Note: Descriptions are shown in the official language in which they were submitted.
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IN VITRO AND IN VIVO MODELS FOR SCREENING
COMPOUNDS TO PREVENT GLUCOCORTICOID-INDUCED BONE
DESTRUCTION
BACKGROUND OF THE INVENTION
Cross-Reference to Related Application
This application claims benefit of Ll.S. provisional application
60/105,805, filed October 27, 1998, now abandoned.
Field of the Invention
The present invention relates generally to bone
physiology. More specifically, the present invention relates to in
vitro and in vivo models for screening compounds to prevent
glucocorticoid-induced bone destruction.
Description of the Related Art
The adverse effects of hypercortisolism on bone have
been recognized for over 60 years (1), but the precise cellular and
molecular basis of these changes has remained elusive. Today, th a
iatrogenic form of the disease has become far more common than
Cushing's syndrome and glucocorticoid-induced osteoporosis is
now third in frequency following post-menopausal and senile
osteoporosis (2).
Bone loss due to glucocorticoid excess is diffuse,
affecting both cortical and cancellous bone, but has a predilection
for the axial skeleton. Spontaneous fractures of the vertebrae or
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ribs are, therefore, often presenting manifestations of the disorder
(3,4). A cardinal feature of glucocorticoid-induced osteoporosis is
decreased bone formation (5). In addition, patients receiving
long-term glucocorticoid therapy sometimes develop collapse of
the femoral head (osteonecrosis), but the mechanism underlying
this is uncertain (6). Decreased bone formation, and in situ death
of isolated segments of the proximal femur suggest that
glucocorticoid excess may alter the birth and death of bone cells.
Defective osteoblastogenesis has been reported to be linked to
reduced bone formation and age-related osteopenia in the SAMP6
mouse (7}. Besides the relationship between aberrant osteoblast
production and osteoporosis, it has been recently shown that a
significant proportion of osteoblasts undergo apoptosis (8), which
raises the possibility that the premature or more frequent
I S occurrence of osteoblast apoptosis could contribute to incomplete
repair of resorption cavities and loss of bone.
Thus, the prior art is deficient in compounds that
possess the advantageous properties of glucocorticoids, namely
anti-inflammatory properties, but do not cause bone loss or
osteoporosis. The present invention provides for methods of
screening compounds to fulfill this long-standing need in the art.
SUMMARY OF THE INVENTION
To demonstrate that glucocorticoid-induced bone
disease is due to changes in the birth or death rate of bone cells, a
murine model of glucocorticoid excess was used as well as bone
biopsy specimens obtained from patients with glucocorticoid-
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induced osteoporosis. This invention demonstrates that
glucocorticoid administration decreases bone formation rate and
bone mineral density accompanied by defective osteoblastogenesis
and osteoclastogenesis in the bone marrow and increases
apoptosis of mature osteoblasts and osteocytes.
One object of the present invention is to provide
methods to screen compounds that retain the anti-inflammatory
properties of glucocorticoids yet do not result in bone loss or -
osteoporosis due to apoptosis of osteoblasts and osteocytes.
In one embodiment of the present invention, there i s
provided a method of screening for compounds that reduce th a
bone deteriorating effects of glucocorticoids, comprising the steps
of: (a) contacting osteoblast and osteocyt.e cells with either a
glucocorticoid alone or a glucocorticoid in combination with a test
compound; and (b) comparing the number of cells undergoing
apoptosis following treatment with the glucocorticoid alone o r
following treatment with the glucocorticoid in combination with
the test compound; wherein a lower number of apoptotic cells
following treatment with the glucocorticoid in combination with
the test compound than with the glucocorticoid alone indicates
that the test compound reduces the bone deteriorating effects of
the glucocorticoid. This embodiment also includes the
aforementioned method, wherein the compound has little effect o n
the anti-inflammatory properties of the glucocorticoid, further
comprising the step of comparing the anti-inflammatory response
of the glucocorticoid in combination with the test compound to the
anti-inflammatory response of the glucocorticoid alone; wherein
essentially equivalent anti-inflammatory responses of th a
glucocorticoid alone and the glucocorticoid in combination with the
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test compound is indicates that the test compound both reduces
the bone deteriorating effects, while retaininD the anti-
inflammatory properties of the glucocorticoid; wherein said anti-
inflammatory response is determined by models of inflammation
selected from the group consisting of the adjuvant-induced
arthritis model and hindlimb inflammation model.
In another embodiment of the present invention, there
is provided a method of screening for glucocorticoid analogs that
possess decreased apoptotic properties towards osteoblast and
osteocyte cells, comprising the steps of: (a) contacting the cells
with either a glucocorticoid or a glucocorticoid analog; and (b)
comparing the number of apoptotic cells following treatment with
the glucocorticoid or the glucocorticoid analog, wherein a lower
number of apoptotic cells following treatment with th a
glucocorticoid analog than with the glucocorticoid indicates that
the glucocorticoid analog possesses decreased apoptotic properties
towards the cells. This embodiment also includes the
aforementioned method, wherein the glucocorticoid analog retains
anti-inflammatory properties, further comprising the step of: (c)
comparing the anti-inflammatory response of the glucocorticoid in
combination with a test compound to the anti-inflammatory
response of the glucocorticoid alone.. wherein essentially
equivalent anti-inflammatory responses of the glucocorticoid
alone and the glucocorticoid in combination with the to s t
compound is indicative of a glucocorticoid analog that possesses
decreased apoptotic properties while retaining anti-inflammatory
properties; wherein said anti-inflammatory response is
determined by models of inflammation selected from the group
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consisting of the adjuvant-induced arthritis model and hindlimb
inflammation model.
