CI*
p†
Ϫ6.3 (3.7)
Ϫ3.7 (3.3)
(Ϫ13.2, 18.3)
0.983
22.4 (7.0)
(12.9, 44.4)
Ͻ0.001
Ϫ18.1 (4.8)
(Ϫ27.6, 3.9)
0.193
Ϫ32.2 (2.6)
(Ϫ41.6, Ϫ10.2)
0.001
—
—
Day 71
Mean (SEM)
95% CI*
p†
Ϫ2.7 (4.1)
1.4 (3.9)
(Ϫ10.5, 18.9)
0.891
13.6 (5.9)
(1.6, 31.1)
0.025
Ϫ19.6 (4.4)
(Ϫ32.5, Ϫ1.2)
0.032
Ϫ23.5 (2.1)
(Ϫ35.5, Ϫ6.1)
0.003
—
—
Day 85
Mean (SEM)
95% CI*
p†
Ϫ4.2 (4.2)
3.8 (4.9)
(Ϫ8.6, 24.7)
0.563
20.0 (6.9)
(7.6, 40.8)
0.002
Ϫ14.4 (2.6)
(Ϫ27.3, 6.9)
0.377
Ϫ21.4 (3.7)
(Ϫ33.8, Ϫ0.5)
0.041
—
—
* Ninety-five percent Dunnett’s adjusted CI for the difference of means relative to placebo; †compared against placebo using Dunnett’s
adjustment for multiple comparison.

356
BEKKER ET AL.
FIG. 4. The effect of OPG
treatment on BSAP, expressed as
percent change from baseline
over time. Data are expressed as
mean and SEM. Dosing occurred
on day 1.
FIG. 5. The effect of OPG
treatment on serum albumin-
adjusted calcium (mg/dl) over
time. Data are expressed as mean
and SEM. Dosing occurred on
day 1.
inactivates mature osteoclasts.(7,18) The sustained effect on
bone resorption suggests that OPG also blocks differentia-
tion of osteoclasts. The delayed decrease in bone formation
marker levels suggests that OPG does not affect bone for-
mation directly but most likely causes a decrease in activa-
tion frequency (or birth rate) of basic multicellular units
(BMUs), the cellular units responsible for bone turnover.
DISCUSSION
This study shows, for the first time, that a single dose of
OPG rapidly decreases bone resorption, as indicated by
bone resorption marker changes, in postmenopausal
women. This result provides convincing evidence that the
OPG/RANKL/RANK regulatory pathway has a major role
in human osteoclast differentiation and activation. The rapid The reason is that bone resorption normally precedes the
onset of action and profound bone antiresorptive effect of coupled bone formation in the usual bone remodeling cycle.
OPG support previous findings in mice that OPG directly An explanation for the increase in BSAP in the 0.3-mg/kg

OSTEOPROTEGERIN IN POSTMENOPAUSAL WOMEN
357
FIG. 6. The effect of OPG
treatment on iPTH (pg/ml) over
time. Data are expressed as mean
and SEM. Dosing occurred on
day 1.
FIG. 7. The effect of OPG
treatment on a second morning
void urinary calcium/creatinine
(mg/g) over time. Data are ex-
pressed as mean and SEM. Dos-
ing occurred on day 1.
OPG group from day 22 onward compared with the placebo markers) but also prevents terminal differentiation of new
group is not obvious. It is unlikely that this is a true osteoclasts (sustained suppression of bone turnover).
biological effect, because, as mentioned, this increase was
not observed with serum osteocalcin.
The other biochemical effects of OPG are consistent
with specific, rapid, and profound suppression of bone
The most likely mechanism of action of OPG is consis- resorption by OPG, prompting appropriate physiological
tent with the findings in preclinical experiments: OPG binds responses to maintain calcium homeostasis. The initial
to RANKL, expressed on osteoblastic or stromal cells, and modest decrease in serum calcium is most likely a result
prevents binding of RANKL to RANK, expressed on preos- of decreased calcium release via bone resorption. The
teoclasts and osteoclasts. This leads to inactivation and increase in PTH secretion is the appropriate physiologi-
apoptosis of osteoclasts (rapid reduction in bone resorption cal response to the reduction in serum calcium concen-

358
BEKKER ET AL.
