This study examined the effects of a sclerostin antibody (Scl-AbIII) alone and in combination with raloxifene on bone formation markers in ovariectomized (OVX) rats. Forty-five female rats were divided into five groups: a sham-operated control group and four OVX groups treated with saline, Scl-AbIII alone, raloxifene alone, or Scl-AbIII and raloxifene combined. Treatment for four weeks with either Scl-AbIII or raloxifene increased bone formation markers, while combination therapy led to greater increases than either treatment alone. The combination therapy showed enhanced effects on bone formation over single agent treatments

1. 46 Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (1): 46-50
INTRODUCTION
Osteoporosis is accepted as a major public health
problem characterized by compromised bone strength,
micro-architectural deterioration of bone tissue and
increased fracture risk with an important socio-economic
burden.1 It is a multifactorial disease, but the most
common form is involutional osteoporosis, which is
associated with advancing age and menopause. Along
with efforts to develop improved antiresorptive agents,
there has been a long-standing goal to develop
therapeutics that can stimulate bone formation, to
increase bone mass, and strength for low bone mass
conditions (e.g. osteoporosis).2
Wnt proteins are signaling molecules that regulate
osteoblasts differentiation into mature osteoblasts. The
binding of Wnt proteins to their specific receptors leads
to stabilization of β-catenin, which translocates to the
nucleus and regulates gene expression. Sclerostin is a
natural Wnt antagonists secreted by osteoblasts/
osteocytes. Sclerostin antibodies (Scl-Abs) have been
shown to neutralize the inhibitory effects of sclerostin on
Wnt/b-catenin signaling and might be an attractive
approach for the development of a novel bone anabolic
agent.3
Estrogen (E2) is involved in the regulation of a number
of molecules that have an effect on osteoclasts. E2 limits
the size of preosteoclast population by apoptosis and
stimulating osteoblastic stromal cells to synthesize more
osteoprotegerine (OPG) that inhibits osteoclast
differentiation and bone resorption. E2 also stimulates
osteoblastic cells to make Transforming Growth Factor-
beta (TGF β) which restrains the expression of cathepsin
K, which is the molecular shovel that osteoclasts used to
dig holes in bone.4 The Selective Estrogen Receptor
Modulators (SERM), such as raloxifene, are non-
steroidal and synthetic class of compounds that
reproduce the positive effects of E2 in bone.5
Ovariectomized (OVX) rat models, mimic changes in
bone metabolism observed in postmenopausal
osteoporosis and have been most commonly used for
the efficacious studies of potential therapeutic agents for
the treatment of osteoporosis.6
The aim of this study was to determine the systemic
effect of sclerostin antibody administration on markers of
bone formation in rats made estrogen deficient (OVX)
and to compare this effect with that of raloxifene and a
combination of sclerostin antibody and raloxifene.
ORIGINAL ARTICLE
Effects of a Combination Therapy of Sclerostin Antibody III and
Raloxifene on Bone Formation Markers in Ovariectomized Rats
Hayam Ibrahim Gad Allam
ABSTRACT
Objective: To determine the systemic effect of sclerostin monoclonal antibody (Scl-AbIII) administration on markers of
bone formation and compare it with a combination of sclerostin antibody and raloxifene.
Study Design: Experimental study.
Place and Duration of Study: Medical College Animal House at King Khaled University Hospital, Riyadh, Saudi Arabia,
from January to November 2014.
Methodology: Forty-five female rats were divided into 5 groups equally; 1 control group and 4 groups of ovariectomized
(OVX) rats: control OVX rats and OVX rats treated by Scl-AbIII, raloxifene or Scl-AbIII+raloxifene one month after
ovariectomy, continued for 4 weeks. At the end of treatment, serum levels of Bone Specific Alkaline Phosphatase (BSAP),
alkaline phosphatase, osteocalcin, Insulin-like Growth Factor-1 (IGF-1), Parathyroid Hormone (PTH), Ca2+ and
phosphorus were measured. Uterus was weighed and body weight change was calculated.