In yet another embodiment of the present invention,
there is provided a method of screening for compounds th a t
stimulate bone development, comprising the steps of: (a)
contacting osteoblast and osteocyte cells with either a
glucocorticoid or a test compound; and (b) comparing the n a m b er
of cells undergoing apoptosis following treatment with the
glucocorticoid or the test compound; wherein a lower number of
apoptotic cells following treatment with the test compound than
with the glucocorticoid indicates that the test compound
stimulates bone development.
In still yet another embodiment of the present
invention, there is provided a method of screening for compounds
that increase bone mineral density, comprising the steps of: (a)
contacting osteoblast and osteocyte cells with either a
glucocorticoid or a test compound; and (b) comparing the number
of cells undergoing apoptosis following treatment with the
glucocorticoid and the test compound; wherein a lower number of
apoptotic cells following treatment with the test compound than
with the glucocorticoid is indicative of a compound that increases
bone mineral density.
In the above-mentioned embodiments, contacting is
selected from the group consisting of in vitnn cell cultures and in
vivo murine animal model and determination of apoptosis is
selected from the group consisting of TLJNEL, DNA fragmentation
and immunohistochemical analysis.
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Other and further aspects, features, and advantages of
the present invention will be apparent from the following
description of the presently preferred embodiments of the
invention. These embodiments are given for the purpose of
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS _
So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions of the invention briefly summarized
above may be had by reference to certain embodiments thereof
which are illustrated in the appended drawings. These drawings
form a part of the specification. It is to be noted, however, that
the appended drawings illustrate preferred embodiments of the
invention and therefore are not to be considered limiting in their
scope.
Figure 1 shows photomicrographs of the effects of
prednisolone on murine vertebral cancellous bone. In panel A, is
a longitudinal, panoramic section from a mouse receiving placebo
and in panel B, a section from a mouse receiving prednisone. The
histomorphometric reading area is outlined. Toluidine blue stain,
original magnification X25.
Figure 2 shows quantification of CFU-OB and
osteoclast progenitors formed in ex vivo bone marrow cell
cultures. Marrow cells were obtained from the femurs of male
mice after 27 d of exposure to placebo (white bars) or 2.1
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mg/kg/d of prednisolone (black barsj. Cells from each mouse
were cultured separately.
Figure 3 shows the effect of prednisolone on murine
osteoblast apoptosis. Osteoblasts were counted in undecalcified
sections of cancellous bone from the vertebral secondary
spongiosa. In panel A, the placebo group is shown and in panel B,
the higher dose prednisolone group. Apoptotic cells in this
experiment were identified using TUNEL and morphometric
features such as nuclear fragmentation and condensation of
chromatin (arrows). Methyl green counterstain viewed with
Nomarski differential interference microscopy, original
magnification X400.
Figure 4 shows the effect of prednisolone on murine
osteocyte apoptosis. The cells were counted in undecalcified
sections of femoral metaphyseal cortical bone. In panel A, th a
placebo group is shown and in panel B, the higher dose
prednisolone group. Apoptotic osteocytes (arrowheads} are seen
in close proximity to normal cells. Methyl green counterstain
viewed with Nomarski differential interference microscopy,
original magnification X630.
Figure 5 shows the effect of chronic prednisone
treatment on apoptosis in human bone. TUNEL-positive
osteoblasts (arrowheads) and osteocytes (arrows) were absent
from normal subjects (Figure 5 A ) but were clearly identified i n
patients with prednisone-induced osteoporosis (Figure SB and
Figure SC). Approximately 5% of the osteocytes and 30% of the
osteoblasts were apoptotic. The photomicrographs are from
transiliac bone biopsy specimens. Methyl green counterstain
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viewed with Nomarski differential interference microscopy,
original magnification X630.
DETAILED DESCRIPTION OF THE INVENTION
Glucocorticoid-induced bone disease is characterized
by decreased bone formation and in situ death of isolated -
segments of bone (osteonecrosis) suggesting that glucocorticoid
excess, the third most common cause of osteoporosis, may affect
the birth or death rate of bone cells thus reducing their numbers.
To examine this, prednisolone was administered to 7-month-old
mice for 27 days and decreased bone density, serum osteocalcin
and cancellous bone area along with trabecular narrowing were
found. These changes were accompanied by diminished bone
formation and turnover, as determined by histomorphometric
analysis of tetracycline-labeled vertebrae, and impaired
osteoblastogenesis and osteoclastogenesis, as determined by ex
vivo bone marrow cell cultures. In addition, the mice exhibited a
3-fold increase in osteoblast apoptosis in vertebrae and showed
apoptosis in 28% of the osteocytes in metaphyseal cortical bone.
As in mice, an increase in osteoblast and osteocyte apoptosis w a s
documented in patients with glucocorticoid-induced osteoporosis.
Decreased production of osteoclasts explains the reduction in bone
turnover while decreased production and apoptosis of osteoblasts
would account for the decline in bone formation and trabecular
width. Furthermore, accumulation of apoptotic osteocytes m a y
contribute to osteonecrosis. These findings provide evidence that
glucocorticoid-induced bone disease arises from changes in the
numbers of bone cells.
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The present invention is directed towards methods of
screening compounds that retain the anti-inflammatory properties
of glucocorticoids while lacking the bone degeneration properties
associated with long-term administration due to apoptosis of
osteoblasts and osteocytes.