tration. This increase in PTH persisted for approximately Oral bisphosphonate treatment may lead to gastrointestinal
28 days. Chronically elevated PTH levels may be dele- intolerance(25,26) and must be taken fasting because of the
terious to the skeleton; oral calcium supplementation in interference of food or drink with intestinal absorption.
longer-term clinical trials may be appropriate to control Calcitonin and SERM treatments modestly decrease bone
the decrease in serum calcium and the elevation in PTH resorption(27,28) in osteoporosis. Oral clodronate and intra-
levels. The decrease in urinary calcium probably is the venous pamidronate, two bisphosphonates, have proven
result of decreased filtered calcium load and increased beneficial in inhibiting bone resorption associated with bone
tubular calcium reabsorption caused by PTH. Similarly, metastases.(29–39) However, their efficacy is modest (ap-
the decrease in serum phosphorus concentration also proximately 20–40% reduction in the incidence of skeletal
probably is caused by reduced release from bone and to events). The oral bioavailability of bisphosphonates is low,
the phosphaturic effects of PTH.
which probably contributes to the limited efficacy in treat-
OPG injections were well tolerated. A few mild injection ing patients with bone metastases. Intravenous pamidronate
site reactions were considered possibly or probably related needs to be administered by infusion over 1–4 h. The need
to the study drug by the physician, who was blinded to study for alternative safe, effective, and convenient therapies for
drug assignment (P. Leese). RANKL-deficient mice, in ad- patients with osteoporosis and other bone diseases persists.
dition to an osteopetrotic phenotype, showed evidence of
Our study has limitations. It was conducted at a single
partial inhibition of early T and B lymphocyte develop- center in a relatively small number of postmenopausal sub-
ment.(8) Although lymphocyte function tests were not con- jects. Broad confirmatory studies in osteoporotic patients
ducted in our study, no evidence of an effect on peripheral are warranted.
T lymphocyte counts (CD3, CD4, and CD8) was found.
In conclusion, this single-dose study in postmenopausal
In an earlier study, OPG was found to be expressed by women indicates that the construct of OPG used in this
many different cell types in Northern blot analysis of human study is a critical regulator of osteoclast development and
tissues.(1) The highest levels were found in lung, heart, activity in humans. OPG causes rapid and sustained inhibi-
kidney, and placenta. In mouse embryos, high expression of tion of bone resorption dose dependently as reflected by the
OPG was found in cartilage rudiments of developing bones, bone resorption marker profile. A single sc OPG dose was
major arteries, gastrointestinal tract, and skin.(1) Transgenic well tolerated. OPG could have wide application as a potent
mice expressing high levels of OPG have severe osteope- antiresorptive therapeutic with a mechanism of action dis-
trosis and secondary splenomegaly(1) but not abnormalities tinct from those of bisphosphonates, calcitonin, or estrogen
of other organs, confirming that the primary role of OPG is and its analogs.
to regulate bone resorption. OPG-deficient mice have os-
teoporosis and medial calcification of major arteries, sug-
gesting that OPG may be protective against arterial calcifi-
cation.(10) The current study confirms that OPG has an
important effect on the human skeleton and although it is
expressed in other tissues, its function, if any, in these
tissues is unclear.
ACKNOWLEDGMENTS
This study was supported with funding from Amgen, Inc.
REFERENCES
Many human bone diseases result from excessive bone
resorption relative to bone formation. This defect can in-
volve the whole skeleton as in osteoporosis or can occur
focally as in lytic bone metastases. This imbalance can
result from changes in hormone levels, as in hyperparathy-
roidism and postmenopausal osteoporosis, or by the action
of locally produced factors, such as cytokines in inflamma-
tory disease or PTH-related protein (PTHrP) in cancer. In
mice, OPG administration is effective in opposing the re-
sorptive effects of PTH, 1,25-hydroxyvitamin D3
[1,25(OH)2D3], TNF-␣, interleukin-1␤ (IL-1␤), and PTHrP,
which together encompass most of the significant osteotro-
pic agents known to be involved in bone diseases.(19,20) If it
is similarly effective against these agents in human disease,
OPG should have wide application as an antiresorptive
agent.
