A Nonprostanoid EP4 Receptor Selective Prostaglandin E2 Agonist Restores Bone Mass and Strength in Aged, Ovariectomized Rats

Article (PDF Available)inJournal of Bone and Mineral Research 21(4):565-75 · May 2006with26 Reads
DOI: 10.1359/jbmr.051110 · Source: PubMed
Abstract
CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia. The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 (PGE2) agonist, CP432, could produce bone anabolic effects in aged, ovariectomized (OVX) rats with established osteopenia. CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to 12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone resorption, and bone strength were determined. Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432 completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3 mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test of femoral shaft compared with both sham and OVX controls. CP432 completely restored trabecular and cortical bone mass and strength in established osteopenic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical surfaces.
A Nonprostanoid EP4 Receptor Selective Prostaglandin E
2
Agonist
Restores Bone Mass and Strength in Aged, Ovariectomized Rats
Hua Zhu Ke,
1
D Todd Crawford,
1
Hong Qi,
1
Hollis A Simmons,
1
Thomas A Owen,
1
Vishwas M Paralkar,
1
Mei Li,
1
Bihong Lu,
1
William A Grasser,
1
Kimberly O Cameron,
1
Bruce A Lefker,
1
Paul DaSilva-Jardine,
1
Dennis O Scott,
1
Qing Zhang,
2
Xiao Yan Tian,
2
Webster SS Jee,
2
Thomas A Brown,
1
and David D Thompson
1
ABSTRACT: CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E
2
agonist.
CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged
ovariectomized rats with established osteopenia.
Introduction: The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4
receptor selective prostaglandin E
2
(PGE
2
) agonist, CP432, could produce bone anabolic effects in aged,
ovariectomized (OVX) rats with established osteopenia.
Materials and Methods: CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to
12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone
resorption, and bone strength were determined.
Results: Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432
completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-
dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing
surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3
mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone
formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and
dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and
OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test
of femoral shaft compared with both sham and OVX controls.
Conclusions: CP432 completely restored trabecular and cortical bone mass and strength in established osteo-
penic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical
surfaces.
J Bone Miner Res 2006;21:565–575. Published online on December 5, 2005; doi: 10.1359/JBMR.051110
Key words: EP4 receptor agonist, bone formation, BMD, bone strength, osteoporosis
INTRODUCTION
O
STEOPOROSIS IS A major health problem worldwide. In
the United States, 10 million individuals have osteo-
porosis, and 18 million more have low bone mass.
(1)
In
addition to the pain associated with osteoporosis, dimin-
ished quality of life, and loss of independence and mortal-
ity, the economic burden on the health care system is very
high.
(1)
The reduction in BMD and bone quality (which
refers to architecture, turnover, damage accumulation, and
mineralization) leads to decreased bone strength and in-
creased number of fractures, typically of the spine, wrist,
and hip.
While the current therapies including bisphosphonates,
hormone replacement therapy (HRT), and selective estro-
gen receptor modulators (SERMs) are effective inhibitors
of bone resorption and bone remodeling,
(2–8)
these agents
do not stimulate bone formation and therefore do not re-
store bone mass and rebuild bone structure. A bone ana-
bolic agent, recombinant human PTH(1-34) [rhPTH(1-34)]
has been shown to possess superior efficacy in increasing
BMD and in reducing fracture risk.
(9)
For example, verte-
bral fracture was reduced 65–69% after 2 years of
rhPTH(1-34) treatment
(9)
compared with an approximate
50% reduction in vertebral fracture with antiremodeling
agents such as bisphosphonates or SERMs.
(2–8)
These clini-
cal results highlight the ability of an anabolic agent to meet
the unmet clinical need in osteoporosis beyond what can be
achieved with antiremodeling therapies. Therefore, there is
great medical need for an improved bone anabolic agent to
Drs Ke, Crawford, Qi, Simmons, Owen, Paralkar, Lu, Grasser,
Cameron, Li, Lefker, Jardine, Thompson, Brown, and Scott are
fill-time Pfizer employees and own Pfizer stock. Dr Jee owns Pfizer
stock. The other authors state that they have no conflicts of interest.
1
Pfizer Global Research and Development, Groton Laboratories, Groton, Connecticut, USA;
2
Radiobiology Division, University of
Utah School of Medicine, Salt Lake City, Utah, USA.
JOURNAL OF BONE AND MINERAL RESEARCH
Volume 21, Number 4, 2006
Published online on December 5, 2005; doi: 10.1359/JBMR.051110
© 2006 American Society for Bone and Mineral Research
565
JO506390 565 575 March
treat patients with established osteoporosis by restoring
bone mass and rebuilding bone structure to prevent further
skeletal fractures.
It has been very well characterized that prostaglandin E
2
(PGE
2
) stimulates both bone resorption and bone forma-
tion but in favor of bone formation, thus increasing bone
mass and bone strength.
(1013)
The pharmacological activi-
ties of PGE
2
are mediated through four cell surface recep-
tor subtypes, EP1EP4.
(14,15)
It is not completely under-
stood which receptor subtype(s) is associated with the
anabolic effect of PGE
2
. Studies have shown that EP2 and
EP4 receptors may play important roles in bone metabo-
lism. It has been reported
(16,17)
that bone resorption in-
duced by PGE
2
is mediated mainly by the EP4 and partially
by the EP2 receptors, whereas EP1 and EP3 receptors did
not play a role. In fetal rat organ cultures, an EP4 receptor
agonist is a more potent stimulator of bone resorption than
an EP2 receptor agonist, but both can stimulate bone for-
mation, supporting the hypothesis that EP2 and EP4 recep-
tors play important roles in bone.
(18)
It has also been re-
ported by Li et al.
(19)
that osteoclastogenesis was impaired
in EP2 receptor knockout mice, suggesting that EP2 recep-
tors play an important role in osteoclast formation. Using
EP1 and EP2 receptor knockout mice, Akhter et al.
(20)
reported that EP1 receptors have minimal influence on
skeletal strength or size, whereas EP2 receptors have a ma-
jor influence on biomechanical properties of bone because
the absence of EP2 receptors resulted in weak bone
strength. Furthermore, we recently reported that a novel,
nonprostanoid EP2 receptor selective agonist stimulates lo-
cal bone formation and enhances fracture healing in rats
and dogs.
(21,22)
These data further support the hypothesis
that the EP2 receptors play a role in bone metabolism. EP4
receptors are expressed in embryonic and neonatal bone
tissue of mice and in bone tissue of young adult rats and
osteoblastic cell lines, and its expression in bone tissues is
upregulated by PGE
2
.
(23,24)
Using rat bone marrow cul-
tures, Weinreb et al. reported
(24)
that the anabolic effect of
PGE
2
is mediated through the EP4 receptors. Mineralized
nodule formation was absent and could not be increased by
treatment with PGE
2
in bone marrow cell cultures derived
from EP4 receptor knockout mice.
(24)
Furthermore, knock-
out of EP4 receptor decreased bone formation and bone
mass, and impaired fracture healing in mice.
