close

Вход

Забыли?

вход по аккаунту

?

Effect of minodronic acid ONO-5920 on bone mineral density and arthritis in adult rats with collagen-induced arthritis.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 48, No. 6, June 2003, pp 1732–1741
DOI 10.1002/art.10987
© 2003, American College of Rheumatology
Effect of Minodronic Acid (ONO-5920) on
Bone Mineral Density and Arthritis in
Adult Rats With Collagen-Induced Arthritis
Itsuro Yamane, Hiroshi Hagino, Toru Okano, Makoto Enokida,
Daisuke Yamasaki, and Ryota Teshima
joint destruction evaluated by radiography of the foot
was reduced in CIA-P rats. The eroded surface was
reduced and the microstructure was maintained in
CIA-P rats compared with CIA-V rats. The mineral
apposition and bone formation rates were not reduced
in the CIA-P rats. In CIA-T rats, however, the inflammation was not suppressed and the inhibitory effect on
bone loss was smaller than that in CIA-P rats.
Conclusion. Minodronic acid suppressed the decrease in BMD and the deterioration of the bone
microstructure caused by arthritis. Prophylactic administration of minodronic acid had a preventive effect on
arthritis at the early stage, although not throughout the
observation period.
Objective. To study the effect of minodronic acid
(ONO-5920) on bone loss and arthritis in rats with
collagen-induced arthritis (CIA) treated according to 2
different schedules.
Methods. Four groups of female Sprague-Dawley
rats (7 months old) were studied: rats without CIA
treated with vehicle (controls), CIA rats treated with
vehicle (CIA-V), CIA rats treated therapeutically
with minodronic acid (CIA-T), and CIA rats treated
prophylactically with minodronic acid (CIA-P).
Minodronic acid was administered orally at 0.2 mg/kg
3 times a week, beginning 2 weeks after initial sensitization in the CIA-T rats and beginning the day after
initial sensitization in the CIA-P rats. Bone mineral
density (BMD) was measured by peripheral quantitative computed tomography in the proximal metaphysis
and diaphysis of the tibia every 2 weeks until week 8,
when the rats were killed. The BMD and bone microstructure of the excised femur were evaluated by dual
x-ray absorptiometry and microfocal computed tomography, respectively. Histomorphometry of the proximal
tibia was also performed.
Results. In CIA-P rats, the incidence of arthritis
and the severity of posterior limb swelling were reduced
early after sensitization, and the decrease in BMD was
prevented throughout the observation period. Bone and
Osteoporosis in patients with rheumatoid arthritis (RA) is an important complication that increases the
risk for fracture. Osteoporosis is classified into two
types: periarticular, as observed near arthritic joints
during the early stage of RA, and generalized, caused by
various systemic factors.
Since its first description by Trentham et al in
1977 (1), the type II collagen–induced arthritis (CIA)
rat model of RA has been used for research on the
pathology and treatment of RA. Through longitudinal
observations of adult rats with CIA, we have demonstrated that the bone mineral density (BMD) of cancellous bone close to the joint begins to decrease early after
the onset of arthritis (2). We concluded that the adult
CIA rat is a useful model of periarticular osteoporosis in
the early stage of RA. We have also reported that
arthritis and bone loss in adult CIA rats are exacerbated
by ovariectomy and suppressed by estrogen replacement
therapy (3).
Bisphosphonates are promising drugs for the
treatment of osteoporosis because of their ability to
increase BMD and prevent fractures. Bisphosphonates
Supported in part by a grant-in-aid from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan (grant
13671509).
Itsuro Yamane, MD, Hiroshi Hagino, MD, Toru Okano, MD,
Makoto Enokida, MD, Daisuke Yamasaki, MD, Ryota Teshima, MD:
Faculty of Medicine, Tottori University, Yonago, Japan.
Address correspondence and reprint requests to Hiroshi
Hagino, MD, Department of Orthopedic Surgery, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8504, Japan. E-mail:
hagino@grape.med.tottori-u.ac.jp.
Submitted for publication August 2, 2002; accepted in revised
form February 11, 2003.
1732
BMD AND MINODRONIC ACID TREATMENT IN RATS WITH CIA
have also been shown to increase BMD in RA complicated by osteoporosis through their suppressive effect on
bone resorption (4). Antiinflammatory effects of
bisphosphonates have been demonstrated in preclinical
(5–17) and clinical (18–20) studies. Minodronic acid
(ONO-5920) is a third-generation bisphosphonate containing an imidazole ring in a side chain. It is one of the
bisphosphonates reported to have the greatest suppressive effect on bone resorption.
The present study was designed to examine the
effect of minodronic acid on bone mineral density and
arthritis and to evaluate the differences in these effects
between prophylactic and therapeutic administration in
adult rats with CIA. Our findings are presented herein.
MATERIALS AND METHODS
Experimental animals. Seven-month-old female
Sprague-Dawley rats (retired breeder animals with a body
weight of 240–350 gm; Shimizu Laboratory Supply, Kyoto,
Japan) were used as the model for adult arthritis in humans.
The animals were given tap water and solid food (calcium
content 1.18 gm/100 gm, phosphorus content 1.09 gm/100 gm,
vitamin D3 content 250 IU/100 gm) (CE-2; Clea Japan, Tokyo,
Japan) ad libitum. The animals were maintained in an animal
room that was illuminated for 12 hours daily (7:00 AM to
7:00 PM) and kept at a room temperature of 24°C. After a
4-week acclimation period, the animals were used for the
experiments.
The animals were divided into 4 groups of approximately equal mean body weight. The first group (n ⫽ 9)
received an injection of vehicle instead of collagen plus vehicle
administration instead of minodronic acid (controls). The
second group (n ⫽ 13) received collagen sensitization plus
vehicle administration instead of minodronic acid (CIA-V).
The third group (n ⫽ 13) received collagen sensitization plus
therapeutic administration of minodronic acid (CIA-T). The
fourth group (n ⫽ 13) received collagen sensitization plus
prophylactic administration of minodronic acid (CIA-P).
