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: email@example.com. 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. 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