Results: Scl-AbIII or raloxifene treatment produced significant increase of serum BSAP, osteocalcin, IGF-1, PTH and Ca2+
levels. Raloxifene, either alone or combined with Scl-AbIII attenuated the decrease in uterus wet weight, and the increase
in body weight seen in OVX rats. Combination therapy of Scl-AbIII, and raloxifene produced significant increase of serum
alkaline phosphatase, osteocalcin and IGF-1 levels than treatment with either Scl-AbIII or raloxifene alone.
Conclusion: Combination therapy of Scl-AbIII and raloxifene is an attractive strategy to enhance bone formation and can
offer better gain over treatment with either one of them alone. Confirmation of these preliminary observations must await
careful long-term studies.
Key Words: Osteoporosis. Sclerostin antibody. Raloxifene. Osteogenesis.
Department of Physiology, College of Medicine, King Saud
University, Riyadh, Saudi Arabia.
Correspondence: Dr. Hayam Ibrahim Gad, Physiology
Department, College of Medicine, King Saud University,
P.O. 2925(29), Riyadh 11461, Saudi Arabia.
E-mail: hayam_gad@hotmail.com
Received: January 23, 2015; Accepted: October 12, 2015.

2. METHODOLOGY
Forty-five female Wistar rats were housed at room
temperature (25±°C) and were allowed water ad libitum.
Their initial weight varied from 225 to 250 grams and
age from 19 to 20 weeks. The experiments were
conducted in accordance with Institutional Review Board
(IRB) at King Khaled University Hospital, Riyadh. The
rats were divided randomly into five groups equally.
Group I included sham operated (intact) control rats.
Group II, III, IV and V were OVX rats received saline,
sclerostin monoclonal antibody III (Scl-AbIII), raloxifene
or a combination therapy of both Scl-AbIII and
raloxifene, respectively.
Rats were anesthetized using ketamine hydrochloride
(120 mg/kg) and xylazine hydrochloride (24 mg/kg)
intramuscularly.7 The experiment for bilateral ovariec-
tomy was done through abdominal incision to expose
the uterus, oviducts and ovaries. The oviduct and blood
vessels supplying the ovaries were tied up, and the
ovaries were removed through an incision. The uterus
and oviducts were put back with the fat tissues around
them, and the incision was closed with suture. In sham
operated rats, an abdominal incision was performed
affecting skin and muscle and peritoneum. No organ
was extirpated or handled. The wound was subse-
quently sealed. The animals were randomized into
groups according to the above protocol.
Treatments of OVX rats were started one month post-
surgery. Twenty-five mg/kg of Scl-AbIII was injected
subcutaneous (S.C.) 2 times per week for 4 weeks.8 Scl-
AbIII stock solution was stored at -80°C. The dosing
solutions were prepared weekly by completely thawing
in a 4°C refrigerator overnight then diluting with saline to
a concentration of 5 mg/ml.
Raloxifene was purchased from Sigma Aldrich Co. and
administered orally at a daily dose of 5mg/kg, for 4
weeks.9 Body weight was measured at the start and at
the end of the experiment. Body weight change was
calculated for each rat.
At the end of the treatment, blood samples (by cardiac
puncture) were collected at 7:00 and 9:00 A.M.,
centrifuged and frozen within one hour, and stored under
identical conditions. The serum samples were assayed
for the levels of estradiol by competitive enzyme
immunoassay.10 Bone specific alkaline phosphatase
(BSAP) using immunoradiometric assay,11 alkaline
phosphates by colorimetric method,12 osteocalcin by
immunoassay,11 insulin like growth factor-1 (IGF-1) by
enzyme linked immunosorbent assay (IGF-1-ELISA
kit),13 parathyroid hormone (PTH) by enzyme amplifier
sensitivity immunoassay (EASIA),14 and calcium and
phosphorous by colorimetric method.15 Uteri were
excised from sacrificed animals and weighed after
trimming associated fat and expressing any luminal fluid.