(25)
These re-
sults led to the hypothesis that EP4 receptors also play an
important role in PGE
2
-induced bone formation. This hy-
pothesis is further supported by the results from in vivo
experiments in rodents using pharmaceutical agents such as
EP4 receptor selective antagonists and agonists.
(2629)
An
EP4 receptorspecific antagonist suppressed the increase in
bone mass induced by PGE
2
in young rats,
(27)
and systemic
administration of selective EP4 receptor agonists increased
bone formation, augmented bone mass and accelerated
bone repair.
(26,28,29)
Our efforts have been focused on iden-
tifying a small molecule, nonprostanoid EP4 receptor se-
lective agonist that would be expected to increase bone
formation and bone mass. These efforts led to the discovery
of CP432 [chemical name: 5-(3-{2S-[3R-hydroxy-4-(3-
trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl}-
propyl)-thiophene-2-carboxylic acid, molecular weight
469.53, see Fig. 1 for structure]. CP432 selectively binds to
the EP4 receptor with a 50% inhibition concentration
(IC
50
) of 8 nM (binding IC
50
for EP1, EP2, and EP3 is 1000,
460, and 1000 nM, respectively). Similarly, CP432 is selec-
tive as an EP4 receptor agonist when measured against
other prostanoid receptors including the prostaglandin D2
(DP), prostaglandin F2 (FP), prostacyclin (IP), and
thromboxane receptors (TP) in the prostanoid receptor
family. CP432 stimulates the EP4 receptormediated sig-
naling by the cAMP pathway with a 50% effective concen-
tration (EC
50
) value of 0.6 nM. The purpose of this study
was to test whether CP432 can stimulate bone formation
and restore bone mass and bone strength in established
severe osteopenic, aged ovariectomized (OVX) rats.
MATERIALS AND METHODS
A total of 70 virgin Sprague-Dawley female rats
(Taconic, Germantown, NY, USA) were used in this study.
Rats were housed singly in 48 × 27 × 20-cm cages at local
vivarium condition (24°C; 12-h light, 12-h dark cycle).
Deionized water was provided to the animals ad libitum.
During the study, the OVX rats were pair-fed with a com-
mercial diet (Purina Laboratory Rodent Chow 5001; Pu-
rina-Mills, St Louis, MO, USA) based on the average
weekly food consumption of the sham control group (20
g/day). This diet contains 0.95% calcium, 0.67% phospho-
rus, and 4.5 IU/g vitamin D
3
. The experiments were con-
ducted according to Pfizer Animal Care and Useapproved
protocols, and animals were maintained in accordance with
the Institute of Laboratory Animal Research Guide for the
Care and Use of Laboratory Animals.
Rats were sham-operated (n 20) or ovariectomized
(OVX, n 50) at 3.5 months of age. Eight and one-half
FIG. 1. Chemical structure of CP432, a nonprostanoid, EP4 re-
ceptor selective agonist. CP432 selectively binds to the EP4 re-
ceptor with a 50% inhibition concentration (IC
50
)of8nMand
stimulates the EP4 receptormediated signaling by the cAMP
pathway with a 50% effective concentration (EC
50
) value of
0.6 nM.
KE ET AL.566
months after surgery, 10 sham-operated and 10 OVX rats
were necropsied and served as basal controls (sham base-
line and OVX baseline, respectively). The remaining 10
sham-operated (sham controls) rats were treated with ve-
hicle (5% ethanol in sterile water, daily subcutaneous in-
jection), and the remaining OVX rats were treated with
vehicle (OVX controls) or CP432 at 0.3, 1.0, or 3.0 mg/kg
body weight/day by daily subcutaneous injection, at the
dosing volume of 0.3 ml/rat/day, for 6 weeks. There were 10
rats in each group. All rats were given subcutaneous injec-
tions of calcein green (10 mg/kg body weight; Sigma), a
fluorochrome bone marker, 12 and 2 days before necropsy
to determine dynamic changes in bone tissue. Rats were
necropsied under anesthesia and killed by CO
2
asphyxia-
tion.
Body weight and general health of the animals
The rats were weighed 1 day before treatment and on the
day of necropsy. The general health of study animals was
monitored daily.
Femoral areal bone mineral measurements
The right femur from each rat was removed at autopsy
and scanned using DXA (QDR 4500/W; Hologic, Waltham,
MA, USA) equipped with Regional High Resolution
Scan software. The scan field size was 5.08 × 1.902 cm,
resolution was 0.0254 × 0.0127 cm, and scan speed was 7.25
mm/s. The femoral scan images were analyzed, and bone
area, areal BMC, and areal BMD of total femora (TF),
distal femoral metaphyses (DFM), and femoral shaft (FS)
were determined as described previously.
(30)
Third lumbar vertebral trabecular bone
histomorphometry
At necropsy, the third lumbar vertebral body was re-
moved, dissected free of soft tissue, fixed in 70% ethanol,
stained with Villanuevas bone stain, dehydrated in graded
concentrations of ethanol, defatted in acetone, and embed-
ded in methyl methacrylate (Fisher Scientific, Fair Lawn,
NJ, USA). Parasagittal sections of third lumbar vertebral
body at thicknesses of 4 and 10 m were cut. The 4-m
sections were further stained with toludine blue and used
for measurement of osteoclast surface and osteoclast num-
ber, whereas the 10-m sections were used for measure-
ment of bone mass, structure, and bone formation related
indices by using an Image Analysis System with software
version 2.2 (Osteomeasure, Atlanta, GA, USA). A tissue
area within 0.5 mm from each dorsal and ventral end was
selected for histomorphometric analysis.
Measurements and calculations related to trabecular
bone volume and structure included trabecular bone vol-
ume, thickness, number, and separation, whereas measure-
ments and calculations related to bone resorption included
osteoclast number and osteoclast surface. Furthermore, the
parameters related to bone formation and turnover in-
cluded mineralizing surface [(double labeling perimeter +
0.5 single labeling perimeter)/total trabecular perimeter ×
100], mineral apposition rate, bone formation rate/bone
volume (BFR/BV), bone formation rate/bone surface
(BFR/BS), and bone formation rate/tissue volume (BFR/
TV). The definitions and formula for calculations of these
parameters were described previously by Parfitt et al.
(31)
and Jee et al.
(32)
Ex vivo pQCT analysis
Excised femurs were scanned by a pQCT machine
(Stratec XCT Research M; Norland Medical Systems, Fort
Atkinson, WI, USA) with software version 5.40. A 1-mm-
thick cross-section of the FS was taken at midpoint of the
femur with a voxel size of 0.10 mm. Volumetric content,
density, and area were determined for both trabecular and
cortical bone as described previously.
(33)
Tibial shaft cortical bone histomorphometry
For cortical bone, undecalcified, Villanueva bone
stained, methyl methacrylate embedded, cross-sections of
the tibial shaft at a thickness of 20 m were prepared. Total
tissue area, periosteal perimeter, cortical bone area, mar-
row cavity area, endocortical perimeter, and dynamic his-
tomorphometric parameters including mineralizing surface,
mineral apposition rate, and bone formation rate were de-
termined on periosteal and endocortical surfaces.