Eight weeks after initial sensitization, the rats were
anesthetized by intraperitoneal injection of ketamine hydrochloride (Ketalar; Sankyo, Tokyo, Japan) plus xylazine (Celactal; Bayer, Leverkusen, Germany) (2:1) at 1 mg/kg, blood was
collected by cardiac puncture, and the animals were killed. The
left femur, left tibia, and both feet were resected for analysis.
This study was performed at the Animal Experiment
Facility, Hospital of Tottori University School of Medicine.
The Animal Experiment Ethical Committee of Tottori University approved the protocol.
Preparation of the CIA model. Under intraperitoneal
anesthesia with ketamine HCl plus xylazine (2:1) at 1 mg/kg of
body weight, 1.0 ml of an emulsion containing 500 mg of
bovine type II collagen in a 0.3% acetic acid solution (catalog
no. K-41; Cosmo Bio, Tokyo, Japan) and 500 mg of Freund’s
incomplete adjuvant (catalog no. 263910; Difco, Detroit, MI)
was injected intracutaneously at 3 sites on the back of each rat.
For additional sensitization, 0.5 ml of the same emulsion was
injected intracutaneously at 2 sites on the posterior aspects of
1733
both hip joints 1 week after initial sensitization. In the controls,
physiologic saline was injected intracutaneously, using the
same volume and methods as for the other 3 groups (2,3).
Administration of study drug. Minodronic acid
(ONO-5920/YM529; chemical name [1-hydroxy-2-(imidazo
[1,2-a]pyridin-3-yl)ethylidene]-bisphosphonic acid monohydrate) was provided by Yamanouchi Pharmaceutical
(Tokyo, Japan). This agent was jointly developed by Ono
Pharmaceutical (Osaka, Japan) and Yamanouchi Pharmaceutical. We prepared a 0.2 mg/kg/5 ml solution of minodronic acid by dissolving minodronic acid with a 0.01N
sodium hydroxide solution and distilled water and diluting
it with 2% (weight/volume) methylcellulose. Minodronic
acid was administered orally using the feeding needle at a
dosage of 0.2 mg/kg 3 times a week for 8 weeks beginning
2 weeks after the initial sensitization in the CIA-T group
and beginning the day after initial sensitization in the
CIA-P group. The same volume of vehicle was administered orally 3 times a week for 8 weeks from the day after
initial sensitization in the control group and the CIA-V
group. The animals fasted for 2 hours before and after each
administration.
Evaluation of arthritis. Rats were examined every 2
weeks until week 8 after initial sensitization for body weight,
arthritis score, and posterior limb swelling. The severity of
inflammation in each limb was evaluated every 2 weeks for the
degree of inflammation, the extent of erythema and edema of
the periarticular tissues, and the enlargement, distortion, or
ankylosis of the joints. Findings were scored on a scale of 0–4
(1), where 0 ⫽ no inflammation, 2 ⫽ unequivocal inflammation of at least 2 joints of the limb or moderate inflammation
of 1 joint, 3 ⫽ severe inflammation of ⱖ1 joint, and 4 ⫽
maximum inflammation of ⱖ1 joint in the limb. The arthritis
score was the sum of the scores for all 4 limbs (maximum
possible score 16) (1). Swelling of the posterior limbs was
evaluated by measuring the ankle width from the medial
malleolus to the lateral malleolus using constant-tension calipers (1). Posterior limb swelling was expressed as the mean of
the values in both limbs.
Radiographic examination. Radiographs of both feet
resected at the time the animals were killed were obtained with
a Sofron model SRO-M50 device (Sofron, Tokyo, Japan).
Destruction of bone and cartilage was classified and scored
according to the method described by Engelhardt et al (21),
which assessed detailed changes in the spongiosa, periosteum,
and compact bone around the arthritic joint. The radiographs
were evaluated by 3 observers who were blinded as to the study
groups. The radiographic score was the mean of the scores
assigned by the 3 observers.
Measurement of BMD. Peripheral quantitative computed tomography (QCT). Volumetric BMD (vBMD; expressed
in mg/cm3) was measured by peripheral QCT (model XCT-960
scanner; Norland-Stratec, Pforzheim, Germany) of the proximal metaphysis (2 mm distal to the growth cartilage) and
diaphysis (5 mm distal to the growth cartilage) of the left tibia
every 2 weeks until week 8 after initial sensitization. The rats were
anesthetized by intraperitoneal injection of 1 mg/kg of ketamine
HCl plus xylazine (2:1), and the metaphysis and diaphysis were
identified by scout scanning. The scan beam was then focused on
the proximal metaphysis of the left tibia, perpendicular to the
bone axis, and the measurement was performed at a voxel size of
1734
YAMANE ET AL
Figure 1. Changes in A, body weight and B, hind paw thickness in rats with collagen-induced
arthritis (CIA) treated with vehicle (CIA-V), with therapeutic administration of minodronic acid
(CIA-T), and with prophylactic administration of minodronic acid (CIA-P), as well as in controls (CONT)
without CIA. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus controls, ⴱⴱ ⫽ P ⬍ 0.0001 versus controls;
❈ ⫽ P ⬍ 0.05 versus CIA-V rats, by Fisher’s protected least significant difference post hoc test.
0.295 mm and a slice thickness of 1 mm. The measurement
parameters were as follows: contmode 2 (for bone contour),
peelmode 20 (for trabecular region), contmode 1 (for cortical
region), and threshold 0.63 for vBMD of cancellous bone and
0.93 for vBMD of cortical bone (22,23).
Dual x-ray absorptiometry. Areal BMD (aBMD;
expressed in gm/cm2) was measured in the entire left femur
and distal one-fifth of the left femur by dual x-ray absorptiometry (model QDR 4500A instrument; Hologic, Waltham,
MA). The femur was placed with its anterior surface upward in
a plastic vessel that had been filled to a depth of 2.5 cm with a
70% alcohol solution. The measurement was performed in the
small-animal mode by adjusting the line spacing to 1.5 mm, the
point resolution to 0.64 mm, and the scan speed to 2.5
mm/seconds.