Statistical analyses were carried out using the Statistical
Package for Social Sciences (SPSS Inc., Chicago, IL,
USA) program for windows version 21. Data were
expressed as mean ±SD. Statistical analysis was
performed by one-way analysis of variance (ANOVA)
with 95% confidence intervals followed by Tukey's
multiple comparison tests. Differences were considered
to be statistically significant at p < 0.05.
RESULTS
OVX-control rats had statistically significant decrease of
estradiol level in comparison with the sham-operated
controls. Neither Scl-AbIII nor raloxifene administration
affected the decrease in estradiol levels (Table I). Both
serum BSAP and alkaline phosphatase levels were
significantly decreased in control OVX rats as compared
to sham-operated controls and OVX rats treated with
Scl-AbIII, raloxifene or Scl-AbIII+Raloxifene (p < 0.001).
Scl-AbIII administration for four weeks increased bone
formation markers, as demonstrated by a 19.8%
increase in serum BSAP and a 24.8% increase in serum
alkaline phosphatase as compared to control OVX rats
(p < 0.001). Similar results were found after 4 weeks of
raloxifene treatment. Combined Scl-AbIII+raloxifene
treatment resulted in greater increase in serum BSAP
and alkaline phosphatase (42.2%, 44.5% respectively
Sclerostin antibody and bone formation markers
Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (1): 46-50 47
Table I: Mean Values ± SD of the measured parameters in controls (group I), ovariectomized rats (OVX) rats (group II), sclerostin antibody III
(Scl-AbIII)-treated OVX rats (group III), Raloxifene-treated OVX rats (group IV), and OVX rats treated with both Scl-AbIII and raloxifene
(group V).
Group I Group II Group III Group IV Group V p-value
Serum estradiol (pmol/l) 20.70 ±1.76 11.17 ±0.98* 11.06 ±0.94* 10.94 ±0.59* 10.91 ±0.71* 0.000
Serum alkaline phosphates (U/l) 351.18 ±43.48 254.18 ±26.77† 317.09 ±28.82 318.67 ±26.81 367.37 ±22.27§ 0.000
Serum osteocalcin (ng/ml) 3.86 ±0.30 2.61 ±0.27† 3.67 ±0.47 3.86 ±0.27 4.38 ±0.44§ 0.000
Serum insulin like growth factor-1 (ng/ml) 268.02 ±8.40 236.33 ±10.76† 263.55 ±6.83 266.42 ±9.66 280.16 ±6.70§§ 0.000
Serum parathyroid hormone (pg/ml) 34.62 ±2.17 26.99 ±3.19† 34.10 ±2.33 35.73 ±3.20 36.36 ±3.30 0.000
Serum calcium (mmol/l) 3.53 ±0.51 2.96 ±0.28†† 3.57 ±0.37 3.60 ±0.41 3.59 ±0.37 0.005
Serum phosphorous (mmol/l) 3.65 ±0.28 4.18 ±0.58‡ 3.49 ±0.29 3.81 ±0.31 3.69 ±0.33 0.005
Body weight changes (g) 76.07 ±2.52 99.61 ±2.50** 98.81 ±2.15** 74.58 ±3.45 72.73 ±3.36 0.000
Uterus wet weight (g) 536.44 ±15.31 128.33 ±4.21^ 129.44 ±4.25^ 198.33 ±4.36^¥ 199.11 ±4.96^¥ 0.000
Significance was considered at p<0.05, 95% confidence interval.
(*) p<0.001 versus group I; (†) p<0.001 versus groups I, III, IV and V; (§) p<0.01 versus group III, p< 0.05 versus group IV; (§§) versus groups I (p<0.05), III (p<0.01), IV (p<0.05);
(††) p<0.05 versus groups I, III, IV and V; (‡) versus groups I (p<0.05) and III (p<0.01); (**) p<0.001 versus groups I, IV and V; (^) p<0.001 versus group I
(¥) p<0.001 versus groups II, III.

3. higher as compared to control OVX rats, p < 0.001).
Moreover, Scl-AbIII+raloxifene-treated rats had higher
BSAP and alkaline phosphatase levels as compared to
rats treated by either Scl-AbIII or raloxifene alone
(Table I, Figure 1).
