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Vol. 302, Issue 1, 205-211, July 2002
Departments of Musculoskeletal Diseases (S.J.H., J.V.-M., M.W.L., M.G., G.S.) and Medicinal Chemistry (W.H.M.), GlaxoSmithKline, King of Prussia, Pennsylvania
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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An excess of thyroid hormone results in increased bone turnover and
loss of bone mass in humans. Exogenous administration of thyroid
hormone to rats has served as a model of human hyperthyroidism in which
antiresorptive therapies have been tested. We have further refined this
model of thyroxine (T4)-induced turnover in the rat. Daily
administration of T4 to aged rats for as short as 1 week resulted in
elevated bone resorption determined by significantly higher urinary
deoxypyridinoline (Dpd) compared with vehicle controls or animals
receiving T4 plus estradiol. Three weeks of daily administration of T4
led to significantly lower bone mineral density compared with untreated
controls or animals receiving T4 plus estradiol. In a follow-up study,
a depot formulation of T4 caused an increase in Dpd identical to that
achieved with a bolus dose. SB-273005 [(4S)-2,3,4,5-tetrahydro-8-[2-[6-(methylamino)-2-pyridinyl] ethoxy]-3-oxo-2-(2,2,2-trifluoroethyl)-1H-2-benzazepine-4- acetic
acid] a potent antagonist of the integrins
v
3 and v5, has been shown previously to inhibit bone resorption in cultures of
human osteoclasts and to protect bone in ovariectomized rats. The
effect of SB-273005 by oral administration was evaluated in this
thyroxine-induced turnover model. Dose-dependent inhibition of
resorption was seen with SB-273005 after 7 days of dosing using Dpd as
a measure of bone resorption. In summary, it has been demonstrated that
the antiresorptive activity of a vitronectin receptor antagonist can be
measured after only 7 days of treatment in this refined rat model of
thyroxine-induced bone turnover. These data suggest that SB-273005 may
be useful for the treatment of metabolic bone diseases, including those
resulting from hyperthyroidism.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Studies have demonstrated that
hyperthyroidism, whether endogenous or exogenous, is associated with
decreased bone density at various skeletal sites and increased risk of
bone fracture (Paul et al., 1988
; Kung et al., 1993; Wejda et al.,
1995). Using histomorphometry, it has been shown that the bone loss is
attributed to an increase in bone turnover associated with a larger
increase in bone resorption than bone formation (Meunier et al., 1972; Perry, 1989; Mosekilde et al., 1990). Studies in the rat have supported
this hypothesis (Eriksen et al., 1985; Yamamoto et al., 1993b).
Biochemical markers of bone turnover have also been shown to be
increased in the rat (Harvey et al., 1991; Kung and Ng, 1994;
Taimela et al., 1994; Ishihara et al., 1997). Taken together, these
results suggest that patients with hyperthyroidism could potentially
benefit from therapy that prevents the increased bone turnover and bone loss.
For bone resorption to occur, osteoclasts must first adhere to the bone
matrix. This adhesive event is mediated by the interaction of the
osteoclast vitronectin receptor
(v3 integrin) with
the RGD tripeptide sequence present in several bone matrix proteins (Clover et al., 1992; Helfrich et al., 1992; Nesbitt et al.,
1993; Shinar et al., 1993). Disruption of this interaction
results in inhibition of resorption both in vitro and in vivo (Fisher
et al., 1993; Yamamoto et al., 1993b, 1998; Crippes et al., 1996). Unlike v3, which is
expressed on mature osteoclasts,
v5 is expressed on
osteoclast precursors and has been proposed to play a role in
osteoclast differentiation (Sago et al., 1999). SB-273005, a
potent antagonist of both of these integrins, inhibits bone resorption
in cultures of human osteoclasts with an IC50 of
11 nM (Lark et al., 2001). This compound also inhibits bone resorption after oral administration in the thyroparathyroidectomized rat and prevents bone loss in ovariectomized (OVX) rats (Lark et al., 2001).