Microstructural analysis of bone using microfocal
computed tomography (micro-CT). Cancellous bone of the
distal metaphysis of the left femur was analyzed by micro-CT
(model MCT-CB100MF scanner; Hitachi Medico Technology,
Tokyo, Japan). The measurement field was a zone perpendicular
to the bone axis, 700 ␮m in the cranial and caudal directions
around a point 2.5 mm proximal to the growth plate. The slice
pitch was 14 ␮m; 100 slices were analyzed. The 2-dimensional
images obtained were converted to 3-dimensional images, and the
bone microstructure was analyzed. The parameters of measurement were bone volume (in mm3) relative to tissue volume (in
mm3) (expressed as a percentage), bone surface (in mm2) relative
to bone volume (in mm3) (expressed per mm), trabecular thickness (in mm), trabecular number (per mm), trabecular separation
(in mm), trabecular bone pattern factor (per mm), and fractal
dimension.
Bone histomorphometry. Bone labeling by intraperitoneal injection with Calcein at 10 mg/kg was performed
twice, at 7 days and 1 day before the rats were killed (schedule
of 1-5-1-1, representing the number of days of the first labeling,
number of days between first and second labeling, number of
days of the second labeling, and the number of days between
second labeling and killing). The proximal tibia resected at the
time of killing was fixed with 10% neutral buffered formalin for
24 hours and dehydrated with 70% alcohol. Villanueva bone
staining was then performed. These bone tissue samples were
embedded in methylmethacrylate resin without decalcification.
The resulting specimen blocks were sectioned in the frontal
plane at a thickness of 5 ␮m with a Jung model K microtome
(Reichert-Jung, Nussloch, Germany).
For histomorphometric analysis, the following features
were measured in the secondary spongiosa extending 1.3–3.9 mm
distally from the proximal growth cartilage of the tibia: bone
volume/tissue volume (expressed as a percentage), osteoid
volume/tissue volume (expressed as a percentage), osteoblast
surface/bone surface (expressed as a percentage), osteoid surface/
bone surface (expressed as a percentage), trabecular thickness (in
␮m), trabecular number (per mm), trabecular separation (in ␮m),
osteoclast surface/bone surface (expressed as a percentage),
eroded surface/bone surface (expressed as a percentage), osteoclast number/bone surface (per mm), mineralizing surface/bone
surface (expressed as a percentage), double-label surface/bone
surface (expressed as a percentage), mineral apposition rate (in
␮m/day), and bone formation rate/bone volume (expressed as a
percentage per year). Each parameter was expressed according to
Table 1. Incidence of arthritis in 7-month-old female SpragueDawley rats*
CIA-V (n ⫽ 13)
CIA-T (n ⫽ 13)
CIA-P (n ⫽ 12)
Week 2
Week 4
Week 6
Week 8
69.2 (9)†
46.2 (6)†
16.7 (2)†
83.3 (11)
92.3 (12)
66.7 (8)
100 (13)
92.3 (12)
83.3 (10)
100 (13)
92.3 (12)
83.3 (10)
* Three groups of rats were sensitized with collagen to produce
collagen-induced arthritis (CIA). One group was treated with vehicle
(CIA-V), the second with therapeutic administration of minodronic
acid (CIA-T), and the third with prophylactic administration of
minodronic acid (CIA-P). Values are the percentage (number) of rats
with CIA.
† P ⬍ 0.05 for comparisons among the 3 groups, by chi-square test.
BMD AND MINODRONIC ACID TREATMENT IN RATS WITH CIA
Table 2.
1735
Arthritis score in the 3 groups of rats with CIA*
CIA-V (n ⫽ 13)
CIA-T (n ⫽ 12)
CIA-P (n ⫽ 10)
Week 0
Week 2
Week 4
Week 6
Week 8
0.0
0.0
0.0
3.0 (0.0, 5.0)
0.5 (0.0, 6.5)
0.0 (0.0, 0.0)
10.0 (3.0, 12.0)
8.0 (5.5, 11.5)
5.0 (3.0, 7.0)
10.0 (6.0, 12.0)
9.0 (6.0, 12.0)
6.5 (3.0, 10.5)
8.0 (6.0, 10.0)
8.5 (5.5, 10.0)
5.5 (4.0, 9.0)
* Values are the median (25th, 75th percentiles). No significant differences were observed between the 3
groups at any time point, as analyzed by Kruskal-Wallis test. See Table 1 for definitions of the treatment
groups.
the classification of Parfitt et al (24) and Jee et al (25). Bone
histomorphometric parameters were measured using a semiautomatic digitizer (System Supply, Nagano, Japan) and a personal
computer (PC-9801; NEC, Tokyo, Japan).
Bone markers. Blood was collected by cardiac puncture
at the time the rats were killed, and serum was isolated by
centrifugation and stored at –20°C. Serum osteocalcin (in ng/ml)
was measured by specific radioimmunoassay based on rat osteocalcin (Biomedical Technologies, Stoughton, MA). Serum levels
of tartrate-resistant acid phosphatase (TRAP; in IU/liter) were
assessed by its enzymatic activity, as determined by a paranitrophenyl phosphate method (SRL, Tokyo, Japan).
Statistical analysis. Fisher’s protected least significant
difference procedure was performed after 2-way analysis of
variance for comparison of data on body weight, posterior limb
swelling, bone histomorphometry, and bone markers among
the study groups. The data obtained for the vBMD of the tibia
measured every 2 weeks until after week 8 using peripheral
QCT were expressed as the percentage of change relative to
the value at initial sensitization (baseline) and were compared
among the groups. The incidence of arthritis was compared
among the 3 CIA groups using the chi-square test. For
comparison of the arthritis score and the radiographic score,
Dunnett’s procedure was performed among the 3 CIA groups
after the Kruskal-Wallis test was performed.