Control OVX rats had significantly lower serum
osteocalcin levels as compared to sham-operated
controls. Combined Scl-AbIII+raloxifene treatment
significantly increased osteocalcin level than treatment
with Scl-AbIII (p < 0.01) or raloxifene (p < 0.05) each
separately. The increases in osteocalcin levels were
67.8%, 47.9%, 40.6% higher in Scl-AbIII+raloxifene
(gp V), raloxifene (gp IV) and Scl-AbIII-treated rats
(gp III) respectively as compared to control OVX rats
(p < 0.001). No significant change could be detected
between Scl-AbIII-treated rats and raloxifene-treated
rats (Table I).
The decrease in serum level of IGF-1 was evident in
control OVX rats as compared to sham-operated
controls and OVX-treated rats by either Scl-AbIII or
raloxifene alone or combined. Combined treatment of
OVX rats with Scl-AbIII and raloxifene significantly
increased IGF-1 level as compared to sham-operated
controls (p < 0.05), OVX treated rats with Scl-AbIII
(p < 0.01) or raloxifene (p < 0.05) each separately
(Table I).
Control OVX rats showed significant decrease of serum
PTH and Ca2+ levels and significant increase of serum
phosphorous levels as compared to sham-operated
controls and OVX rats treated with either Scl-AbIII,
raloxifene or Scl-AbIII+raloxifene. PTH levels increased
significantly with Scl-AbIII (26.34%), raloxifene (32.38%)
or Scl-AbIII+raloxifene (34.72%) as compared to control
OVX rats (p < 0.001). Combined treatment of OVX rats
with Scl-AbIII+raloxifene did not affect the increase of
Ca2+ or the decrease of phosphorous levels achieved by
treatment of either of Scl-AbIII or raloxifene alone
(Table I).
After 4 weeks of observation, body weight increased in
all groups. However, when comparing the different study
groups, the weight gain of control OVX rats and those
rats treated with Scl-AbIII rats at the end of the study
period was more pronounced than that of sham-
operated controls (99.61 ±2.50, 98.81 ±2.15 vs. 76.07
±2.52 g, respectively). Raloxifene therapy, either alone
or combined with Scl-AbIII provided to OVX rats, kept
weight changes comparable with those seen in sham-
operated controls (74.58 ±3.45, 72.73 ±3.36 vs. 76.07
±2.52 g, respectively, Table I).
Bilateral ovariectomy induced a substantial decrease of
uterine wet weight in relation to the sham-operated
controls, expected results of a lowered E2 level, and
was 23.92% of the uterine weight of the intact control.
Although administration of Scl-AbIII to OVX rats did not
affect uterine wet weight; administration of raloxifene,
either alone or combined with Scl-AbIII, attenuated the
decrease of uterus mass in the OVX rats (36.97%,
37.12% respectively of the uterine weight of the sham-
operated controls (Table I).
DISCUSSION
The present study showed an increase in serum BSAP
and alkaline phosphatase after 4 weeks’ administration
of either Scl-AbIII or raloxifene. While, combined
treatment with Scl-AbIII and raloxifene resulted in more
increase in BSAP and serum alkaline phosphatase as
compared to non-treated OVX rats (p < 0.001).
The marked improvements in bone formation markers in
Scl-AbIII-treated OVX rats provide the evidence that
inhibition of sclerostin can enhance bone formation,
which may be directly related to the effects of reversing
sclerostin's inhibitory effect on osteoblast activity. The
mechanism by which sclerostin negatively regulates
bone formation is an area of continuing investigation.