The present invention is further directed towards
methods of screening compounds that promote bone regeneration
by inhibiting the apoptosis of osteoblasts and osteocytes.
As used herein, the terms "glucocorticoid"
and
"glucocorticoid analog" is defined as
substances that bind to the
glucocorticoid receptor.
As used herein, the term ''apoptosis" refers
to
programmed cell death with nuclear fragmentation
and cell
shrinkage as detected by morphological criteria and Terminal
Uridine Deoxynucleotidal Transferase End Labeling (TUNEL)
Nick
staining.
As used herein, the terms "anti-inflammatory
response" or "anti-inflammatory propert~~'"refers to preventing
the induction of cytokines and other
events that lead to T cell
activation. Several models of inflammationare routinely used
in
the art, including the adjuvant-inducedarthritis model and
hindlimb inflammation model which are well known to those
having ordinary skill in this art (54,
55).
As used herein, the term "bone mineral density"
refers
to bone mass as defined by Dual-Energy X-Ray Absorbtiometry
(DEXA).
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The following examples are given for the purpose of
illustrating various embodiments of the invention and are not
meant to limit the present invention in any fashion:
EXAMPLE 1
~r imals
Male Swiss Webster mice (Charles River Laboratories,
Stone Ridge, NY) were electronically tagged (Biomedic Data System
Inc., Maywood, NJ) and kept in plastic cages (3-5 animals p a r
cage) under standard laboratory conditions with a 12 hr dark, 12
hr light cycle and a constant temperature of 20°C and humidity of
48%. All mice were fed on a standard rodent diet (Agway RMH
3000, Arlington Heights, IL) containing 22% protein, 5% fat, 5%
fiber, 6% ash, 3.5 Kcal/g, 1.0 IU vitamin D3/g, 0.97%o calcium and
0.85% phosphorus with water ad libituni. The animals and food
supply were weighed at one week intervals throughout the
experiment. Studies were approved by the UAMS Division of
Laboratory and Animal Medicine.
EXAMPLE 2
Glucocorticoid administration--experimental design
Bone mineral density (BMD) determinations were done
at two week intervals to identify the peak adult bone mass of the
mice, which was reached between 5 and 6 months-of-age (9).
Animals at peak bone mass were used to avoid obscuring the
negative impact of glucocorticoid excess on bone mineral density
by the confounding effects of increased linear and radial growth.
Before the experiment began, bone mineral density m a a s a r a m a n t s
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were repeated to allocate the animals into groups (n = 4 - 5) with
equivalent spinal density values. The mice (7-mo-old) received
placebo or prednisolone, a synthetic glucocorticoid. analog that
does not require hepatic hydroxylation and has minimal
mineralocorticoid activity, thus eliminating the need for potassium
supplementation or sodium restriction (10,11). Implantation of
pellets releasing 0.5 mg/kg/d of prednisolone (the no effect dose)
did not decrease bone mineral density. Therefore, two doses w ere
used, 0.7 mg/kg/d (lower dose) and 2.1 mg/kg/d (higher dose),
chosen from pilot studies to bracket the dose (1.4 mg/kg/d) that
invariably causes densitometric evidence of bone loss. These
doses were administered for 27 days by subcutaneous
implantation of slow-release pellets (Innovative Research of
America, Sarasota, FL). Bone mineral density measurements were
obtained at the beginning of the experiment and 27 days post-
implantation. For dynamic histomorphometric measurements,
tetracycline HCl (30 mg/kg body weight) was given
intraperitoneally 17 and 23 days post-implantation. After 2 7
days, the mice were sacrificed, serum and urine specimens were
taken, bone marrow aspirates were obtained from the right femur
for ex vivo marrow cell cultures and the left femur and lumbar
vertebrae were prepared for histomorphometric analysis. Livers
were examined for fatty infiltration as a sign of prednisolone
toxicity. The weight of the seminal vesicles (mg/100 g body
weight) was used as an index of the androgen status of the
animals (12). To help interpret these measurements, a separate
group of animals was orchidectomized (n = 5).
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EXAMPLE 3
Bone densitometry
Dual-energy X-ray absorptiometry (DEXA) was used to
determine global (whole body minus the head), spinal a n d
hindquarters bone mineral density in live mice (7,9). The scans
done at 27 days after pellet implantation were analyzed using the
'Compare' technique, in which the evaluation is based on the exact
positioning and region of interest placement of the baseline scan.
Accuracy of the DEXA measurements was demonstrated by the
strong linear relationship between ash weight and bone mineral
content at each region (7). Over the 18 months, the coefficient of
variation for the bone mineral density of a plastic-embedded
whole mouse skeleton was 3.0% (n = 146).
EXAMPLE 4
Serum and urine biochemical measurements
Serum osteocalcin was measured b y
radioimmunoassay using a goat anti-murine osteocalcin and
murine osteocalcin as tracer and standard (Biomedical
Technologies, Stoughton, MA). Urinary free deoxypyridinoline
excretion was determined by a microtiter competitive enzyme
immunoassay (Pyrilinks-D, Metra Biosystems, Mountain View, CA}
and was expressed as a ratio to the urinary creatinine.