For body weight, posterior limb swelling, BMD, bone
histomorphometry, arthritis score, and radiographic score,
only data for the rats that developed arthritis were used. The 3
rats that did not develop arthritis (1 in the CIA-T group and 2
in the CIA-P group) and the 1 animal that died during the
anesthetic procedure (in the CIA-P group) were excluded from
the analyses. Statistical analysis was performed using StatView
software (version 5.0; SAS Institute, Cary, NC).
RESULTS
Changes in body weight. The body weight decreased significantly at weeks 4, 6, and 8 in rats that
developed arthritis, regardless of whether they received
minodronic acid (Figure 1A). No significant differences
among the 3 collagen-sensitized groups were observed.
Incidence of arthritis. The incidence of arthritis
at week 8 was 100% in CIA-V rats, 92.3% in CIA-T
rats, and 83.3% in CIA-P rats. The differences among
the 3 groups were significant at week 2 (P ⬍ 0.05)
(Table 1).
Posterior limb swelling was most severe in the
CIA-V rats at week 4, but subsided thereafter (Figure
1B). In CIA-P rats, posterior limb swelling was significantly milder than that in CIA-V rats at week 2 (P ⬍
Figure 2. Percentage change in bone mineral density (BMD) of the tibial metaphysis. A, BMD of cancellous
bone. B, BMD of cortical bone. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus controls; ⴱⴱ ⫽ P ⬍ 0.0001
versus controls; ❈ ⫽ P ⬍ 0.05 versus CIA-V rats; ❈❈ ⫽ P ⬍ 0.0001 versus CIA-V rats; # ⫽ P ⬍ 0.05 versus
CIA-T rats, by Fisher’s protected least significant difference post hoc test. See Figure 1 for definitions of the
treatment groups.
1736
YAMANE ET AL
Figure 3. Percentage change in bone mineral density (BMD) of the tibial diaphysis. A, BMD of
cancellous bone. B, BMD of cortical bone. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus controls;
❈ ⫽ P ⬍ 0.05 versus CIA-V rats, by Fisher’s protected least significant difference post hoc test. See Figure
1 for definitions of the treatment groups.
0.05) and was slightly milder than that in CIA-V rats and
in CIA-T rats throughout the observation period.
The arthritis score had reached the maximum by
week 6 in all groups of rats (Table 2). This score was
lower in CIA-P rats than in CIA-V and CIA-T rats at all
points after sensitization, although the differences were
not statistically significant.
Volumetric BMD of the tibial metaphysis. Cancellous bone. In CIA-V and CIA-T rats, vBMD of
cancellous bone began to decrease with the onset of
arthritis and became significantly lower than that in
controls at week 4 and after (P ⬍ 0.05 for all time points)
(Figure 2A). In CIA-T rats, the vBMD of cancellous bone
did not decrease after week 6, and became significantly
higher than that in CIA-V rats at week 8 (P ⬍ 0.05). In
CIA-P rats, the vBMD also decreased with the onset of
arthritis, but no significant difference compared with the
controls was noted throughout the observation period. The
vBMD in CIA-P rats was significantly higher than that in
CIA-V and CIA-T rats beginning at week 4 (P ⬍ 0.05 for
all time points).
Cortical bone. In CIA-V rats, the vBMD of
cortical bone was decreased at week 8, which was
delayed compared with the decrease in cancellous bone
(Figure 2B). However, the vBMD of cortical bone was
not significantly different in the minodronic acid–treated
groups compared with the CIA-V group until week 8
(P ⬍ 0.05).
Volumetric BMD of the tibial diaphysis. Cancellous bone. In CIA-V rats, the vBMD of cancellous bone
Figure 4. Bone mineral density (BMD) of A, the entire left femur and B, the distal one-fifth of
the left femur. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus controls; ❈ ⫽ P ⬍ 0.05
versus CIA-V rats, by Fisher’s protected least significant difference post hoc test. See Figure 1 for
definitions of the treatment groups.
BMD AND MINODRONIC ACID TREATMENT IN RATS WITH CIA
1737
Figure 5. Comparison of the 3-dimensional architecture of the cancellous bone at the distal femoral
metaphysis in A, control rats without collagen-induced arthritis (CIA), B, rats with CIA treated with
vehicle (CIA-V), C, rats with CIA treated with therapeutic administration of minodronic acid (CIA-T),
and D, rats with CIA treated with prophylactic administration of minodronic acid (CIA-P).
was significantly decreased compared with the controls
at week 8 (P ⬍ 0.05) (Figure 3A). This value was not
significantly different in the minodronic acid–treated
groups compared with the controls, but it was significantly higher in the CIA-P group than in the CIA-V
group (P ⬍ 0.05).
Table 3. Microstructure of cancellous bone at the distal one-fifth of the left femur, as measured by microfocal computed tomography in all 4 study
groups*
Control (n ⫽ 9)
CIA-V (n ⫽ 13)
CIA-T (n ⫽ 12)
CIA-P (n ⫽ 10)
BV/TV (%)
BS/BV (/mm)
Tb.Th (mm)
Tb.N (/mm)
Tb.Sp (mm)
TBPF (/mm)
Fractal
dimension
27.60 ⫾ 2.61
18.87 ⫾ 5.51†
20.73 ⫾ 4.03‡
22.89 ⫾ 3.54‡§
49.31 ⫾ 3.40
62.69 ⫾ 8.87‡
61.54 ⫾ 9.95‡
57.77 ⫾ 6.30‡
0.041 ⫾ 0.003
0.033 ⫾ 0.004†
0.033 ⫾ 0.005‡
0.035 ⫾ 0.004‡
6.78 ⫾ 0.39
5.72 ⫾ 1.06‡
6.22 ⫾ 0.60
6.58 ⫾ 1.06§
0.116 ⫾ 0.012
0.154 ⫾ 0.037‡
0.137 ⫾ 0.019
0.127 ⫾ 0.022§
20.88 ⫾ 4.37
39.96 ⫾ 15.99‡
37.89 ⫾ 13.84‡
30.98 ⫾ 13.09
2.421 ⫾ 0.022
2.365 ⫾ 0.071
2.394 ⫾ 0.051
2.387 ⫾ 0.041
* Four groups of rats were evaluated: control rats without collagen-induced arthritis (CIA) and CIA rats treated with vehicle (CIA-V), with
therapeutic administration of minodronic acid (CIA-T), or with prophylactic administration of minodronic acid (CIA-P). Values are the mean ⫾ SD.