Bone antiresorptive agents such as estrogen, bisphospho-
nates, calcitonin, and selective estrogen receptor modulators
(SERMs) are used clinically to decrease bone turnover.
Each of these treatments has limitations. Estrogen treatment
is effective in osteoporosis but often is not well tolerated,(21)
increases the risk of endometrial cancer if used without
progesterone,(22) and may increase the risk of breast cancer
(although controversy still exists regarding this risk).(23,24)
1. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS,
Luethy R, Nguyen HQ, Wooden S, Bennett L, Boone T,
Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL,
Trail G, Sullivan J, Davy E, Bucay N, Renshaw GL, Hughes
TM, Hill D, Pattison W, Campbell P, Boyle WJ 1997 Osteo-
protegerin: A novel secreted protein involved in the regulation
of bone density. Cell 89:309–319.
2. Tsuda E, Goto M, Mochizuki SI, Yano K, Kobayashi F,
Morinaga T, Higashio K 1997 Isolation of a novel cytokine
from human fibroblasts that specifically inhibits osteoclasto-
genesis. Biochem Biophys Res Commun 234:137–142.
3. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC,
Tometsko ME, Roux ER, Teepe MC, DuBose RF, Cosman D,
Galibert L 1997 A homologue of the TNF receptor and its
ligand enhance T-cell growth and dendritic-cell function. Na-
ture 390:175–179.
4. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M,
Kalachikov S, Cayani E, Bartlett FS, Frankel WN, Lee SY,
Choi Y 1997 TRANCE is a novel ligand of the tumor necrosis
factor receptor family that activates c-Jun N-terminal kinase in
T cells. J Biol Chem 272:25190–25194.
5. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Bur-
gess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H,
Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX,
Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney
J, Boyle WJ 1998 Osteoprotegerin ligand is a cytokine that

OSTEOPROTEGERIN IN POSTMENOPAUSAL WOMEN
359
regulates osteoclast differentiation and activation. Cell 93: 19. Hofbauer LC, Dunstan CR, Spelsberg TC, Riggs BL, Khosla S
165–176.
1998 Osteoprotegerin production by human osteoblast lineage
cells is stimulated by vitamin D, bone morphogenetic
protein-2, and cytokines. Biochem Biophys Res Commun 250:
776–781.
6. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M,
Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A,
Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N,
Suda T 1998 Osteoclast differentiation factor is a ligand for
osteoprotegerin/osteoclastogenesis-inhibitory factor and is
identical to TRANCE/RANKL. Proc Natl Acad Sci 95:3597–
3602.
7. Burgess TL, Qian YX, Kaufman S, Ring BD, Van G, Cappar-
elli C, Kelley M, Hsu HL, Boyle WJ, Dunstan CR, Hu S,
Lacey DL 1999 The ligand for osteoprotegerin (OPGL) di-
rectly activates mature osteoclasts. J Cell Biol 145:527–538.
8. Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli
C, Morony S, Oliveira-dos SA, Van G, Itie A, Khoo W,
Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ,
Penninger JM 1999 OPGL is a key regulator of osteoclasto-
genesis, lymphocyte development and lymph-node organogen-
esis. Nature 397:315–323.
9. Li J, Sarosi I, Yan XQ, Morony S, Capparelli C, Tan HL,
McCabe S, Elliot R, Scully S, Van G, Kaufman S, Juan SC,
Sun Y, Tarpley J, Martin L, Christensen F, McCabe J, Ko-
stenuik P, Hsu H, Fletcher F, Dunstan CR, Lacey DL, Boyle
WJ 2000 RANK is the intrinsic hematopoietic cell surface
receptor that controls osteoclastogenesis and regulation of
bone mass and calcium metabolism. Proc Natl Acad Sci USA
97:1566–1571.
10. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Cap-
parelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ,
Simonet WS 1998 Osteoprotegerin-deficient mice develop
early onset osteoporosis and arterial calcification. Genes Dev
12:1260–1268.
11. Yano K, Tsuda E, Washida N, Kobayashi F, Goto M, Harada A,
Ikeda K, Higashio K, Yamada Y 1999 Immunological character-
ization of circulating osteoprotegerin/osteoclastogenesis inhibi-
tory factor: Increased serum concentrations in postmenopausal
women with osteoporosis. J Bone Miner Res 14:518–527.