The present study had two purposes: to establish a short-term model of thyroid-induced osteopenia in the rat and to evaluate the effect of a vitronectin receptor antagonist, SB-273005, in this high turnover model. For the latter, the model was shortened from the standard 21 days previously used by most investigators to 7 days through the use of biochemical markers of bone turnover. Rats were made thyrotoxic by exogenous administration of L-thyroxine (T4), and bone turnover was elevated. A depot formulation of T4 was employed to eliminate daily dosing of T4. We then evaluated the effect of SB-273005 given concomitantly with T4 on biochemical markers of bone turnover.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Model Development. All procedures were approved by the Animal Care and Use Committee of GlaxoSmithKline, and animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Eight-month-old virgin female Sprague-Dawley rats (Charles River Laboratories, Raleigh, NC) were used for all three experimental protocols. The animals were randomly sorted into groups of eight based on body weight.
The first protocol was designed to investigate the effects of thyroid hormone on bone markers and bone mineral density (BMD) in rats. For this experiment, we followed a method published previously (Yamamoto et al., 1993b). All animals were dosed subcutaneously once daily for 24 days starting at day 0 with either vehicle (0.003 N NaOH in saline), T4
(Sigma-Aldrich, St. Louis, MO) bolus at 250 µg/kg in vehicle,
or T4 plus estradiol (2.5 mg, 90-day time-release subcutaneous pellet
of 17-estradiol was purchased from Innovative Research of America,
Sarasota, FL). Blood was collected in the morning on days 
4 and 18 for the analysis of osteocalcin. Additional blood samples were
collected prior to dosing and at 0.5, 2, 8, and 24 h
postadministration from the T4 control group for the determination of
triiodothyronine (T3) and T4 levels on days 0 and 18. Peripheral
quantitative computed tomography (pQCT) was performed on six rats per
group, 1 week prior to treatment and just prior to termination of the
dose period. Twenty-four-hour urine samples were collected on days 5,
7, 14, and 21. On day 24, the rats were euthanized by
CO2 inhalation.
A second experiment was performed to determine the appropriate dose of
subcutaneous depot T4 that would be required to achieve the same
elevation in bone turnover that was achieved with the T4 bolus. The
animals were dosed subcutaneously for 17 days starting at day 0 with
one of the following: vehicle (0.003 N NaOH in saline); T4 bolus at 250 µg/kg/day in vehicle; or T4 (21-day subcutaneously implanted
time-release pellet at a dose of 25, 50, or 100 mg obtained from
Innovative Research of America). T3 and T4 were measured in blood
collected at the same time each day on days 1, 3, 7, and 14 from all
animals receiving T4 by pellet. Urine was collected on days 8, 7, and
14. Additional blood samples were collected in the morning on days 1
and 17 for the determination of osteocalcin. pQCT scans were performed
3 days prior to treatment and 1 day before termination.
Effect of Vitronectin Receptor Antagonist, SB-273005, on Bone
Resorption in T4-Induced Turnover Model in the Rat.
A third
experiment was performed to evaluate the ability of the osteoclast
vitronectin receptor antagonist, SB-273005, to inhibit the T4-induced
increase in bone turnover. The animals were divided into five groups: a
vehicle group (oral 1% methylcellulose, pH 3.5; Sigma-Aldrich) and
four groups that received a 50-mg T4 pellet (subcutaneously implanted
as in protocol 2). The four T4 groups were also treated orally once
daily as follows: a vehicle group or SB-273005 at 3, 10, or 30 mg/kg in
vehicle. The animals were treated for 8 days starting on day 0. Urine
was collected on days 1 and 7. Blood for the determination of
osteocalcin levels was collected on day 8.
Compound.
SB-273005 was synthesized in the Department of
Medicinal Chemistry at GlaxoSmithKline (Miller et al., 2000).