(31,32)
Biomechanical testing of the fifth lumbar vertebral
body and FS
Biomechanical testing of the fifth lumbar vertebra (LV5)
and FS was performed using an Instron 5500 servo-electric
testing machine and associated Merlin II software (software
version 4.05; Instron, Canton, MA, USA). A compression
test was used to determine the ultimate strength, stiffness,
and toughness for LV5. A three-point bending test was
used to determine the maximal load to failure and stiffness
for FS. A detailed description of these tests has been pre-
viously reported.
(11)
Statistics
Data are expressed as mean ± SE. Statistics were calcu-
lated using StatView 4.0 packages (Abacus Concepts,
Berkeley, CA, USA). The ANOVA test was used for all
groups necropsied at the end of the study, and Fishers
protected least significant difference (PLSD) test was used
to compare the differences between each group. The dif-
ference between the groups at 6 weeks and basal controls
was determined by Students t-test. p < 0.05 was considered
a significant difference.
RESULTS
Effects on body weight and general health
of animals
At the beginning of treatment (8.5 months after surgery),
the average body weight of the sham rats was 404 g,
whereas the OVX rats weighed 550 g. During the 6-week
study period, rats in the sham control group gained (body
weight at necropsy minus body weight at the beginning of
treatment) an average of 9.7 g, and rats in the OVX control
group gained an average of 4.4 g. In OVX rats treated with
CP432, the average change in body weight was +4.6, −16.4,
EP4 AGONIST RESTORES BONE IN OVX RATS 567
and 29 g for 0.3, 1, and 3 mg/kg/day, respectively. How-
ever, at the end of the study, body weight did not differ
significantly between OVX rats treated with vehicle and
OVX rats treated with all doses of CP432. No diarrhea or
hair loss was observed in OVX rats treated with CP432. No
other obvious side effects were observed during the study.
Effects on femoral areal bone mineral
measurements
Bone area of the TF, DFM, and FS did not differ signifi-
cantly among sham baseline, OVX baseline, sham controls,
and OVX controls (Table 1). Compared with OVX con-
trols, bone area of the DFM in OVX rats treated with
CP432 at 1 and 3 mg/kg/day was significantly higher by
5.5% and 5.2%, respectively. Compared with sham base-
line, OVX for 8.5 months induced significant decreases in
BMC (16%, 17%, and 14% for TF, DFM, and FS, re-
spectively) and BMD (15%, 17%, and 14% for TF,
DFM, and FS, respectively; Table 1). No continuous de-
crease in BMC and BMD was found in OVX rats treated
with vehicle compared with OVX baseline. At 0.3 mg/kg/
day, CP432 partially restored BMC and BMD of the TF
and completely restored BMC and BMD of the FS,
whereas it had no significant effect on BMC and BMD of
the DFM compared with OVX controls (Table 1). OVX
rats treated with CP432 at 1 and 3 mg/kg/day had signifi-
cantly higher BMC and BMD of the TF, DFM, and FS
compared with OVX baseline and OVX controls. At 1 mg/
kg/day, OVX rats treated with CP432 had significantly
higher BMC of the FS compared with sham controls
(+14%). Compared with sham controls, there was a signifi-
cant increase in BMC and BMD of the TF (+12% and +6%,
respectively) and FS (+21% and +15%, respectively) in
OVX rats treated with CP432 at 3 mg/kg/day. These data
indicate that CP432 not only completely prevented bone
loss but also added extra bone to the total femur and femo-
ral shafts (cortical bone site).
Effects on trabecular bone histomorphometry of the
third lumber vertebral body
Histomorphometric analysis showed that OVX for 8.5
months induced significant decreases in trabecular bone
volume (BV/TV, 40%), thickness (Tb.Th, 13%), and
number (Tb.N, 31%), and significant increases in osteo-
clast number (OCN/BS, +159%) and osteoclast surface
(OCS/BS, +162%) compared with sham controls at baseline
(Table 2). At 8.5 months after surgeries, there was no sig-
nificant difference in mineralizing surface (MS/BS), mineral
apposition rate (MAR), and bone formation rates (BFR/
TV, BFR/BV, BFR/BS) between sham and OVX rats. No
further bone loss was found between baseline OVX and
OVX rats treated with vehicle for 6 weeks.
Treatment with CP432 in OVX rats at all doses induced
significant increases in BV/TV compared with baseline
OVX and OVX rats treated with vehicle (Table 2). Com-
pared with OVX baseline, treatment with CP432 at 0.3, 1,
and 3 mg/kg/day significantly increased BV/TV by 38%,
66%, and 38%, respectively. Similarly, when compared
with OVX rats treated with vehicle, treatment with CP432
TABLE 1. DXA ANALYSIS OF WHOLE FEMUR
Parameters
Sham OVX Sham OVX OVX + CP432
Baseline Baseline Vehicle Vehicle 0.3 mg 1 mg 3 mg
Total femur
Bone area (cm
2
) 1.996 ± 0.020 1.955 ± 0.032 1.968 ± 0.020 2.005 ± 0.037 2.013 ± 0.026 2.073 ± 0.037
†‡
2.073 ± 0.037
†‡
BMC (g) 0.507 ± 0.012 0.424 ± 0.010* 0.492 ± 0.011 0.443 ± 0.015*
0.480 ± 0.013c 0.548 ± 0.026
‡§
0.548 ± 0.017*
†‡§
BMD (g/cm
2
) 0.254 ± 0.005 0.217 ± 0.003* 0.250 ± 0.003 0.220 ± 0.004*
0.238 ± 0.005
‡§
0.264 ± 0.009
‡§
0.264 ± 0.004
†‡§d
Distal femoral metaphysis
Bone area (cm
2
) 0.275 ± 0.003 0.272 ± 0.005 0.280 ± 0.003 0.276 ± 0.004 0.281 ± 0.003 0.291 ± 0.005*
‡§
0.290 ± 0.005*
†‡§
BMC (g) 0.055 ± 0.002 0.045 ± 0.002* 0.053 ± 0.002 0.049 ± 0.001*
0.051 ± 0.001
0.058 ± 0.003
‡§
0.054 ± 0.002
‡§
BMD (g/cm
2
) 0.198 ± 0.006 0.164 ± 0.005* 0.190 ± 0.005 0.176 ± 0.003*
0.184 ± 0.004
0.199 ± 0.006
‡§
0.187 ± 0.003
‡§
Femoral shaft
Bone area (cm
2
) 0.231 ± 0.004 0.231 ± 0.005 0.230 ± 0.003 0.236 ± 0.004 0.244 ± 0.004*
†‡
0.246 ± 0.004*
†‡
0.242 ± 0.007
BMC (g) 0.0561 ± 0.001 0.048 ± 0.001* 0.057 ± 0.002 0.051 ± 0.002
0.057 ± 0.002
‡§
0.065 ± 0.003*
†‡§
0.069 ± 0.003*
†‡§
BMD (g/cm
2
) 0.243 ± 0.005 0.209 ± 0.003* 0.246 ± 0.005 0.216 ± 0.006*
0.232 ± 0.005
‡§
0.263 ± 0.012
‡§
0.284 ± 0.008*
†‡§
Mean ± SE.