12. Matsuzaki K, Udagawa N, Takahashi N, Yamaguchi K, Ya-
suda H, Shima N, Morinaga T, Toyama Y, Yabe Y, Higashio
K, Suda T 1998 Osteoclast differentiation factor (ODF) in-
duces osteoclast-like cell formation in human peripheral blood
mononuclear cell cultures. Biochem Biophys Res Commun
246:199–204.
13. Shalhoub V, Faust J, Boyle WJ, Dunstan CR, Kelley M,
Kaufman S, Scully S, Van G, Lacey DL 1998 Osteoprotegerin
and osteoprotegerin ligand effects on osteoclast formation
from human peripheral blood mononuclear cell precursors.
J Cell Biochem 72:251–261.
14. Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Spelsberg
TC, Riggs BL 1999 Estrogen stimulates gene expression and
protein production of osteoprotegerin in human osteoblastic
cells. Endocrinology 140:4367–4370.
15. Garnero P, Sornay-Rendu E, Chapuy M-C, Delmas P 1996
Increased bone turnover in late postmenopausal women is a
major determinant of osteoporosis. J Bone Miner Res 11:337–
349.
20. Morony S, Capparelli C, Lee R, Shimamoto G, Boone T,
Lacey DL, Dunstan CR 1999 A chimeric form of osteoprote-
gerin inhibits hypercalcemia and bone resorption induced by
IL-1 beta, TNF-alpha, PTH, PTHrP, and 1,25(OH)(2)D-3.
J Bone Miner Res 14:1478–1485.
21. Speroff L, Rowan J, Symons J, Genant H, Wilborn W 1996
The comparative effect on bone density, endometrium, and
lipids of continuous hormones as replacement therapy
(CHART study): A randomized controlled trial. JAMA 276:
1397–1403.
22. Grady D, Gebretsadik T, Kerlikowske K, Ernster V, Petitti D
1995 Hormone replacement therapy and endometrial cancer
risk: A meta-analysis. Obstetrics Gynecol 85:304–313.
23. Lindsay R, Bush TL, Grady D, Speroff L, Lobo RA 1996
Therapeutic controversy: Estrogen replacement in menopause.
J Clin Endocrinol Metab 81:3829–3838.
24. Colditz GA, Hankinson SE, Hunter DJ, Willett WC, Manson
JE, Stampfer MJ, Hennekens C, Rosner B, Speizer FE 1995
The use of estrogens and progestins and the risk of breast
cancer in postmenopausal women. N Engl J Med 332:1589–
1593.
25. de Groen PL, Lubbe DF, Hirsch LJ, Daifotis A, Stephenson W,
Freedholm D, Pryor TS, Seleznick MJ, Pinkas H, Wang KK
1996 Esophagitis associated with the use of alendronate.
N Engl J Med 335:1016–1021.
26. Ettinger B, Pressman A, Schein J 1998 Clinic visits and
hospital admissions for care of acid-related upper gastrointes-
tinal disorders in women using alendronate for osteoporosis.
Am J Man Care 4:1377–1382.
27. Overgaard K, Hansen MA, Jensen SB, Christiansen C 1992
Effect of salcatonin given intranasally on bone mass and
fracture rates in established osteoporosis: A dose-response
study. BMJ 305:556–561.
28. Delmas PD, Bjarnason NH, Mitlak BH, Ravoux AC, Shah AS,
Huster WJ, Draper M, Christiansen C 1997 Effects of ralox-
ifene on bone mineral density, serum cholesterol concentra-
tions, and uterine endometrium in postmenopausal women.
N Engl J Med 337:1641–1647.
29. Siris ES, Sherman WH, Baquiran DC, Schlatterer JP, Osser-
man EF, Canfield RE 1980 Effects of dichloromethylene
diphosphonate on skeletal mobilization of calcium in multiple
myeloma. N Engl J Med 302:310–315.
30. Lahtinen R, Laakso M, Palva I, Virkkunen P, Elomaa I 1992
Randomised, placebo-controlled multicentre trial of clodronate
in multiple myeloma. Lancet 340:1049–1052.