EXAMPLE 5
Bone histomorphometric anal sis
The distal femora and lumbar vertebrae were fixed in
4°C Millonig's phosphate-buffered 10% formalin, pH 7.4,
embedded undecalcified in methyl methacrylate and stained
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(7,9,13). The histomorphometric examination was done with a
computer and digitizer tablet (OsteoMetrics Inc. Version 3.00,
Atlanta, GA) interfaced to a Zeiss Axioscope (Carl Zeiss, Inc.,
Thornwood, NY) with a drawing tube attachment. All cancellous
measurements were two-dimensional, confined to the secondary
spongiosa and made at X400 magnification (numerical aperture
0.75). The terminology and units used are those recommended b y
the Histomorphometry Nomenclature Committee of the American
Society for Bone and Mineral Research (14). The trabecular width
and osteoid width were measured directly. Trabecular spacing
and number were calculated (15). Only TRAPase-positive cells
were included in the osteoclast perimeter. The rate of bone
formation (pm'/p.m/d) and turnover (%/d) were calculated (7).
EXAMPLE 6
Detection and quantification of osteoblasts and osteoclasts in ex
vivo bone marrow cultures
One femur from each mouse was flushed with 5 ml of
phenol red-free aMEM (Gibco BRL, Gaithersburb, MD) containing
10% FBS (Hyclone, Logan, UT) to obtain marrow cells. After the
cells were rinsed and resuspended to obtain a single cell
suspension, the nucleated cell count was determined using a
Coulter Counter. Cells from each animal were cultured separately.
The number of colony-forming unit-fibroblast (CFU-F)
and CFU-osteoblast (CFU-OB) present in the bone marrow
preparations were determined (16-18). Briefly, cells were seeded
at 1.5 x 106 per 10 cm' well for the determination of CFU-F
number and maintained for 10 days in phenol red-free aMEM
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containing 15% preselected FBS, 50 p.M ascorbic acid and 10 mM (3-
glycerophosphate (Sigma Chemical Co, St. Louis, MO) with one-half
of the medium replaced after 5 days. After fixation in neutral
buffered formalin and staining with hematoxylin, colonies
containing a minimum of 20 fibroblastoid cells were enumerated.
Cells were seeded at 2.5 x 106 cells per 10 cm'- well for the
determination of CFU-OB number and cultured for 25-28 days as
described above for CFU-F. After fixation in 50% ethanol and 18%
formaldehyde, cultures were stained using Von Kossa's method to
visualize and enumerate colonies containing mineralized bone
matrix.
Osteoclast formation in bone marrow cultures was
assessed in replicate cultures (4-6 from each animal) maintained
for 9 days in the presence of aMEM, 10% FBS and 10 nM
1.25(OH)2D3 (7). Briefly, marrow cells were cultured at 1.5 x 106
per 2 cm'- well on 13 mm round Thermanox disks and maintained
for 8 days in the presence of 10% FBS in aMEM supplemented
with 10~g M 1.25{OH)ZD3 (provided by Dr. Milan Uskokovic,
Hoffman-LaRoche, Nutley, NJ). At the end of the experiment, cells
were processed for the autoradiographic detection of bound 'ZSI-
calcitonin ('25I-CT} and stained for tartrate-resistant acid
phosphatase. Because many osteoclasts in murine bane possess
only one nucleus (7), it is impossible to distinguish between
preosteoclasts and mononuclear osteoclasts in ex vivo cultures of
murine bone marrow cells. Therefore, mononucleated and
multinucleated cells that both bind ''-5I-CT and express TRAPase
were designated as osteoclastic cells. The number of osteoclasts
formed in this assay is a reflection of the number of osteoclast
progenitors present in the bone marrow aspirate and the n a m b a r
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of stromal/osteoblastic support cells that form during the culture
period.
The number of CFU-F colonies, CFU-OB colonies, a n d
osteoclastic cells formed from the marrow cells of each animal w a s
expressed as the number per femur, which was calculated b y
multiplying the number of colonies or osteoclasts obtained per 106
cells seeded at the initiation of the cultures by the total number of
marrow cells obtained from the animal.
EXAMPLE 7
Measurement of ap optosisin undecalcified sections
bone
Sections were mounted on silane-coated
glass slides
(Scientific Device Lab, c., Des Plains, deplasticized
In IL), and
incubated in 10 mM buffer, pH 7.6,
citrate in a microwave
oven a t
98C for 5 minutes.Slides were then incubatedwith 0.5% pepsin
for 30 minutes at 37C. Apoptotic cells detected by the
were
TUNEL reaction (transferase-mediated biotin-dUTP nick end-
labeling) using Klenow terminal deoxynucleotidyl transferase
(Oncor, Gaithersburg, MD) in sections counterstaine d with 1%
methyl green. The TUNEL reaction was noted within
cell nuclei
and the cells whose nuclei were clearly brown from the
peroxidase-labeled anti-digoxigenin antibody insteadof the blue-
green from the methyl green were interpreted as positive.
Plastic-embedded sections of weaned rat mammary tissue were
used as a positive control. Negative cantrols
were made b y
omitting the transferase. Morphological changes characteristic
of
apoptosis were examined carefully to minimize ambiguity
regarding the interpretation of results. With these precautions,
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TUNEL has been unequivocally associated with apoptosis (19). I n
addition, TUNEL has been used with DNA fragmentation a n d
immunohistochemical studies to demonstrate apoptosis of
osteoblastic cells and osteoblasts both in vitro and in vivo (8,20).
Apoptosis was also assessed in transiliac bone biopsy specimens
taken from two patients with glucocorticoid-induced osteoporosis
(22- and 36-yr-old, receiving 15 to 25 mg/d of prednisone for 3
to 6 yr) and from 12 age-, sex- and race-matched controls (13).