P values were determined by Fisher’s protected least significant difference post hoc test. BV ⫽ bone volume; TV ⫽ tissue volume; BS ⫽ bone
surface; Tb.Th ⫽ trabecular thickness; Tb.N ⫽ trabecular number; Tb.Sp ⫽ trabecular separation; TBPF ⫽ trabecular bone pattern factor.
† P ⬍ 0.0001 versus controls.
‡ P ⬍ 0.05 versus controls.
§ P ⬍ 0.05 versus CIA-V rats.
1738
YAMANE ET AL
Table 4. Bone histomorphometry of the proximal tibia in all 4 study groups*
Controls (n ⫽ 9)
CIA-V (n ⫽ 13)
CIA-T (n ⫽ 12)
CIA-P (n ⫽ 10)
BV/TV (%)
OV/TV (%)
Ob.S/BS (%)
OS/BS (%)
Tb.Th (␮m)
Tb.N (1/mm)
Tb.Sp (␮m)
22.87 ⫾ 2.72
15.79 ⫾ 6.37‡
17.29 ⫾ 3.68‡
22.49 ⫾ 5.88†§
0.093 ⫾ 0.043
0.099 ⫾ 0.060
0.087 ⫾ 0.059
0.092 ⫾ 0.070
4.70 ⫾ 2.46
5.15 ⫾ 1.74
4.54 ⫾ 0.99
4.05 ⫾ 1.68
23.32 ⫾ 4.39
27.02 ⫾ 5.79
20.24 ⫾ 6.93†
19.04 ⫾ 6.19†
67.26 ⫾ 7.74
50.95 ⫾ 11.99‡
52.49 ⫾ 6.24‡
61.49 ⫾ 12.32†§
3.42 ⫾ 0.40
3.04 ⫾ 0.68
3.28 ⫾ 0.48
3.64 ⫾ 0.44
228.87 ⫾ 32.70†
292.68 ⫾ 79.88
258.53 ⫾ 47.27
217.12 ⫾ 38.73†
Cortical bone. Volumetric BMD of cortical bone
was not significantly reduced in any of the groups
throughout the observation period (Figure 3B).
Areal BMD of the femur. The aBMD of the
entire left femur was not significantly different
among the 4 groups (Figure 4A). However, the
aBMD in the distal one-fifth of the left femur was
significantly lower in the CIA-V group than in the
other 3 groups (P ⬍ 0.05). There was no significant
reduction in the aBMD in the minodronic acid–
treated groups compared with the control group
(Figure 4B).
Microstructure of cancellous bone at the distal
femur. In the CIA-V group, the trabecular structure
became thin and distorted compared with that in the
control group (Figure 5). In the CIA-T and CIA-P
groups, the trabecular structure was maintained. In
particular, prophylactic administration of minodronic
acid had a greater effect on preservation of the trabecular architecture than did therapeutic administration of
the drug.
The bone volume/tissue volume was significantly
lower in the collagen-sensitized groups (CIA-V, CIA-T,
and CIA-P) than in the control group (P ⬍ 0.05 for all
comparisons), but significantly higher in the CIA-P
group than in the CIA-V group (P ⬍ 0.05) (Table 3).
The bone surface/bone volume was significantly higher,
but the trabecular thickness was significantly lower, in
the 3 collagen-sensitized groups than in the control
group (P ⬍ 0.05 for all comparisons). The trabecular
number was significantly higher in the control and
CIA-P groups than in the CIA-V group (P ⬍ 0.05 for
each comparison). Trabecular separation and trabecular
bone pattern factor were significantly lower in the
control group than in the CIA-V group (P ⬍ 0.05 for
each comparison), but were not significantly different in
the control and the CIA-P groups.
Figure 6. Radiographic appearance of the right foot of A, control
rats without collagen-induced arthritis (CIA), B, rats with CIA
treated with vehicle (CIA-V), C, rats with CIA treated with therapeutic administration of minodronic acid (CIA-T), and D, rats with
CIA treated with prophylactic administration of minodronic acid
(CIA-P).
BMD AND MINODRONIC ACID TREATMENT IN RATS WITH CIA
1739
Table 4. (cont’d)
Oc.S/BS (%)
ES/BS (%)
Oc.N/BS (/mm)
MS/BS (%)
DLS/BS (%)
MAR (␮m/day)
BFR/BV (%/year)
1.11 ⫾ 0.37
1.87 ⫾ 0.95
1.52 ⫾ 0.65
1.27 ⫾ 0.87
7.26 ⫾ 2.05
11.11 ⫾ 2.97‡
7.93 ⫾ 2.75†
6.95 ⫾ 3.08†
1.64 ⫾ 0.54
2.92 ⫾ 1.32‡
2.00 ⫾ 0.74†
1.79 ⫾ 0.82†
12.69 ⫾ 3.76
16.28 ⫾ 6.98
12.50 ⫾ 5.41
13.93 ⫾ 4.92
4.11 ⫾ 2.69
6.94 ⫾ 5.34
3.27 ⫾ 3.23
3.91 ⫾ 2.96
0.857 ⫾ 0.200
0.916 ⫾ 0.261
0.659 ⫾ 0.266
0.779 ⫾ 0.147
122.95 ⫾ 51.54†
223.51 ⫾ 110.37
124.36 ⫾ 71.62†
146.86 ⫾ 89.55†
* Four groups of rats were evaluated as described in Table 3. Values are the mean ⫾ SD. P values were determined by Fisher’s protected least
significant difference post hoc test. BV ⫽ bone volume; TV ⫽ tissue volume; OV ⫽ osteoid volume; Ob.S ⫽ osteoblast surface; BS ⫽ bone surface;
OS ⫽ osteoid surface; Tb.Th ⫽ trabecular thickness; Tb.N ⫽ trabecular number; Tb.Sp ⫽ trabecular separation; Oc.S ⫽ osteoclast surface; ES ⫽
eroded surface; Oc.N ⫽ osteoclast number; MS ⫽ mineralizing surface; DLS ⫽ double-label surface; MAR ⫽ mineral apposition rate; BFR ⫽ bone
formation rate.