31. McCloskey EV, MacLennan IC, Drayson MT, Chapman C,
Dunn J, Kanis JA 1998 A randomized trial of the effect of
clodronate on skeletal morbidity in multiple myeloma. MRC
Working Party on Leukaemia in Adults. Br J Haematol 100:
317–325.
32. Paterson AHG, Powles TJ, Kanis JA, McCloskey E, Hanson J,
Ashley S 1993 Double-blind controlled trial of oral clodronate
in patients with bone metastases from breast cancer. J Clin
Oncol 11:59–65.
33. Kanis JA, Powles T, Paterson AHG, McCloskey EV, Ashley S
1996 Clodronate decreases the frequency of skeletal metasta-
ses in women with breast cancer. Bone 19:663–667.
34. Diel IJ, Solomayer EF, Costa SD, Gollan C, Goerner R,
Wallwiener D, Kaufmann M, Bastert G 1998 Reduction in
new metastases in breast cancer with adjuvant clodronate
treatment. N Engl J Med 339:357–363.
35. Berenson JR, Lichtenstein A, Porter L, Dimopoulos MA, Bor-
doni R, George S, Lipton A, Keller A, Ballester O, Kovacs MJ,
Blacklock HA, Bell R, Simeone J, Reitsma DJ, Heffernan M,
16. Lewis LL, Shaver JF, Woods NF, Lentz MJ, Cain KC, Hertig
V, Heidergott S 2000 Bone resorption levels by age and
menopausal status in 5,157 women. Menopause 7:42–52.
17. Ju HSJ, Leong S, Brown B, Stringer MA, Leigh S, Scherrer C,
Shepard K, Jenkins D, Knudsen J, Cannon R 1997 Comparison
of analytical performance and biological variability of three
bone resorption assays. Clin Chem 43:1570–1576.
18. Hakeda Y, Kobayashi Y, Yamaguchi K, Yasuda H, Tsuda E,
Higashio K, Miyata T, Kumegawa M 1998 Osteoclastogenesis
inhibitory factor (OCIF) directly inhibits bone-resorbing ac-
tivity of isolated mature osteoclasts. Biochem Biophys Res
Commun 251:796–801.

360
BEKKER ET AL.
Seaman J, Knight RD 1996 Efficacy of pamidronate in reduc-
ing skeletal events in patients with advanced multiple my-
eloma. N Engl J Med 334:488–493.
breast cancer and lytic bone lesions: A randomized, placebo-
controlled trial. J Clin Oncol 17:846–854.
39. Hortobagyi GN, Theriault RL, Lipton A, Porter L, Blayney D,
Sinoff C, Wheeler H, Simeone JF, Seaman JJ, Knight RD,
Heffernan M, Mellars K, Reitsma DJ 1998 Long-term preven-
tion of skeletal complications of metastatic breast cancer with
pamidronate. J Clin Oncol 16:2038–2044.
36. Berenson JR, Lichtenstein A, Porter L, Dimopoulos MA, Bor-
doni R, George S, Lipton A, Keller A, Ballester O, Kovacs M,
Blacklock H, Bell R, Simeone JF, Reitsma DJ, Heffernan M,
Seaman J, Knight RD 1998 Long-term pamidronate treatment
of advanced multiple myeloma patients reduces skeletal
events. Myeloma Aredia Study Group. J Clin Oncol 16:593–
602.
37. Hortobagyi GN, Theriault RL, Porter L, Blayney D, Lipton A,
Sinoff C, Wheeler H, Simeone JF, Seaman J, Knight RD,
Heffernan M, Reitsma DJ 1996 Efficacy of pamidronate in
reducing skeletal complications in patients with breast cancer
and lytic bone metastases. N Engl J Med 335:1785–1791.
38. Theriault RL, Lipton A, Hortobagyi GN, Leff R, Glueck S,
Stewart JF, Costello S, Kennedy I, Simeone J, Seaman JJ,
Address reprint requests to:
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Knight RD, Mellars K, Heffernan M, Reitsma DJ 1999 Pam- Received in original form March 1, 2000; in revised form August
idronate reduces skeletal morbidity in women with advanced 5, 2000; accepted September 11, 2000.