Two longitudinal sections were examined from each patient and
control subject. Osteoblasts were identified as cuboidal cells lining
the osteoid-covered trabecular perimeter (7,9,13). Osteocytes
were identified inside lacunae in mineralized bone.
EXAMPLE 8
Statistics
Differences in the bone densitometry values were
determined using the percentage change in BMD from baseline.
Dose response relations were tested by one-way ANOVA. To
further evaluate changes in bone histomorphometry, a Student's t
test was used to assess for significant differences between group
means, after testing for equivalence of variances and normal
distribution of data. The significance of the relative frequency of
apoptotic cells was determined with the x'- statistic. P values less
than 0.05 were considered significant (21 ).
2 5 EXAMPLE 9
Demonstration of bone loss in mice receiving-prednisolone
In mice implanted with the higher dose of
prednisolone, global and spinal BMD at 27 days were significantly
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lower than those found in the mice that were implanted with
placebo pellets (TABLE I). The decrease in global bone mineral
density was dose dependent (P <0.05). Demonstrating the
expected propensity for the axial skeleton, glucocorticoid-induced
loss of bone mineral density was less conspicuous at the
hindquarters. The levels of serum osteocalcin, a marker of
osteoblast activity, were decreased more than 50% w h a n
compared to placebo, while urinary deoxypyridinoline excretion
was not significantly different between the groups (TABLE I).
These effects were not due to changes in food intake, body weight
or androgen status (TABLE II). In addition, hepatic fatty
infiltration was absent.
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TABLE I
Bone Mineral Density (BMD~ and Serum and Urine Biochemical
Measurements in Prednisolone-treated Mice
Measurement lacebo 0.7 2.1 mg/kg,/d
mgLg/d
Global BMD (% change) -2.7 t 2.1 -5.0 2.2* -6.6 1.9t
Spinal BMD (% change) -3.1 t 3.0 -6.8 3.2 -8.7 3.5*
Hindquarters BMD
(% change) 0.4 10.4 -3.8 8.0 -3.4 6.9
Osteocalcin (p,g/L) 93.8 11.5 27.7* 46.4 13.8
63.0 t
Deoxypyridinoline
lu,M/mM creatinine) 78 3 + 9 3 63 14.7 81.5 11.3
6
+
Data shown are the mean ~ SD from 5-7 animals. *P <0.05 v s
placebo; 1'P < 0.005 vs placebo.
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TABLE II
Food Intake Bodv Weight and Seminal Vesicle Weight i n
Prednisolone-treated Mice
~Vleasurement Placebo 0.7 m /-~k~/d 2.1 mg/k~/d.
Food Intake (g/d) 3.4 ~ 0.6 3.6 ~ 0.2 3.7 t 0.4
Body Weight (g} 37.9 ~ 6.0 33.8 ~ 4.3 32.2 ~ 4.2
Seminal Vesicle Weight (mg/100 g
bodv wei Qhtl 74 6 + 14 6 92 7 + 8 7 83.1 ~ 6.9
Data=mean ~ SD. Seminal vesicle weight in a orchidectomized
control was 11.3 ~ 3.1 mg/100 g body weight, P < 0.001 vs treated
mice.)
EXAMPLE 10
l~ffects of glucocorticoid administration on vertebral bone
histomo,r~hometrv
Consistent with the bone mineral density results, i n
the animals receiving the higher dose, there was a 40~/o decline i n
the vertebral cancellous bone area and a 23% decline in trabecular
width (P <0.01) (TABLE III). In both prednisolone groups, there
was a trend towards increased trabecular spacing and there w a s
decreased trabecular number in the lower dose group indicating
that some trabecular profiles were entirely resorbed.
2~ In the higher dose group, osteoid area decreased b y
29%, osteoid perimeter by 34% and osteoid width by 27% (P
<0.01). A trend toward decreased osteoblast and osteoclast
perimeters was found in the animals receiving the higher dose.
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a a., a i v,~yy~~JJ9'
There was, however, a 3-fold increase in the empty erosion
cavities (devoid of osteoclasts) or reversal perimeter. The
tetracycline-based histomorphometry showed that prednisolone
administration caused a 26% decrease in the mineralizing
S perimeter (P <0.05). In addition, a dose-dependent decrease in
the mineral appositional rate was noted (P <0.05); this decline w a s
22% with the lower dose and 40% with the higher dose. _
Furthermore, there was a 53% decrease in the rate of bone
formation with the higher dose (P < 0.01 ), which correlated wi th
the vertebral cancellous bone area (r = 0.57, P <0.05), indicating
that the glucocorticoid-induced decreases in bone area were
associated with a reduction in the rate of bone formation. Bone
turnover, expressed as a percentage of the bone area per day, also
decreased in a dose-dependent manner (P <0.05).
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TABLE III
Vertebral Cancellous Bone Histomornhometrv in Swiss Webster
Mice After 27 Days of Prednisolone
Administration
Histomorphometric
Determination Placebo 0.7 mm~/kg/d2.1 m~/kg/d
Bone area/Tissue area(%) 10.4 1.4 6.9 2.1 6.3 1.7'~
Trabecular width (p.m ) 48.0 2.4 48.6 4.3 37.1
4.4~-
.