* p < 0.05 vs. baseline sham.
p < 0.05 vs. Sham-vehicle.
p < 0.05 vs. Baseline OVX.
§
p < 0.05 vs. OVX vehicle.
KE ET AL.568
at 0.3, 1, and 3 mg/kg/day significantly increased BV/TV by
41%, 84%, and 56%, respectively. The increased BV/TV
was accompanied with an increase in Tb.Th and Tb.N and
a decrease in trabecular separation (Tb.Sp) in OVX rats
treated with CP432 at all doses (Table 2). At both the base-
line and the end of the study, OVX rats had significantly
higher osteoclast number and osteoclast surface compared
with their respective sham controls (Table 2), indicating
that bone resorption was higher in the OVX rats after an
extend length of time (8.5 and 10 months after surgery).
Treatment with CP432 at all doses significantly decreased
bone resorption parameters (OCN/BS and OCS/BS) com-
pared with both OVX baseline and OVX controls (from
50% to 65%), with no clear dose-response between 0.3
and 3 mg/kg/day. Bone formation parameters, BS/MS,
MAR, BFR/TV, BFR/BV, and BFR/BS, increased signifi-
cantly in the OVX rats treated with CP432 at all doses
compared with sham baseline, OVX baseline, sham con-
trols, and OVX controls (Table 2). No clear dose-response
was observed for MS/BS, whereas there was a dose-
dependent increase in MAR, BFR/TV, BFR/BV, and BFR/
BS in OVX rats treated with CP432. For example, MS/BS
increased significantly by 122%, 114%, and 140% in OVX
rats treated with CP432 at 0.3, 1, or 3 mg/kg/day, respec-
tively, compared with OVX controls. However, BFR/TV
increased significantly by 206%, 295%, and 396% in OVX
rats treated with CP432 at 0.3, 1, or 3 mg/kg/day, respec-
tively, compared with OVX controls. These results show
that CP432 increases trabecular bone formation by increas-
ing osteoblast number and osteoblast activity in these aged
OVX rats.
Effects on trabecular bone strength of the LV5
In LV5, OVX for 8.5 months induced significant de-
creases in ultimate strength (28%), stiffness (38%), elas-
tic modulus (39%), and toughness (38%) compared with
the sham baseline group (Fig. 2). No further decrease in
these parameters was observed in OVX rats treated with
vehicle compared with OVX baseline. Ultimate strength,
stiffness, and toughness in OVX rats treated with CP432 at
all doses increased significantly compared with OVX rats
treated with vehicle (Fig. 2). CP432 at 1 and 3 mg/kg/day
significantly increased toughness in OVX rats compared
with sham controls (Fig. 2D). Elastic modulus increased
significantly in OVX rats treated with CP432 at 0.3 and 1
mg/kg/day compared with OVX controls (Fig. 2C). These
data indicate that CP432 completely restored bone strength
in LV5 (a trabecular bone site) back to sham control levels
in established osteopenic, OVX rats.
Effects on cortical BMC and BMD (pQCT analysis
of femoral shaft)
OVX for 8.5 months (OVX baseline) induced significant
decreases in total BMC, total BMD, cortical BMC, cortical
BMD, cortical bone area, and cortical thickness, and a sig-
TABLE 2. SELECTED TRABECULAR BONE HISTOMORPHOMETRIC PARAMETERS OF THIRD LUMBAR VERTEBRAL BODY (LV3)
Parameters
Sham OVX Sham OVX OVX + CP432
Baseline Baseline Vehicle Vehicle 0.3 mg 1 mg 3 mg
Trabecular bone
volume (%) 34.2 ± 1.65 20.4 ± 1.15* 31.4 ± 1.13 18.5 ± 1.44*
26.1 ± 2.26
‡§
34.0 ± 2.85
‡§
28.8 ± 1.83
‡§
Trabecular
thickness (mm) 102 ± 3.53 89.4 ± 4.32* 100.5 ± 3.45 97.7 ± 3.72 112.2 ± 8.1
144.3 ± 6.1*
†‡§
124.0 ± 3.9*
†‡§
Trabecular
number (/mm) 3.33 ± 0.11 2.31 ± 0.15* 3.15 ± 0.14 1.90 ± 0.15*
2.31 ± 0.12
†‡§
2.35 ± 0.17*
†§
2.31 ± 0.09*
†§
Trabecular
separation (mm) 200 ± 10.8 357 ± 25.0* 222 ± 11.1 462 ± 48.4*
331 ± 26.5*
†§
311 ± 49.4*
†§
316 ± 22.0*
†§
Mineralizing
surface/BS (%) 15.4 ± 1.51 18.5 ± 1.71 17.1 ± 1.37 21.6 ± 0.88 47.8 ± 2.04*
†‡§
46.2 ± 2.28*
†‡§
51.8 ± 2.15*
†‡§
Mineral apposition
rate (mm/day) 0.53 ± 0.07 0.54 ± 0.03 0.67 ± 0.03 0.80 ± 0.04*
†‡
0.91 ± 0.03*
†‡§
1.23 ± 0.09*
†‡§
1.35 ± 0.07*
†‡§
Bone formation
rate/BV (%/year) 51.5 ± 7.6 66.5 ± 3.0 69.5 ± 5.55 108.8 ± 8.0*
243.2 ± 17.9*
†‡§
248.4 ± 28.6*
†‡§
351.2 ± 28.0*
†‡§
Bone formation
rate/BS
(mm/day × 100) 8.76 ± 1.30 9.73 ± 0.59 11.48 ± 1.06 17.25 ± 1.03*
43.7 ± 2.63*
†‡§
57.0 ± 5.09*
†‡§
70.2 ± 4.42*
†‡§
Bone formation
rate/TV (%/year) 18.3 ± 2.91 13.7 ± 1.22 21.8 ± 1.73 19.9 ± 1.59
61.0 ± 3.95*
†‡§
78.8 ± 6.74*
†‡§
99.0 ± 8.05*
†‡§
Osteoclast
surface/BS (%) 0.78 ± 0.08 2.05 ± 0.25* 0.63 ± 0.06 2.46 ± 0.18*
1.01 ± 0.10
†‡§
0.86 ± 0.07
†‡§
1.15 ± 0.05
†‡§
Osteoclast
number/BS (#/mm) 0.17 ± 0.03 0.45 ± 0.05* 0.16 ± 0.02 0.50 ± 0.03*
0.22 ± 0.02*
†‡§
0.19 ± 0.009
‡§
0.23 ± 0.01*
†‡§
Mean ± SE.
* p < 0.05 vs. baseline sham.
p < 0.05 vs. Sham-vehicle.
p < 0.05 vs. Baseline OVX.
§
p < 0.05 vs. OVX vehicle.