† P ⬍ 0.05 versus CIA-V rats.
‡ P ⬍ 0.05 versus controls.
§ P ⬍ 0.05 versus CIA-T rats.
Bone histomorphometry of the proximal tibia.
Bone volume/tissue volume and trabecular thickness
were significantly higher in the control and CIA-P
groups than in the CIA-V and CIA-T groups (P ⬍ 0.05
for all comparisons) (Table 4). Trabecular separation was significantly lower in the control and CIA-P
groups than in the CIA-V group (P ⬍ 0.05 for each
comparison). The eroded surface/bone surface, osteoclast number/bone surface, and bone formation rate/
bone volume values were significantly lower in the
control and the minodronic acid–treated (CIA-T and
CIA-P) groups than in the CIA-V group (P ⬍ 0.05 for all
comparisons). The osteoid surface/bone surface value
was significantly lower in the minodronic acid–treated
groups than in the CIA-V group (P ⬍ 0.05 for both
comparisons).
Markers of bone metabolism. The level of
osteocalcin was 14.5 ⫾ 2.3 ng/ml (mean ⫾ SD) in the
control group, 22.8 ⫾ 8.0 ng/ml in the CIA-V group, 17.5 ⫾
4.8 ng/ml in the CIA-T group, and 17.8 ⫾ 6.1 ng/ml in the
CIA-P group. The osteocalcin level was significantly higher
in the CIA-V group than in the control group (P ⬍ 0.05).
The mean ⫾ SD serum level of TRAP was 14.8 ⫾ 3.5,
17.0 ⫾ 8.6, 15.4 ⫾ 7.7, and 11.6 ⫾ 4.0 IU/liter, respectively,
in the control, CIA-V, CIA-T, and CIA-P groups. The
TRAP level was highest in the CIA-V group, but there
were no statistically significant differences among the study
groups.
Findings of radiographic examination. Joint destruction was observed in all the collagen-sensitized
groups. However, the CIA-V group demonstrated more
marked atrophy of trabecular bone and more extensive
lysis of cortical bone than did the CIA-P group (Figure
6). The median radiographic scores were 12.5 (25th and
75th percentiles 6.4, 12.8) in the CIA-V group, 11.1
(25th and 75th percentiles 8.3, 12.9) in the CIA-T group,
and 9.3 (25th and 75th percentiles 3.4, 11.3) in the
CIA-P group. The difference between the CIA-V and
CIA-P groups was significant (P ⬍ 0.05).
DISCUSSION
Minodronic acid has been experimentally demonstrated to have anti–bone resorption activity that is more
than 10,000 times stronger than that of etidronate and
10–100 times stronger than that of alendronate (26). A
clinical trial in Japan of minodronic acid administered
orally at dosages of 0.5, 1.0, and 1.5 mg/day (⬃0.01–0.03
mg/kg/day) in patients with osteoporosis showed that
each dosage was efficacious (27). In an experiment in
which minodronic acid was administered orally at dosages of 0.02, 0.1, and 0.5 mg/kg/day in animal models of
osteoporosis, the decrease in BMD was suppressed
significantly and dose dependently, but the values for the
bone remodeling parameters were closest to those in the
control group at a dosage of 0.1 mg/kg/day (28). The
dosage used in the present study (0.2 mg/kg 3 times a
week, administered orally after fasting) was therefore
⬃3–10 times higher than the optimum dosage for the
treatment of osteoporosis in humans, but we considered
it to be appropriate for experiments in animals. We also
used retired breeder rats as experimental animals. In
controls, the body weight and BMD remained constant
throughout the study. Therefore, the influence of growth
on changes in BMD and bone turnover was small in this
study.
The results of micro-CT of the distal femoral
metaphysis and bone histomorphometry of the proximal
tibial metaphysis indicated that the decrease in BMD in
the CIA-V group was due to an increase in the eroded
surface and a decrease in the trabecular thickness. The
osteoid surface and bone formation rate, however, were
increased. These results, suggesting a high bone turn-
1740
over rate near the joint in rats with CIA, were similar to
those in a carrageenan-induced arthritis model (29,30).
Bisphosphonate has been reported to suppress
decreases in BMD near the joints in animals used as
arthritis models (7,9,12,16,31,32). This effect is considered to be a result of the suppression of bone resorption,
which is enhanced by arthritis (31,32), and the prevention of the decrease in the mineralized trabecular bone
area (7). In the present study, prophylactic administration of minodronic acid to CIA rats suppressed the
increases in the eroded surface and osteoid surface and
maintained the trabecular thickness and trabecular bone
pattern factor, thereby preventing a decrease in BMD
throughout the observation period. In addition, the
mineral apposition rate and bone formation rate were
not significantly different between CIA-P rats and controls. Therefore, minodronic acid was found to suppress
the enhancement of bone resorption due to arthritis and
to maintain the BMD and bone microstructure without
inhibiting mineralization.