Trabecular spacing (p.m ) 423 69 712 546
125
302
Trabecular number (per mm) 1.44 0.47 1.77 0.33
1.66 O.b
Osteoid area/Bone area 2.1 0.2 2.2 0.8 1.5 0.2'~
(%)
Osteoid perimeter/Bone perimeter
(%) I5.1 2.1 15.8 5.1 9.9 1.1 ~'
Osteoid width (p.m ) 2.6 0.4 2.0 0.3 1.9 0.3*
Osteoblast perimeter/Bone perimeter
(%) 1.2 0.9 2.2 0.2 0.5 0.4
Osteoclast perimeter/Bone perimeter
(%) 2.7 1.1 2.6 0.5 1.1 1.7
Reversal perimeter/Bone perimeter
2.5 2.3 3.2 2.2 7.2 1.1~
Mineralizing perimeter/Bone perimeter
(%) 12.9 ~ 0.5 13 .9 ~ 5 .6 9.5 ~ 2.5
Mineral appositional rate
(p,m/d) 1.23 ~ 0.11 0.96 ~ 0.11* 0.74 ~ 0.20fi
Bone formation rate/Bone perimeter
(~,m2/(m/d) 0.15 ~ 0.020.13 ~ 0.04 0.07 ~ 0.03fi
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Bone turnover f %/d) 0.68 ~ 0.09 0.46 ~ 0.12* 0.24 ~ 0.11 t
Data shown are the mean ~ SD. There are 4-5 animals per group. *P
<0.05 vs. placebo; tP <0.01 vs. placebo.
EXAMPLE 11
Effects of alucocorticoid administration on osteoblastogenesis and
osteoclastogenesis
In bone marrow cell cultures from the animals
receiving the higher dose, there was no significant change in CFU-F
colonies ( 1250 t 374 vs. 698 ~ 104, NS). However, the number of
CFU-OB colonies decreased by 86% (375 ~ 257 SD vs. 54 ~ 14, P
<0.05) and the number of osteoclastic cells formed in response to
1.25(OH)ZD3 in ex vivo marrow cultures decreased by 65% (1387 ~
920 vs. 492 ~ 311, P <0.05) (Figure 2).
EXAMPLE 12
Effects of ~lucocorticoid administration on apoptosis
Counting a total of 973 osteoblasts, there was a 3-fold
increase in osteoblast apoptosis in the vertebral cancellous bone of
mice receiving the higher dose of prednisolone when compared to
controls (2.03% ~ 0.34 vs. 0.66% ~ 0.07, P <O.OS). Morphological
changes typical of apoptosis accompanied the TUNEL-positive
osteoblasts and included sharply defined, condensed chromatin
plastered against the nuclear membrane, nuclear fragmentation
and cell shrinkage (Figure 3A and 3B).
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In addition, prednisolone caused the appearance of
apoptotic osteocytes in cortical bone sections taken from femora
(Figure 4A and 4B). Whereas none of the osteocytes exhibited
apoptotic features in the control animals, 28% of 131 cortical
osteocytes were apoptotic in the animals receiving the higher
dose. Osteocyte apoptosis was restricted to small groups of cells i n
the center of the femoral metaphyseal cortex and were absent
from vertebral cortical bone. The apoptotic osteocytes were
identified in close proximity to normal osteocytes, in contrast to
the large homogenous areas of dead and dying cells typical of cell
necrosis. An increase in apoptotic hypertrophic chondrocytes a n d
bone marrow cells was also noted in mice receiving either dose of
prednisolone. Osteoclast apoptosis was not observed.
EXAMPLE 13
Demonstration of apoptotic osteoblasts and osteocvtes in patients
with ~l_ucocorticoid-induced osteoporosis
In transiliac bone biopsies taken from two patients,
TUNEL-positive osteoblasts and osteocytes were clearly identified
in both (Figure SB and SC} but were absent from specimens taken
from 12 age-, sex- and race-matched controls (Figure SA). As in
the murine model, bone histomorphometry from these two
patients showed the changes expected with chronic glucocorticoid
therapy (5): reduced cancellous bone area (11.1 and 8.8%, normal
is 22.4 ~ 1.2 SEM), decreased trabecular width (62 and lI8 Vim,
normal is 161 ~ 9), decreased osteoblast perimeter (2.1 and 2.3%,
normal is 7.6 ~ 0.4), decreased osteoclast perimeter (0 and 0.4%,
normal is 0.9 ~ 0.2), increased reversal perimeter ( 13.~ and 15.4%,
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normal is 6.9 ~ 0.7) and diminished bone formation rate (0.02 and
0.05 p.m'-/~.m/d, normal is 0.095 ~ 0.012). In the cancellous bone
of these specimens, approximately 5% of the osteocytes and 30% of
the osteoblasts were apoptotic. Apoptosis of osteoclasts or cortical
osteocytes was not observed. A transiliac bone biopsy represents
a much smaller sample of the human skeleton than the murine
femur and lumbar vertebrae represent of the mouse skeleton.
Therefore, it is not surprising that the percentage of apoptotic
osteoblasts and osteoclasts was different in the human and murine
specimens.
EXAMPLE 14
~arly effects on bone resorption
To directly establish whether glucocorticoids initially
accelerate bone resorption in the mouse, the vertebral cancellous
bone histology were examined in an additional group of somewhat
younger mice (5-mo-old) after 7 days administration of the higher
dose of prednisolone or placebo (n - 5). It ~ was found that
whereas prednisolone caused a 59% decrease in the osteoblast
perimeter (5.2% ~ 1.5 SD vs. 2.1 ~ l.l, 1? <0.005), the osteoclast
perimeter increased 96% (0.51 % ~ 0.34 vs. 1.00 ~ 0.41, P <0.05).