EP4 AGONIST RESTORES BONE IN OVX RATS 569
nificant increase in endocortical circumference compared
with sham baseline (Table 3). These differences were main-
tained at the end of the study compared with OVX rats
treated with vehicle and sham rats treated with vehicle,
indicating no further bone loss occurred in OVX rats during
the treatment period. There was a dose-dependent increase
in cortical and marrow trabecular bone in OVX rats treated
with CP432 (Table 3; Fig. 3). Dose-dependent increases in
the thickness of cortex and the formation of new marrow
trabecular bone were found in the OVX rats treated with
CP432 (Fig. 3A). At the two highest dose groups, massive
new marrow trabecular bone was observed. Total bone con-
tent in OVX rats treated with CP432 at 0.3 mg/kg/day in-
creased significantly by 8.7% compared with OVX controls
and did not differ significantly compared with sham con-
trols, indicating that CP432 at this dose completely restored
the total BMC back to sham control level (Table 3). Total
BMC in OVX rats treated with CP432 at 1 and 3 mg/kg/day
increased significantly compared with both sham and OVX
controls (+12 to +28%), suggesting that CP432 at these
doses not only restored total BMC back to sham levels but
also added extra bone to this skeletal site (Table 3). Simi-
larly, cortical BMC, cortical bone area, and cortical thick-
ness were dose-dependently increased, whereas endocorti-
cal circumference was dose-dependently decreased in OVX
rats treated with CP432 compared with both sham and
OVX controls (Table 3), suggesting that CP432 induced
endocortical bone formation and formed new endocortical
bone. Total tissue area and periosteal circumference in-
creased significantly in the OVX rats treated with CP432 at
1 mg/kg/day compared with OVX controls. Cortical BMD
decreased dose-dependently in OVX rats treated with
CP432 compared with OVX and sham controls (Table 4).
This may be caused by the formation of massive new cor-
tical bone induced by this agent, thus the proportion of new
bone increased after treatment. In CP432-treated OVX
rats, trabecular BMD in the femoral shaft increased signifi-
cantly and dose-dependently by 36%, 109%, and 156% for
0.3, 1, and 3 mg/kg/day, respectively, compared with OVX
controls (Table 3).
Effects on tibial shaft cortical
bone histomorphometry
Qualitatively, double-labeled surface was observed in
only 1 of 10 rats on the periosteal surface and 0 of 10 on the
endocortical surfaces for the sham baseline. In the OVX
baseline, double-labeled surface was observed in 4 of 10
rats on periosteal surface and 10 of 10 rats on endocortical
surfaces. Four of 10 and 8 of 10 rats possessed double-
labeled surface on the periosteal and endocortical surfaces,
respectively, for sham rats treated with vehicle. For OVX
rats treated with vehicle, there were 8 of 10 and 10 of 10 rats
that possessed double-labeled surface on the periosteal and
endocortical surfaces, respectively. OVX rats treated with
CP432 at all doses possessed double-labeled surface on
both periosteal and endocortical surfaces with the excep-
tion of two rats at the 0.3-mg dose group on periosteal
surface. At 3 mg/kg/day, CP432 dramatically increased the
double-labeled surface and interlabel distance on the peri-
osteal surface compared with sham and OVX controls.
Treatment with CP432 dose-dependently increased endo-
cortical labeled surface and interlabeled distance compared
with both sham and OVX controls, indicating CP432 at all
doses administered stimulates bone formation on these sur-
faces (Fig. 3B). We also observed that CP432 induced new
marrow trabecular bone formation in a dose-dependent
manner in the marrow cavity of tibial shafts where there
were very little trabeculae in sham and OVX controls.
Newly formed marrow trabecular bone was observed in all
OVX rats treated with CP432 at all three doses, whereas
newly formed marrow trabecular bone was not observed in
FIG. 2. Ultimate strength, stiffness, elastic
modulus, and toughness of fifth lumber ver-
tebral body from sham and OVX rats at
the beginning of treatment (B-Sham and
B-OVX), sham rats treated with vehicle
(Sham), and OVX rats treated with vehicle
(OVX) or CP432 at 0.3, 1, and 3 mg/kg/day.
Complete restoration of bone strength was
observed with CP432 treatment. Mean ± SE.
a
p < 0.05 vs. B-Sham;
b
p < 0.05 vs. Sham;
c
p
< 0.05 vs. B-OVX;
d
p < 0.05 vs. OVX.
KE ET AL.570
any of the sham and OVX control rats (Fig. 3B). These
newly formed trabeculae were labeled with calcein green,
indicating an active bone mineralization on these surfaces
at necropsy (Fig. 3B). Furthermore, treatment with CP432
in OVX activated bone remodeling in the inner third of the
cortex as evidenced by increasing labeling surfaces and re-
sorption cavity in these area (Fig. 3B).
Quantitatively, OVX baseline had significantly larger
marrow cavity area and higher periosteal MAR and BFR/
BS and endocortical MS/BS, MAR, and BFR/BS compared
with sham baseline (Table 4). Compared with their respec-
tive baseline controls, sham or OVX rats treated with ve-
hicle for 6 weeks had higher periosteal and endocortical
MAR and BFR/BS and endocortical MS/BS. We have no
TABLE 3. PQCT ANALYSIS OF FEMORAL SHAFTS (FS)
Parameters
Sham OVX Sham OVX OVX + CP432
Baseline Baseline Vehicle Vehicle 0.3 mg 1 mg 3 mg
Total bone content
(mg/mm) 11.9 ± 0.28 10.7 ± 0.2* 11.6 ± 0.26 10.7 ± 0.32*
11.6 ± 0.26
‡§
13.1 ± 0.51
†‡§
13.7 ± 0.37*
†‡§
Total bone density
(mg/cm
3
) 1091 ± 12.5 994 ± 16.2* 1123 ± 13.4 995 ± 17.7*
1018 ± 31.7 1110 ± 39.0
‡§
1198 ± 25.9*
†‡§
Total tissue area
(mm
2
) 10.9 ± 0.26 10.8 ± 0.28 10.4 ± 0.23 10.8 ± 0.27 11.5 ± 0.35
11.8 ± 0.24*
†‡§
11.4 ± 0.31
Cortical bone content
(mg/mm) 11.8 ± 0.26 10.6 ± 0.19* 11.7 ± 0.27 10.7 ± 0.33*
11.5 ± 0.25
13.0 ± 0.55*
†‡§
13.8 ± 0.41*
†‡§
Cortical bone density
(mg/cm
3
) 1415 ± 3.7 1390 ± 3.9* 1423 ± 2.9 1395 ± 3.9*
1380 ± 7.7*
1342 ± 19.4*
†‡§
1298 ± 15.8*
†‡§
Cortical bone area
(mm
2
) 8.32 ± 0.18 7.65 ± 0.14
a
8.18 ± 0.18 7.68 ± 0.23 8.34 ± 0.18
‡§
9.75 ± 0.52*
†‡§
10.7 ± 0.43*
†‡§
Cortical thickness
(mm) 0.96 ± 0.01 0.86 ± 0.01* 0.99 ± 0.02 0.86 ± 0.02*
0.93 ± 0.04 1.20 ± 0.12*
†‡§
1.51 ± 0.12*
†‡§
Trabecular density
(mg/cm
3
) 140.6 ± 4.69 114.1 ± 4.0* 164.2 ± 8.7
*
128.6 ± 2.8*
174.6 ± 19.4
‡§
269.9 ± 45.8*
†‡§
328.5 ± 41.7*
†‡§
Periosteal
circumference
(mm) 11.7 ± 0.14 11.6 ± 0.15 11.4 ± 0.13 11.6 ± 0.15 12.0 ± 0.18
12.2 ± 0.12*
†‡§
12.0 ± 0.16
Endocortical
circumference
(mm) 5.65 ± 0.13 6.24 ± 0.19* 5.20 ± 0.13 6.20 ± 0.17*
6.19 ± 0.39
4.60 ± 0.73
‡§
2.52 ± 0.76*
†‡§
Mean ± SE.