Biochemical Analyses. Urinary deoxypyridinoline (Dpd) was analyzed by enzyme-linked immunosorbent assay (Pyrilinks-D; Quidel, Santa Clara, CA). Urinary creatinine was measured using a Monarch 2000 analyzer (Instrumentation Laboratory Co., Lexington, MA). Serum osteocalcin was analyzed by radioimmunoassay (Biomedical Technologies, Stoughton, MA). Serum T3 and T4 were measured by enzyme-linked immunosorbent assay (Trinity Biotech USA, Jamestown, NY).
Bone Mineral Density Measurements. Volumetric BMD was determined by pQCT using the Stratec/Norland Research M (Orthometrix Inc., White Plains, NY). Quality control of the instrument was carried out each day prior to and after sample analysis by scanning a cone phantom of known density. Scans were made in vivo of the left proximal tibia. Animals were anesthetized with isoflurane inhalant. A three-dimensional, 0.5-mm slice was taken through the proximal tibial metaphysis at a point 15% of the length between the tibia-fibula junctions and closer to the knee. Settings for the mask were as follows: object length, 200 mm; voxel size, 0.1 mm; diameter, 40 mm; speed, 3 mm/sec; number of blocks, 2; scout view speed, 30 mm/sec; and scout view distance between lines, 0.5 mm. BMD, bone mineral content, and cross-sectional area were determined for the total, trabecular, subcortical, and cortical regions. Analysis was as follows: Calcbd (density and area calculations for the trabecular, total, and subcortical regions) was set at contour mode 2, peel mode 2, inner threshold of 800 mg/cm3, and Cortbd (cortical bone density and area determinations) was set at separation mode 2 with a threshold of 800 mg/cm3.
Statistical Analysis. Osteocalcin (except for protocol 3), Dpd, and BMD data were analyzed by repeated measures analysis of variance (ANOVA) (Statistica version 5.1; Statsoft Inc., Tulsa, OK). For these data, we compared the change from baseline to the final time point between treated groups and the appropriate control. In addition, two other statistical tests were performed on the urinary Dpd data from protocol 3: a linear trends test and a simple ANOVA using SAS (version 8.01; SAS Institute, Inc., Cary, NC). All other data were analyzed by Student's t test (Microsoft Excel; Microsoft, Redmond, WA).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animal Health. The animals in all three experiments appeared in good general health. T4-treated animals, however, did lose a significant (p < 0.05) amount of body weight over the course of all three studies compared with vehicle controls (approximately 9, 7, and 5% for studies 1, 2, and 3, respectively). In addition, approximately 20% of the 50-mg T4 pellet group and nearly all of the 100-mg T4 pellet group exhibited edema around the site of the T4 pellet.
Model Development.
To verify and extend previously reported
results, we evaluated daily bolus injection of T4 in intact rats. Bolus
administration of T4 on day 0 resulted in a rapid rise in serum T4
levels that remained elevated for at least 24 h (Fig.
1). Subsequently, a rise in T3 levels was
observed, which remained elevated from at least 0.5 to 8 h after
T4 administration. On day 18, levels for both T3 and T4 were similar to
day 0 levels (data not shown).
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Effect of Vitronectin Receptor Antagonist, SB-273005, on Bone Resorption in T4-Induced Turnover Model in the Rat. As shown above, the increased bone turnover and level of bone loss resulting from treatment with the 50-mg pellet of T4 were comparable to those following T4 daily bolus injection. Furthermore, in both experiments, the elevation of bone resorption after 7 days of treatment resulted in measurable loss of trabecular BMD at later time points. Based on these data, the effect of a vitronectin receptor antagonist, SB-273005, on bone turnover was evaluated in this system. To do this, SB-273005 was coadministered with T4 for a period of 8 days, and biochemical markers of bone turnover were evaluated.