Summary
The choice of the mouse fox these studies was based o n
its validity as a model of the bone loss associated with loss of sex
steroids (9,22) and with senescence (7), but the mouse also h a s
several advantages over other animals (TABLE IV). In the mouse,
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glucocorticoid administration consistently induces axial, greater
than appendicular, bone loss without weight loss or
hypogonadism, accompanied by histological indices of impaired
osteoblast function, thus reproducing the major features of the
human disease (2-5). Although the doses used in the studies
described herein were higher in relation to body weight than in
humans, they were only mildly higher than the dose determined
by serial bone densitometry to have no effect and were consistent
with the much higher metabolic clearance of glucocorticoids a n d
other compounds in laboratory animals than in humans (35-37).
Nonetheless, the similarity of the glucocorticoid-induced increases
in apoptotic cells and bone histomorphometric features in mice
and humans indicates that the observations in the mouse are not
due to pharmacological differences.
I 5 The effects of glucocorticoids were examined after 2 7
days, a period equivalent in the mouse to about 3 to 4 years in
humans. Thus, these findings represent long-term, rather than
acute effects. Although a significant correlation was found
between the severity of the cancellous bone loss and the extent of
reduction in bone formation, several other lines of evidence imply
that some of the observed bone loss was due to an early increase
in bone resorption which had subsided by the time of
examination. First, there was suggestive evidence of complete loss
of some trabeculae (TABLE III). Second, based on the bone
turnover measured in the placebo group which must be close to
the rate found in all the animals at the beginning of the study,
even with total suppression of bone formation, the initial rate of
bone turnover could have accounted only for an exponential
decline in cancellous bone area of 18%, whereas a 40% decrease
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was observed. Finally, an early increase in osteoclast perimeter
was confirmed by histomorphometric examination of vertebral
cancellous bone after 7 days of prednisolone administration.
By 27 days of prednisolone administration, bone
resorption fell to, or below, normal, as indicated by the downward
trend in the osteoclast perimeter, normal urinary
deoxypyridinoline excretion and profound decrease i n
osteoclastogenesis. The persistent increase in erosion cavities
devoid of osteoclasts, measured as the reversal perimeter, merely
indicates delayed bone formation (38), and has been previously
observed in glucocorticoid-treated patients (5,39). Consequently,
the present invention emphasizes the relevant findings at 27 days
to chronic, rather than short-term, glucocorticoid administration to
humans.
Vertebral cancellous bone in adult mice undergoes
sequential, coupled bone remodeling that is qualitatively similar
to that occurring in human bone (7,9). Many of the changes i n
cellular, osteoid and tetracycline-based histological indices
induced by glucocorticoid administration can be accounted for b y
a reduction in the activation frequency of bone remodeling, the
main determinant of the rate of bone turnover (40), which is a n
inevitable consequence of the substantial decrease in
osteoclastogenesis that was observed. Although a reduction in
bone turnover will not by itself cause bone loss, the decrease in
trabecular width, which was the major structural change
observed. is usually the result of incomplete cavity repair. This is,
at least in part, due to inadequate osteoblast recruitment, either
from diminished production or ineffective migration to the bone
surface (40). The reduction in osteoblastogenesis was of sufficient
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magnitude to explain the decrease in bone formation rate, and
would also have contributed to the inadequate osteoblast
recruitment and consequent decline in trabecular width. Thus, the
inhibitory effect of glucocorticoids on early bone cell progenitors
S in the bone marrow can account for many of the in v i v o
observations.
The data herein also bear on recent ideas concerning
the relationships between early osteoblast and osteoclast
progenitors in the bone marrow. Although mature osteoclasts and
osteoblasts are needed successively at each bone surface site that
is being remodeled, these cells are needed simultaneously as th a
basic multicellular unit (which is the instrument of bone
remodeling) progresses through or across the surface of bone (41 ).
The necessary parallel production of executive cells is
accomplished by signals that originate from early members of t h a
stromal cell-osteoblast family, which support in various ways the
production of mononuclear preosteoclasts in the bone marrow
(42). The demonstration herein of a marked reduction in the
numbers of both CFU-OB and osteoclast progenitors derived from
ex vivo bone marrow cell cultures makes it likely that
glucocorticoid administration inhibits the proliferation and/or
differentiation of the stromal cell-osteoblast family at an early
stage, leading to a reduction in the number of mature, matrix-
secreting osteoblasts as well as the osteoblastic cells that support
osteoclast development. A direct inhibitory effect of
glucocorticoids on osteoclast precursor proliferation is not
excluded by the data herein, but would be less easy to reconcile
with the finding of an early increase in the osteoclast perimeter.
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Some osteoblasts become osteocytes and some become
lining cells, but these fates combined do not account for all th a
osteoblasts initially present. Although migration along or away
from the bone surface is possible, death has always seemed the
most likely alternative fate (43). Osteoblasts in remodeling bone
undergo apoptosis with a frequency sufficient to account for most
or all of those missing (8). Based on the dynamic
histomorphometry at the marine vertebral secondary spongiosa
and a wall width of about 15 ~,m (7,9,14), the mean active life
span of an osteoblast was calculated on cancellous bone b y
dividing wall width by the mineral appositional rate. From this
calculation, the mean active lifespan of a marine asteoblast is
about 12 days or 288 hours. The prevalence of osteoblast
apoptosis in the present study was 0.0066 in the placebo group.