* p < 0.05 vs. baseline sham.
p < 0.05 vs. Sham-vehicle.
p < 0.05 vs. Baseline OVX.
§
p < 0.05 vs. OVX vehicle.
FIG. 3. (A) pQCT images of femoral shafts
from sham rats treated with vehicle (Sham),
OVX rats treated with vehicle (OVX), or
CP432 at 0.3, 1 or 3 mg/kg/day. Dose-
dependent increase in cortical thickness and
marrow trabecular bone was found associ-
ated with CP432 treatment. (B) Photographs
taken from the cross-sections of tibial shafts
of sham-operated rats treated with vehicle
(Sham), OVX rats treated with vehicle
(OVX), or CP432 at 0.3, 1 and 3 mg/kg/day.
Increased double-labeled surface (in green)
and interlabel distance (arrows) was found in
OVX rats treated with CP432 on endocorti-
cal surfaces (arrows) in comparison with ve-
hicle treated sham or OVX rats. Dose-
dependent increase in trabecular bone
formation (TB, labeled with calcein green)
was found with CP432 treatment in bone
marrow (BM) cavity of tibial shafts. Further-
more, CP432 activated bone remodeling (ar-
rowheads) in the inner third of the cortex
(Ct). Villanueva bone stain, 20-m-thick
cross-sections.
EP4 AGONIST RESTORES BONE IN OVX RATS 571
Fig 3 live 4/C
explanation for these changes. One possible reason may be
the handling of these rats while dosing, because these rats
had not been handled for 8.5 months. There was a signifi-
cant increase in marrow cavity area, periosteal and endo-
cortical bone formation parameters, and endocortical bone
resorption parameters, and a significant decrease in cortical
bone area in OVX controls compared with sham controls at
the end of the study (Table 4).
Treatment with CP432 dose-dependently increased total
tissue area, cortical bone area, and periosteal and endocor-
tical bone formation parameters, and decreased endocorti-
cal bone resorption parameter (Table 4). Compared with
OVX controls, total tissue area increased significantly by
13%, 23%, and 30% in OVX rats treated with CP432 at 0.3,
1, and 3 mg/kg/day, respectively. Similarly, cortical bone
area increased significantly by 16%, 30%, and 41% in OVX
rats treated with CP432 at 0.3, 1, and 3 mg/kg/day, respec-
tively, compared with OVX controls. Total tissue area and
cortical bone area were significantly higher in OVX rats
treated with all doses of CP432 compared with sham con-
trols, with the exception of cortical bone area at the 0.3
mg/kg/day (Table 4). No significant difference was found in
the marrow cavity area in OVX rats treated with CP432
compared with OVX controls.
There was a dose-dependent increase in periosteal MS/
BS, MAR, and BFR in OVX rats treated with CP432
(Table 4). At the highest dose, all of these parameters were
significantly increased in CP432-treated OVX rats com-
pared with sham and OVX baseline and at the end of study,
indicating CP432 stimulates periosteal bone formation. En-
docortical MS/BS, MAR, and BFR were significantly in-
creased at all doses of CP432-treated OVX rats compared
with sham and OVX controls at baseline and at the end of
study (Table 4). Furthermore, the endocortical eroded pe-
rimeter, an index of bone resorption, decreased signifi-
cantly at all doses of CP432-treated OVX rats compared
with sham and OVX controls at baseline and at the end of
study (Table 4).
Effects on femoral shaft cortical bone strength
OVX for 8.5 months induced significant decreases in
maximal load (28%), ultimate strength (17%), stiffness
(51%), and toughness (29%) in FS compared with base-
line sham controls (Fig. 4). Similarly, at the end of the
study, OVX rats had lower maximal load, ultimate strength,
stiffness, and toughness compared with sham controls.
There was no significant difference found between OVX
controls and OVX rats treated with CP432 at 0.3 mg/kg/
day. However, these parameters were completely restored
back to sham control levels after treatment with CP432 at 1
and 3 mg/kg/day (Fig. 4). These data indicate that CP432
TABLE 4. SELECTED CORTICAL BONE HISTOMORPHOMETRIC PARAMETERS OF TIBIAL SHAFT (TS)
Parameters
Sham OVX Sham OVX OVX + CP432
Baseline Baseline Vehicle Vehicle 0.3 mg 1 mg 3 mg
Total tissue area
(mm
2
) 5.13 ± 0.12 5.21 ± 0.13 4.97 ± 0.10 4.77 ± 0.16 5.40 ± 0.12
†§
5.87 ± 0.11*
†‡§
6.21 ± 0.23*
†‡§
Marrow cavity area
(mm
2
) 0.75 ± 0.05 1.02 ± 0.06
*
0.77 ± 0.04 0.99 ± 0.09*
1.01 ± 0.08*
0.97 ± 0.07*
0.88 ± 0.06
Cortical bone area
(mm
2
) 4.37 ± 0.10 4.19 ± 0.09 4.20 ± 0.08 3.78 ± 0.11*
†‡
4.39 ± 0.07
§
4.90 ± 0.07*
†‡§
5.33 ± 0.20*
†‡§
Periosteal mineralizing
surface (%) 23.5 ± 2.80 25.39 ± 3.18 20.4 ± 2.64 32.39 ± 4.67
24.67 ± 4.60 33.8 ± 3.83*
52.0 ± 6.71*
†‡§
Periosteal mineral
apposition rate
(m/day) 0.00 ± 0.00 0.28 ± 0.12* 0.30 ± 0.12* 0.74 ± 0.13*
†‡
0.73 ± 0.12*
†‡
1.01 ± 0.06*
†‡
1.46 ± 0.11*
†‡§
Periosteal bone
formation rate
(m/day × 100) 0.00 ± 0.00 8.17 ± 3.76* 8.23 ± 3.51* 27.32 ± 6.52*
†‡
20.0 ± 5.02* 35.6 ± 5.62*
†‡
80.8 ± 15.5*
†‡§
Endocortical
mineralizing surface
(%) 18.1 ± 2.01 29.0 ± 2.16* 45.2 ± 1.87* 67.3 ± 2.78*
†‡
92.3 ± 3.8*
†‡§
86.8 ± 4.41*
†‡§
89.7 ± 3.38*
†‡§
Endocortical mineral
apposition rate
(m/day) 0.00 ± 0.00 0.80 ± 0.05* 0.59 ± 0.07* 0.96 ± 0.06*
1.35 ± 0.07*
†‡§
1.34 ± 0.06
a
*
†‡§
1.65 ± 0.13*
†‡§
Endocortical bone
formation rate
(m/day × 100) 0.00 ± 0.00 23.0 ± 1.65* 26.3 ± 3.36* 65.6 ± 6.71*
†‡
125.5 ± 8.81*
†‡§
116.4 ± 8.14*
†‡§
150.0 ± 14.6*
†‡§
Endocortical eroded
surface/BS (%) 6.43 ± 0.78 7.44 ± 0.70 2.25 ± 0.32* 3.25 ± 0.96*
0.15 ± 0.15*
†‡§
0.14 ± 0.14*
†‡§
1.14 ± 0.41*
†‡§
Mean ± SE.