Baseline Dpd levels were not significantly different from one another (ANOVA). On day 7, a doubling of Dpd levels over vehicle controls was observed with a 50-mg T4 pellet (Fig. 7A). Coadministration of SB-273005 with T4 decreased Dpd levels dose dependently with reductions of 26, 40, and 68% for the 3, 10, and 30 mg/kg oral doses, respectively. This dose dependence was verified through a linear trends analysis of day 7 raw data. A probability value of 0.0007 for all three doses and a probability value of 0.0277 for the two lowest doses were observed. In addition, repeated measures analysis of the baseline and day 7 data confirmed significant inhibition by SB-273005 at the 30 mg/kg dose (p < 0.01). The 50-mg T4 pellet increased serum osteocalcin levels over vehicle controls on day 8 (Fig. 7B). SB-273005 had no significant effect on serum osteocalcin levels.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Treatment of rats with excess thyroid hormone for 3 weeks produced
increased T3 levels. This led to increases in urinary Dpd (resorption)
and serum osteocalcin (osteoblast activity) and, subsequently, a loss
of trabecular bone in the proximal tibia. Cotreatment of T4 and
estradiol prevented the increased turnover and bone loss associated
with levothyroxine therapy. The marker changes were observed as early
as 7 days and were predictive of bone mass changes at 3 weeks. Taken
together, these data show that bone markers are an excellent predictor
of changes in bone mass. These data are in agreement with clinical data
showing the predictive value of markers in hyperthyroidism (Siddiqi et
al., 1997) and in osteoporosis following treatment (Ravin et al., 1999; Delmas et al., 2000).
A second study demonstrated that a 50-mg T4 pellet produced a response in bone over 2 weeks similar to that with the T4 bolus. The use of the pellets is a convenience that eliminates daily dosing of the T4. The two lower dose pellets were very consistent in the day-to-day systemic levels of T3 that were produced.
Having confirmed that excess thyroid hormone in rats induces high bone turnover and cancellous bone loss, the antiresorptive effect of the osteoclast vitronectin receptor antagonist, SB-273005, on T4-induced turnover changes was examined. The observation that SB-273005 inhibited the increase in Dpd associated with T4 administration suggests that the compound inhibited bone resorption. No difference was seen in serum osteocalcin levels by SB-273005, indicating that the compound had no effect on mature osteoblast function during this short duration of treatment.
Previous reports (Ongphiphadhanakul et al., 1993; Kung and Ng,
1994; Ishihara et al., 1997) have shown that excess thyroid hormone in
rats causes increased levels of the bone formation markers, osteocalcin
and alkaline phosphatase, and the resorption markers,
tartrate-resistant acid phosphatase, pyridinoline, and Dpd.
Ongphiphadhanakul et al. (1993) have shown that after 3 weeks of
treatment in male hyperthyroid rats with a bisphosphonate, femoral mRNA
levels of tartrate-resistant acid phosphatase and alkaline phosphatase
were reduced compared with T4 controls. The current study is, however,
the first report of inhibition in as little as 7 days of a bone
resorption marker by an antiresorptive agent. Such a short-term model
is an advance over previous study designs with respect to evaluation of
antiresorptive therapy.
The efficacy of SB-273005 in the prevention of bone loss has been
observed in several animal models. SB-273005 reduced resorption by
inhibiting the parathyroid hormone-stimulated calcemic response of
hypocalcemic thyroparathyroidectomized rats, and in the OVX rat
SB-273005 reduced the bone resorption marker, Dpd, and prevented bone
loss in the lumbar vertebrae (Lark et al., 2001). In a study by
Engleman et al. (1997), a peptide mimetic of the vitronectin receptor
was administered intravenously in the rat, and inhibited the
OVX-induced increase in urinary pyridinyl cross-links and prevented
trabecular bone loss. In both of these studies, an inhibition of
OVX-induced bone loss was preceded by an inhibition of a marker of
osteoclastic bone resorption. Therefore, if the current study was
extended beyond 8 days, it is expected that the reduction in Dpd would
lead to a reduction in the loss of bone mass.
Treatment of thyrotoxicosis in humans restores bone metabolism to
normal (Macleod et al., 1993) and leads to an increase in BMD (Rosen
and Adler, 1992). However, several clinical studies have shown that
despite effective treatment for hyperthyroidism, bone loss recovery may
be incomplete (Toh et al., 1985; Mosekilde et al., 1990; Franklyn et
al., 1994; Lupoli et al., 1996). Since reduction in BMD is a risk
factor for subsequent bone fracture, antiresorptive therapy may prove beneficial.