The following relationship was applied:
tAP/288 = 0.0066/f AP,
where tAP is the mean duration (in hours) of the DNA
fragmentation phase of apoptosis that is detected by TUNEL, and
f AP 1S the fraction of osteoblasts that undergoes apoptosis a n d
based on a value of tAp of about 3 hours, determined previously
for regenerating liver (44), the corresponding value for fAp in the
placebo group is 0.6. Thus, the low prevalence of apoptosis in the
placebo group is consistent with studies of human bone that 5 0-
70% of osteoblasts undergo apoptosis, and that only a minority
become osteocytes or lining cells (43).
In the animals receiving the higher dose of
prednisolone, the prevalence of apoptosis was 0.0203. With
prednisolone administration, phagocytosis of the apoptotic cells
would be suppressed and it was estimated that tAP could b a
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doubled (45). Wall width was reduced to about 8 ~.m and mineral
appositional rate to 0.74 ~,m/d, so that the active lifespan of a n
osteoblast is about 260 hours. In these circumstances, the
corresponding value for fAP in the prednisolone group is 0.9.
Although there is some uncertainty to the assumptions used for
these estimates, the approach does help explain the data and
disclose the devastating impact of glucocorticoid excess o n
osteoblast survival. The higher proportion of osteoblasts showing
features of apoptosis in glucocorticoid-treated mice and human
subjects could indicate no more than prolongation of the time
needed for completion of the process, but it is more likely that
glucocorticoids induce apoptosis, either prematurely in cells
already destined for this fate or in cells otherwise destined to
become lining cells or osteocytes. In either case, the mean active
lifespan of osteoblasts would be shortened and less bone formed.
Thus, the reduction in bone formation by glucocorticoids could b a
due to increased death as well as decreased birth of osteoblasts.
Osteocytes are long-lived but not immortal cells. I n
human rib cortical bone, their lifespan has been estimated a t
about 20 years (47); if bone remains unremodeled for a longer
time, the osteocytes die, as revealed by empty lacunae and
hypermineralized perilacunar bone, referred to as micropetrosis
(48). Osteocyte death in cancellous bone, indicated by absence of
lactic dehydrogenase activity, increases in prevalence with age in
the upper femur but not in the vertebrae (49), probably because
of the higher bone turnover in the spine. Empty lacunae a n d
enzyme absence can reveal the fact, but not the mode, of death.
Osteocyte apoptosis has recently been detected in human iliac
cancellous bone and its prevalence was increased b y
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pharmacological induction of estrogen deficiency (19). The
present invention demonstrated that chronic glucocorticoid
administration, both to mice and to human patients, likewise
increases the prevalence of osteocyte apoptosis. The proportion of
apoptotic osteocytes was much higher than of osteoblasts,
reflecting the unique unavailability of osteocytes for phagocytosis
because of their anatomic isolation from scavenger cells, and the
need for extensive degradation to small molecules to dispose of
the cells through the narrow canaliculi. As a result, the process is
prolonged and affected cells accumulate.
The network of osteocytes probably participates in the
detection of microdamage and the transmission of signals that lead
to its repair by remodeling (50). Disruption of the network b y
osteocyte apoptosis could compromise this mechanism, leading to
microdamage accumulation and increased bone fragility (51 ).
Second, chronic glucocorticoid administration sometimes leads to
so-called aseptic or avascu1ar necrosis of bone (6). Glucocorticoid-
induced osteocyte apoptosis, a cumulative and unrepairable
defect, would explain the correlation between total dose a n d
incidence of avascu1ar necrosis of bone (53) and its occurrence
after glucocorticoid administration had ceased.
In conclusion, the present invention has demonstrated
that the mouse is a valid and informative model of glucocorticoid-
induced bone disease, not confounded by weight loss or sex-
steroid deficiency, and that many of the effects of chronic
glucocorticoid administration on bone can be explained b y
decreased birth of osteoblast and osteoclast precursors and
increased apoptosis of mature osteoblasts and osteocytes.
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TABLE IV
Confounding Factors with Glucocorticoid Administration
Animals Factors
Rats (23,24) Paradoxical increase in cancellous bone mass*,
decreased food intake and weight.
Rabbits and Inconsistent changes in bone density and
cancellous
dogs (25-27) bone area, weight loss, hepatic fatty infiltration.
Ewes (28-30) Histological changes resemble glucocorticoid-
treated patients but corresponding changes in
bone density and cancellous bone area are
inconsistent.
*Glucocorticoids inhibit bone resorption and promote apoptosis in
rat osteoclasts in vitro (31), whereas bone resorption is stimulated
in neonatal mouse calvaria (32). Glucocorticoids stimulate bone
nodule formation from rat calvarial cells i~z vitro (33) but inhibit
differentiation in a murine osteoblastic cell line (34).
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3. Fitzpatrick, L.A. 1994. Glucocorticoid-induced osteoporosis.
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Any patents or publications mentioned in this
specification are indicative of the levels of those skilled in the art
to which the invention pertains. Further, these patents and
publications are incorporated by reference herein to the same
extent as if each individual publication was specifically and
individually indicated to be incorporated by reference.
One skilled in the art will appreciate readily that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those
objects, ends and advantages inherent herein. The present
examples, along with the methods, procedures, treatments,
molecules, and specific compounds described herein are presently
representative of preferred embodiments, are exemplary, and are
not intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art which
are encompassed within the spirit of the invention as defined b y
the scope of the claims .
34