* p < 0.05 vs. baseline sham.
p < 0.05 vs. Sham-vehicle.
p < 0.05 vs. Baseline OVX.
§
p < 0.05 vs. OVX vehicle.
KE ET AL.572
completely restored bone strength in the FS back to sham
control levels in established osteopenic, OVX rats.
DISCUSSION
This study clearly shows that a nonprostanoid EP4 re-
ceptor selective PGE
2
agonist stimulates bone formation on
trabecular, periosteal, and endocortical surfaces and inhib-
its bone resorption on trabecular and endocortical surfaces
in OVX rats with established osteopenia. In addition, this
agonist also activates intracortical bone remodeling on the
inner third of cortex. These surface specific effects led to a
complete restoration of trabecular and cortical bone mass
and bone strength. At the higher doses, the EP4 agonist not
only restores but also adds extra bone to these OVX rats.
The common side effects associated with PGE
2
treatment
such as diarrhea and hair loss were not observed in the
OVX rats treated with this EP4 agonist. These data support
the hypothesis that the EP4 receptor plays an important
role in PGE
2
-induced bone anabolism and suggest that EP4
receptor agonists may have therapeutic potential for man-
agement of medical conditions with low bone mass such as
osteoporosis.
Although we did not have a PGE
2
group as a positive
control in this study, the bone anabolic effects observed for
CP432 were similar to those reported for PGE
2
.
(1013)
CP432 stimulates bone formation through increasing osteo-
blast recruitment (increased mineralizing surface) and ac-
tivity (increased mineral apposition rate) on periosteal, en-
docortical, and trabecular surfaces. For instance, CP432 at 3
mg/kg/day significantly increases mineralizing surface by
60%, 33%, and 140% and mineral apposition rate by 97%,
72%, and 70% on periosteal, endocortical, and trabecular
surfaces, respectively, compared with vehicle-treated OVX
controls. These effects led to new bone deposition on peri-
osteal surfaces (17% increase in total tissue area in tibial
shaft), endocortical surface (Fig. 3), and trabecular surfaces
(56% increase in trabecular bone volume). Histological ex-
amination showed that the increased formation mode of
bone modeling is the mechanism responsible for bone gain
on the periosteal surface because there was very minimal
bone resorption on this surface in 12-month-old female rats
that had been OVX for 9.5 months. On the other hand,
bone formation induced by CP432 was found on both
smooth and scalloped surfaces, indicating both formation
mode of bone modeling and remodeling-dependent bone
formation was activated on endocortical and trabecular sur-
faces. Furthermore, this EP4 agonist also stimulated new
bone formation in the marrow cavity of tibial and femoral
shafts (Figs. 3B) where there was little or no trabecular
bone in sham or OVX control rats, suggesting that the pre-
existing bone surface might not be required for EP4 agonist
induced bone formation. This bone anabolic action could
provide substantial benefits for a therapy that intends to use
for established severely osteoporotic patients because these
patients might have very minimal preexisting trabecular
bone surfaces. On intracortical surfaces where minimal
modeling and remodeling occurred in sham and OVX con-
trol rats, treatment with CP432 activated bone remodeling
in the inner third of the cortex where a very active bone
formation (labeling surface) was observed (Fig. 3B). Acti-
vating bone remodeling on intracortical surface may have
negative impact on cortical bone mass and strength. Our
data obtained from the cortical bone sites (DXA and pQCT
analysis of femoral shaft, histomorphometry analysis of
tibial shaft, and bone strength testing of the femoral shafts)
revealed that cortical BMC, area, and strength were signifi-
cantly increased in CP432-treated rats compared with ve-
hicle-treated controls. This indicates either there was no
negative impact, or, if there was negative impact, it was
overcome by dramatic increases in periosteal and endocor-
tical new bone deposition and marrow trabecular bone for-
FIG. 4. Maximal load, ultimate strength,
stiffness, and toughness of femoral shaft
from sham and OVX rats at the beginning of
treatment (B-Sham and B-OVX), sham rats
treated with vehicle (Sham), and OVX rats
treated with vehicle (OVX) or CP432 at 0.3,
1, and 3 mg/kg/day. Complete restoration of
bone strength was observed with CP432
treatment. Mean ± SE.
a
p < 0.05 vs. B-Sham;
b
p < 0.05 vs. Sham;
c
p < 0.05 vs. B-OVX;
d
p
< 0.05 vs. OVX.
EP4 AGONIST RESTORES BONE IN OVX RATS 573
mation. Similar findings were reported for human biosyn-
thetic PTH(1-34). PTH increased intracortical bone
remodeling accompanied by concurrent increases in bone
at the periosteal and endocortical surfaces. The combina-
tion of these effects resulted in an increase in cortical bone
strength in rabbit model.
(34)
It has been reported that PGE
2
stimulates both bone
resorption and formation in long bone culture in vitro,
(35)
and similar effects were observed in rat studies in vivo.
(10)
In this study, we found that bone resorption parameters on
both trabecular and endocortical surfaces were significantly
decreased after 6 weeks of treatment in OVX rats with
CP432. The differential effects on bone resorption between
PGE
2
and an EP4 receptor selective agonist may be caused
by the activity at the other receptor subtypes. For example,
activation of the EP2 receptor may be involved with bone
resorption activity associated with PGE
2
. The supporting
evidence for this hypothesis is that osteoclastogenesis was
impaired in EP2 receptor knockout mice.
(19)
Thus, activat-
ing the EP2 receptor may increase osteoclast formation as-
sociated with PGE
2
treatment. However, there is also evi-
dence suggesting that the EP4 receptor mediates PGE
2
-
induced bone resorption in mice.
(16,17,36)
Therefore, using
the results from genetic inactivation of EP subtypes in mice
may not explain PGE
2
s bone effects in rat models. An-
other explanation for this differential effect could be caused
by the duration of administration as shown by Jee and
Ma.