Other antiresorptive therapies have been tested in hyperthyroid
patients with mixed success. In a study that supports the effect of
estradiol observed here, Franklyn et al. (1995) reported that estrogen
replacement therapy abolished the reduction in femoral and vertebral
BMD in postmenopausal women with previous thyrotoxicosis and subsequent
T4 therapy. In studies by Kung and Yeung (1996) and Jodar et al.
(1997), intranasal calcitonin provided no additional benefit over
calcium or attainment of a euthyroid state, respectively. However,
alendronate, a bisphosphonate, increased BMD and decreased serum
osteocalcin levels in pre- and postmenopausal hyperthyroid women
treated with methimazole, an antithyroid agent (Lupoli et al., 1996).
In conclusion, we have established a short-term model of thyroid
hormone-induced osteopenia. The resorption inhibitors, estradiol and
the v3 and
v5 antagonist
SB-273005, were able to inhibit T4-induced turnover. Although the
present study did not examine whether the vitronectin receptor
antagonist, SB-273005, could restore the established bone loss in a rat
model of hyperthyroidism, the findings indicate that a vitronectin
receptor antagonist may be of potential benefit for thyrotoxic patients
with high bone turnover osteoporosis.
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Acknowledgments |
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We thank Dr. Aili Cheng for the statistical evaluation of the linear trends test and the ANOVA test concerning SB-273005 and Dpd levels.
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Footnotes |
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Accepted for publication February 28, 2002.
Received for publication October 10, 2001.
Address correspondence to: Dr. Sandra J. Hoffman, GlaxoSmithKline, Mail Code UW2109, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406. E-mail: Sandra_J_Hoffman{at}gsk.com
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Abbreviations |
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SB-273005, (4S)-2,3,4,5-tetrahydro-8-[2-[6-(methylamino)-2-pyridinyl]ethoxy]-3-oxo-2-(2,2,2-trifluoroethyl)-1H-2-benzazepine4acetic acid; OVX, ovariectomized; BMD, bone mineral density; T4, thyroxine; T3, triiodothyronine; pQCT, peripheral quantitative computed tomography; Dpd, deoxypyridinoline; ANOVA, analysis of variance.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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3 integrin inhibits osteoclast-mediated bone resorption in the thyroparathyroidectomized rat.
Endocrinology
137:
918-924[Abstract].v3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo.
J Clin Investig
99:
2284-2292[Medline].v5 is expressed on avian osteoclast precursors and regulated by retinoic acid.
J Bone Miner Res
14:
32-38[CrossRef][Medline].This article has been cited by other articles:
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) and T3 (
) following
subcutaneous injection of T4 (250 µg/kg) on day 0. Bleed times were
predose, and 0.5, 2, 8, and 24 h postadministration. Data are
presented as mean ± S.E. of eight animals per group.
) on urinary Dpd (A) and serum osteocalcin
(B) in rats. The animals received T4 (250 µg/kg) or vehicle once
daily by subcutaneous injection for 24 days. 17
, p < 0.01, 
) over a 21-day period on serum T4
(A) and T3 (B) in rats. The animals received vehicle once daily by
subcutaneous injection for 17 days. Animals were bled the same time of
day on all sample days. Data presented are mean ± S.E. of eight
animals per group. 
) over a 21-day period
on urinary Dpd (A) and serum osteocalcin (B) in rats. The animals
received T4 bolus (250 µg/kg) or vehicle once daily by subcutaneous
injection for 17 days. The baseline levels of Dpd/creatinine for the
groups were: vehicle, 0.23 ± 0.03 nmol/mg; T4 bolus, 0.19 ± 0.03 nmol/mg; and T4 pellets, 0.17 ± 0.02, 0.19 ± 0.02, and
0.17 ± 0.02 nmol/mg for the 25-, 50-, and 100-mg pellets,
respectively. The serum for osteocalcin levels was collected on days