The production of inflammatory cytokines, such
as tumor necrosis factor ␣ , interleukin-1, and
interleukin-6 in synovial tissue and bone marrow is
reported to increase in patients with RA as well as in
animal models of arthritis (33–36). These inflammatory
cytokines may induce inflammatory reactions and promote osteoclast differentiation (37,38) as well as cause a
decrease in BMD (33,34,39). Bisphosphonate appears to
act on osteoclasts by directly inducing apoptosis and
indirectly suppressing osteoclast functions via other cells
(40). Inflammatory cytokines are suggested to be involved in this indirect action (26,34). Recently, a
bisphosphonate was reported to suppress the production
of cytokines and to have antiinflammatory effects in
arthritic rats (12,15). However, these antiinflammatory
effects vary with the species of experimental animal, the
kind of bisphosphonate evaluated, and the dosage of
bisphosphonate administered (14).
In the present study, the incidence of arthritis
and the severity of posterior limb swelling were reduced
early after sensitization, and destruction of bone and
joint was reduced on radiographic examination of
the foot 8 weeks after sensitization, in the CIA-P group
but not in the CIA-T group. It has been reported that
levels of inflammatory cytokines begin to increase before the onset of arthritis (32,34). Therefore, our findings suggest that the administration of minodronic acid
beginning before the onset of arthritis suppressed the
incidence of arthritis and reduced the severity of arthritis, inhibiting the increase in inflammatory cytokine
YAMANE ET AL
levels. However, the mechanism of this effect remains to
be clarified.
In conclusion, we found that minodronic acid
suppressed the decrease in BMD and deterioration of
the bone microstructure caused by arthritis. The suppressive effect of minodronic acid on the decrease in
BMD was ascribed to suppression of bone resorption,
unaccompanied by a disturbance in bone mineralization.
Prophylactic administration of minodronic acid had a
stronger suppressive effect on the decrease in BMD than
did therapeutic administration. Prophylactic administration of minodronic acid had a preventive effect on the
incidence of arthritis and the severity of posterior limb
swelling at the early stage of arthritis, but beyond the
early stage, there were no significant differences among
CIA groups. These results indicate that minodronic acid
is effective for the treatment of osteoporosis complicating RA.
ACKNOWLEDGMENTS
Ono Pharmaceutical Company and Yamanouchi Pharmaceutical Company supplied the minodronic acid, and we
acknowledge their support. We also acknowledge Kiichi
Nonaka for advice on peripheral QCT analysis, staff of Hitachi
Medico Technology Company for performing the micro-CT
analysis, and staff of the Niigata Bone Science Institute for
assistance in preparing and staining tissue sections.
REFERENCES
1. Trentham DE, Townes AS, Kang AH. Autoimmunity to type II
collagen: an experimental model of arthritis. J Exp Med 1977;146:
857–68.
2. Enokida M, Yamasaki D, Okano T, Hagino H, Morio Y, Teshima
R. Bone mass changes of tibial and vertebral bones in young and
adult rats with collagen-induced arthritis. Bone 2001;28:87–93.
3. Yamasaki D, Enokida M, Okano T, Hagino H, Teshima R. Effects
of ovariectomy and estrogen replacement therapy on arthritis and
bone mineral density in rats with collagen-induced arthritis. Bone
2001;28:634–40.
4. Eggelmeijer F, Papapoulos SE, van Paassen HC, Dijkmans BAC,
Valkema R, Westedt ML, et al. Increased bone mass with pamidronate treatment in rheumatoid arthritis: results of a three-year
randomized, double-blind trial. Arthritis Rheum 1996;39:396–402.
5. Nakamura M, Ando T, Abe M, Kumagai K, Endo Y. Contrast
between effects of aminobisphosphonate and non-aminobisphosphonate on collagen-induced arthritis in mice. Br J Pharmacol
1996;119:205–12.
6. Takaoka Y, Nagai H, Mori H, Takahashi N. The effect of
TRK-530 on experimental arthritis in mice. Biol Pharm Bull
1997;20:1147–50.
7. Osterman T, Virtamo T, Lauren L, Kippo K, Pasanen I, Hannuniemi R, et al. Slow-release clodronate in prevention of inflammation and bone loss associated with adjuvant arthritis. J Pharmacol
Exp Ther 1997;280:1001–7.
8. Osterman T, Kippo K, Lauren L, Hannuniemi R, Sellman R.
Effect of clodronate on established adjuvant arthritis. Rheumatoid
Int 1994;14:139–47.
BMD AND MINODRONIC ACID TREATMENT IN RATS WITH CIA
9. Muller K, Wiesenberg I, Jaeggi K, Green JR. Effects of the
bisphosphonate zoledronate on bone loss in the ovariectomized
and in the adjuvant arthritic rat. Arzneimittelforschung 1998;48:
81–6.
10. Flora L. Comparative antiinflammatory and bone protective effects of two diphosphonates in adjuvant arthritis. Arthritis Rheum
1979;22:340–6.
11. Osterman T, Kippo K, Lauren L, Pasanen I, Hannuniemi R,
Sellman R. A comparison of clodronate and indomethacin in the
treatment of adjuvant arthritis. Inflamm Res 1997;46:79–85.
12. Takanashi M, Funaba Y, Ito M, Kawabe N, Nakadate-Matsushita
T. Inhibitory effects of TRK-530 on rat adjuvant arthritis. Pharmacology 1998;56:242–51.
13. Osterman T, Kippo K, Hannuniemi R, Sellman R. Effect of
clodronate on established collagen-induced arthritis in rats. Inflamm Res 1995;44:258–63.
14. Zhao H, Shuto T, Hirata G, Iwamoto Y. Aminobisphosphonate
(YM175) inhibits bone destruction in rat adjuvant arthritis. J Orthop Sci 2000;5:397–403.
15. Takahashi M, Koike J, Kawabe N, Nakadate-Matsushita T. Inhibitory effect of TRK-530 on inflammatory cytokines in bone
marrow of rats with adjuvant arthritis. Pharmacology 1998;56:
237–41.