One body of research supports the hypothesis that
sclerostin inhibits Wnt-β-catenin signaling by interacting
with Wnt coreceptors and thus impairing osteoblast
differentiation and function.16 Consistent with the
present findings is the human clinical study of Padhi
et al.17 who found that a single dose of a humanized
sclerostin monoclonal antibody in healthy men and
postmenopausal women resulted in a dose-dependent
increase in the concentrations of the bone-formation
markers (BSAP and osteocalcin) and decreased the
bone-resorption marker (serum C-telopeptide). In
addition, Tasci et al. found high serum alkaline
phosphatase in raloxifene treated osteoporotic rats.18
Serum levels of osteocalcin are regarded as sensitive
and specific marker of osteoblastic activity and the rate
of bone formation. Hence, the finding of the present
Hayam Ibrahim Gad Allam
48 Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (1): 46-50
Figure 1: Effect of sclerostin antibody III (Scl-AbIII) and raloxifene on serum
bone specific alkaline phosphatase in controls (group I), ovariectomized rats
(OVX) rats (group II), sclerostin antibody III (Scl-AbIII)-treated OVX rats
(group III), Raloxifene-treated OVX rats (group IV) and OVX rats treated with
both Scl-AbIII and raloxifene (group V). All values are represented as
mean±S.D. * = p<0.001 versus all other groups, £ = p<0/001 versus groups III
and IV.

4. work of reduced serum osteocalcin in OVX rats suggests
that reduced osteoblast activity may be responsible for
the osteoporosis. Evidence of a stimulatory effect of
Scl-AbIII or raloxifene on serum osteocalcin level was
apparent following their administration for 4 weeks.
Combined Scl-AbIII+raloxifene treatment was more
effective than Scl-AbIII treatment alone in improving
serum osteocalcin levels. This is in accordance with the
study of Li et al. who reported significant increase of
serum osteocalcin, recruitment and functional longevity
of osteoblasts after Scl-Ab treatment.6
Humans with inherited sclerostin deficiency have
increased bone mass and are resistant to fracture.19
However, the extent to which sclerostin might regulate
bone formation and bone mass in a normal aged
skeleton is not clear. The data of the current study
describes the powerful anabolic response to sclerostin
inhibition in OVX rats and clearly shows that sclerostin
functions as a pivotal negative regulator of bone
formation.
The evidence for an antiresorptive effect with Scl-Ab
administration was observed in ovariectomy-induced
bone loss rats6 and gonad-intact female monkey
models.8 Alaee et al. showed enhanced bone repair
following treatment of the rat femoral defect model with
Scl-Ab.20 Furthermore, their results showed significantly
44% higher osteocalcin levels at 2 weeks of Scl-Ab
treatment compared to the vehicle group. Tian
demonstrated significant increase of bone mass by Scl-
Ab treatment of immobilization/disuse rat model
compared with their controls.21 Moreover, Sinder et al.
reported that treatment of mouse model of osteogenesis
imperfecta for 5 weeks with Scl-Ab had increased bone
mass, cortical bone strength and induced bone
formation on surfaces that are normally quiescent or
resorptive in rapidly growing mice.22 Taken together,
these findings suggested that antibody-mediated
blockade of sclerostin represents a promising new
therapeutic approach for the anabolic treatment of
immobilization-induced osteopenia and a therapy during
fracture healing to improve bone formation.23
Several reports suggest modulating effect of raloxifene
on production of various factors involved in
osteoclastogenesis and osteoclast survival, resulting in
suppression of bone resorption.24 In this study, the
treatment combined with Scl-AbIII and raloxifene
significantly increased BSAP, osteocalcin and IGF-1
levels in comparison with Scl-AbIII treatment alone.
Raloxifene is a unique agent that apparently possesses
sufficient intrinsic activity to act like an agonist in bone
and liver, but is a relatively pure antagonist in uterine
tissue. In this study, raloxifene caused statistically
significant increase in uterine weight in relation to the
OVX controls. This effect may be related to the slight
hypertrophy of the myometrium and endometrial stroma,
which, in previous work, has been attributed to water
retention.25
CONCLUSION
Combination therapy of Scl-AbIII and raloxifene can
offer better gain over treatment with either one of them
alone. These results support the potential of Scl-AbIII
and raloxifene as attractive strategy to enhance bone
formation. Confirmation of these preliminary
observations must await careful long-term studies.
Acknowledgment: This research project was supported
by a grant from the “Research Center of the Female
Scientific and Medical Colleges”, Deanship of Scientific
Research, King Saud University.
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