(10)
They found that bone resorption was increased for
a short period of time (the first 710 days) and returned to
normal or below normal associated with PGE
2
treatment in
rats. The study duration for this study was 6 weeks. It is not
known if bone resorption could have increased through ac-
tivation of bone remodeling on endocortical and trabecular
bone surfaces associated with EP4 agonist at the beginning
of the study. When rats were autopsied at week 6, most of
the resorption surface has been followed by bone forma-
tion. We did not measure the urinary and serum marker of
bone resorption in this study; thus, further investigation will
be needed to confirm the antiresorptive action of CP432. A
more detailed time-course study is required to determine
the effects of EP4 receptor agonists on bone resorption
during the transient and steady state. The direct effect of
this EP4 agonist on osteoclast formation, activation, or ap-
optosis in vitro has not been done and will require further
study.
The effects on BMD and bone strength of this nonpros-
tanoid EP4 receptor agonist, CP432, were similar to those
reported for a prostanoid EP4 receptor selective agonist,
ONO-4819, in OVX rats, although no detailed data regard-
ing its effect on osteoblast bone formation and osteoclast
bone resorption in OVX rats was reported for ONO-
4819.
(25)
However, in a rat immobilization model, ONO-
4819 significantly increased osteoid surface and osteoblast
surface,
(25)
which indicated its stimulatory effect on bone
formation in rats. The results from these two studies in rat
models, combined with a report that shows an EP4 receptor
selective antagonist can reverse PGE
2
-induced bone forma-
tion in rats,
(27)
reveal that the EP4 receptor is responsible
for PGE
2
-induced bone formation in rats.
In summary, we found that a newly discovered EP4 re-
ceptor selective agonist, CP432, stimulates bone formation
through increased osteoblast number and activity on peri-
osteal, endocortical, and trabecular surfaces and inhibits
bone resorption on endocortical and trabecular surfaces,
leading to new bone deposition on all these bone surfaces
with a complete restoration of bone mass and bone strength
in established osteopenic, aged OVX rats. At higher doses,
this EP4 agonist not only restores the lost bone mass and
strength but also adds extra bone to the rat skeleton. Our
data support the hypothesis that the EP4 receptor mediates
the bone anabolic effects of PGE
2
. The effects of this and
other EP4 agonists on other organs such as heart and kid-
ney and their detailed safety profile need to be studied.
CP432, an EP4 receptor selective PGE
2
agonist, induced
a positive remodeling on trabecular and endocortical sur-
faces and a renewed modeling on trabecular, endocortical,
and periosteal surfaces. Therefore, it clearly belongs to the
classification of anabolic drug proposed by Riggs and
Parfitt,
(37)
if its surface-specific action and efficacy can be
confirmed in osteoporotic patients.
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Address reprint requests to:
Hua Zhu Ke, MD
Amgen Inc.
One Amgen Center Drive
Mail Stop 29-M-A
Thousand Oaks, CA 91320, USA
E-mail: hke@amgen.com
Received in original form June 30, 2005; revised form September
21, 2005; accepted November 29, 2005.
EP4 AGONIST RESTORES BONE IN OVX RATS 575
    • "EP2 and EP4 receptor knockout mice have been shown to have impaired fracture healing and impaired bone resorption.(18,19) An EP2-selective agonist induced bone healing in beagles(20); similarly, an EP4-selective agonist has been used in a rat model of bone repair with positive effects.(21,22) Recent work in our laboratory showed that another EP4-selective agonist accelerated the delayed fracture healing in aged mice and compensated for the reduced fracture healing observed in COX-2−/− mice.(23,24) "
    [Show abstract] [Hide abstract] ABSTRACT: As a downstream product of cyclooxygenase 2 (COX-2), prostaglandin E(2) (PGE(2)) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro-computed tomographic (µCT), and biomechanical measures. EP1(-/-) mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1(-/-) mice also had an earlier appearance of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1(-/-) mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE(2) receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing.
    Full-text · Article · Apr 2011
    • "CP-533,536, a nonprostanoid PGE2 E2 receptor agonist, was found to stimulate new bone formation on trabecular, endocortical, and periosteal surfaces in intact rat bones and to increase callus size, density, and strength after fracture [48]. When administered subcutaneously, CP432, a PGE2 E4 receptor agonist, was found to stimulate bone formation through increased osteoblast recruitment and activity on periosteal, endocortical, and trabecular surfaces in OVX rats [42]. These anabolic effects of CP432 were found on both smooth and scalloped endocortical and trabecular surfaces, indicating both bone modeling-and remodeling-dependent bone formation were activated. "
    [Show abstract] [Hide abstract] ABSTRACT: The definition of bone quality is evolving particularly from the perspective of anabolic agents that can enhance not only bone mineral density but also bone microarchitecture, composition, morphology, amount of microdamage, and remodeling dynamics. This review summarizes the molecular pathways and physiologic effects of current and potential anabolic drugs. From a MEDLINE search (1996-2010), articles were identified by the search terms "bone quality" (1851 articles), "anabolic agent" (5044 articles), "PTH or parathyroid hormone" (32,229 articles), "strontium" or "strontium ranelate" (283 articles), "prostaglandin" (77,539 articles), and "statin" or "statins" (14,233 articles). The search strategy included combining each with the phrase "bone quality." Another more limited search aimed at finding more novel potential agents. Parathyroid hormone is the only US Food and Drug Administration-approved bone anabolic agent in the United States and has been the most extensively studied in in vitro animal and human trials. Strontium ranelate is approved in Europe but has not undergone Food and Drug Administration trials in the United States. All the studies on prostaglandin agonists have used in vivo animal models and there are no human trials examining prostaglandin agonist effects. The advantages of statins include the long-established advantages and safety profile, but they are limited by their bioavailability in bone. Other potential pathways include proline-rich tyrosine kinase 2 (PYK2) and sclerostin (SOST) inhibition, among others. The ongoing research to enhance the anabolic potential of current agents, identify new agents, and develop better delivery systems will greatly enhance the management of bone quality-related injuries and diseases in the future.
    Article · Dec 2010
    • "However, ONO-4891 at the higher doses then that required for bone formation caused diarrhoea, hypotension and the thickening of intestinal epithelium in rodents. Another non-prostanoid EP4 receptor-selective PGE 2 agonist that has been characterised for its effects on bone is CP-734,432 [7]. It increases osteogenesis in vitro, as demonstrated by a dose-dependent increase in mineralised nodule formation in rat bone marrow cell cultures. "
    [Show abstract] [Hide abstract] ABSTRACT: Prostaglandins, PGE(2) in particular, have diverse actions on various organs, including inflammation, bone healing, bone formation, embryo implantation, induction of labour and vasodilatation, among others. However, systemic side effects have limited their clinical utility. The pharmacological activities of PGE(2) are mediated through four G protein-coupled receptor subtypes, EP1-EP4. Recent studies have shown that EP2 and EP4 receptors play important roles in regulating bone formation and resorption. EP2 and EP4 receptor-selective agonists have been shown to stimulate local or systemic bone formation, augment bone mass and accelerate the healing of fractures or bone defects in animal models upon local or systemic administration, thus, potentially offering new therapeutic options for enhancing bone formation and bone repair in humans. This review will focus on the studies related to bone formation and bone healing in the EP receptor knockout (KO) mice and the EP2 or EP4 receptor-selective agonist treated animal models.
    Full-text · Article · Jan 2008
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