16. Francis MD, Hovancik K, Boyce RW. NE-58095: a diphosphonate
which prevents bone erosion and preserves joint architecture in
experimental arthritis. Int J Tissue React 1989;11:239–52.
17. Dunn CJ, Doyle DV, Willoughby DA. Investigation of the acute
and chronic anti-inflammatory properties of diphosphonate using
a broad spectrum of immune and non-immune inflammatory
reactions. Drug Dev Res 1993;28:47–55.
18. Eggelmeijer F, Papapoulos SE, van Paassen HC, Dijkmans BAC,
Breedveld FC. Clinical and biochemical response to single infusion
of pamidronate in patients with active rheumatoid arthritis: a
double blind placebo controlled study. J Rheumatol 1994;21:
2016–20.
19. Maccagno A, Di Giorgio E, Roldan EJ, Caballero LE, Perez
Lloret A. Double blind radiological assessment of continuous oral
pamidronic acid in patients with rheumatoid arthritis. Scand
J Rheumatol 1994;23:211–4.
20. Tan PLJ, Ames R, Yeoman S, Ibbertson HK, Caughey DE. Effects
of aminobisphosphonate infusion on biochemical indices of bone
metabolism in rheumatoid arthritis. Br J Rheumatol 1989;28:
325–8.
21. Engelhardt G, Homma D, Schnitzler C. Meloxicam: a potent
inhibitor of adjuvant arthritis in the Lewis rat. Inflamm Res
1995;44:548–55.
22. Gasser JA. Assessing bone quantity by pQCT. Bone 1995;17 Suppl
4:145S–54S.
23. Sato M. Comparative x-ray densitometry of bones from ovariectomized rats. Bone 1995;17 Suppl 4:157S–62S.
24. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H,
Meunier PJ, et al. Bone histomorphometry: standardization of
nomenclature, symbols, and units. J Bone Miner Res 1987;2:
595–610.
25. Jee WS, Mori S, Li XJ, Chan S. Prostaglandin E2 enhances cortical
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
1741
bone mass and activates intracortical bone remodeling in intact
and ovariectomized female rats. Bone 1990;11:253–66.
Fleisch H. Bisphosphonates: preclinical. In: Fleisch H, editor.
Bisphosphonates in bone disease: from the laboratory to the
patient. New York: Academic Press; 2000. p. 27–66.
Morii H, Nishizawa Y, Taketani Y, Nakamura T, Itabashi A,
Mizunuma H, et al. A randomized controlled trial with ONO-5920
(minodronate/YM529) in Japanese patients with postmenopausal
osteoporosis [abstract]. J Bone Miner Res 2002;17 Suppl 1:S471.
Yoshida Y, Morita A, Kitamura K, Inazu M, Okimoto N, Okazaki
Y, et al. Responses of trabecular and cortical bone turnover and
bone mass and strength to bisphosphonate YH529 in ovariohysterectomized beagles with calcium restriction. J Bone Miner Res
1998;13:1011–22.
Virtama P, Helela T, Kalliomaki JL. Osteoporosis in rheumatoid
arthritis: a follow-up study. Acta Rheumatol Scand 1968;14:
276–84.
Moran EL, Fornasier TL, Bogoch TR. Pamidronate prevents bone
loss associated with carrageenan arthritis by reducing resorptive
activity but not recruitment of osteoclasts. J Orthop Res 2000;18:
873–81.
Podworny NV, Kandel RA, Renlund RC, Grynpas MD. Partial
chondroprotective effect of zoledronate in a rabbit model of
inflammatory arthritis. J Rheumatol 1999;26:1972–82.
Hayashida K, Ochi T, Fujimoto M, Owaki H, Shimaoka Y, Ono K,
et al. Bone marrow changes in adjuvant-induced and collageninduced arthritis: interleukin-1 and interleukin-6 activity and
abnormal myelopoiesis. Arthritis Rheum 1992;35:241–5.
Cantatore FP, Acquista CA, Pipitone V. Evaluation of bone
turnover and osteoclastic cytokines in early rheumatoid arthritis
treated with alendronate. J Rheumatol 1999;26:2318–23.
Fujimoto M, Hayashida K, Ochi T, Owaki H, Shimaoka Y,
Okamura M, et al. Fluctuation of interleukin-1 and -6 activity in
bone marrow serum in collagen-induced arthritis in rats. Biomed
Res 1992;13:243–51.
Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M,
Kotake S, et al. Tumor necrosis factor alpha stimulates osteoclast
differentiation by a mechanism independent of the ODF/RANKLRANK interaction. J Exp Med 2000;17:275–86.
Azuma Y, Kaji K, Katogi R, Takeshita S, Kudo A. Tumor necrosis
factor-alpha induces differentiation of and bone resorption by
osteoclasts. J Biol Chem 2000;18:4858–64.
Fujimoto M, Ochi T, Owaki H, Wakitani S, Suzuki R, Takai M, et al.
Elevated activity of interleukins-1, -2, and -3 in the bone marrow of
collagen-induced arthritic rats. Biomed Res 1988;9:401–7.
Szekanecz Z, Halloran MM, Volin MV, Woods JM, Strieter RM,
Haines KG III, et al. Temporal expression of inflammatory
cytokines and chemokines in rat adjuvant-induced arthritis. Arthritis Rheum 2000;43:1266–77.
Bogoch E, Gschwend N, Bogoch B, Rahn B, Perren S. Juxtaarticular bone loss in experimental inflammatory arthritis. J Orthop
Res 1988;6:648–56.
Rogers MJ, Frith JC, Luckman SP, Coxon FP, Benford HL,
Monkkonen J, et al. Molecular mechanisms of action of bisphosphonates. Bone 1999;24 Suppl 5:73S–9S.
Документ
Категория
Без категории
Просмотров
2
Размер файла
211 Кб
Теги
acid, induced, 5920, ono, density, bones, adults, effect, arthritis, rats, collagen, mineraly, minodronic
1/--страниц
Пожаловаться на содержимое документа