Effect of clodronate treatment on established bone loss in ovariectomized rats.

The ability of clodronate to prevent bone loss and weakening of bone strength was studied in adult rats with established osteopenia. Six-month-old female Sprague Dawley rats were randomized into 13 groups. One group was killed at the start of the study, nine groups were ovariectomized (ovx), and three groups sham-operated (sham). After 4 months, the ovx rats were given either clodronate or vehicle subcutaneously (s.c.), once a week for 3 or 6 months, the cumulative doses of both dosing regimens being 36, 84, and 300 mg/kg. Clodronate reduced the increase in bone turnover as evidenced by serum osteocalcin and urinary deoxypyridinoline. Cancellous bone loss was more severe in distal femur than in lumbar vertebral body already at 4 months after ovx. Cortical osteopenia of femoral middiaphysis was significant at 7 and 10 months after operation and was in accordance with the impaired bending strength of the femoral shaft. In the tibia, the bending strength was, by contrast, increased at each timepoint after ovx. In distal femur, higher values of cancellous bone volume (BV/TV) were found after 6 months of clodronate treatment than in ovx/vehicle-treated rats. In lumbar vertebrae, only the lowest dose of clodronate slightly counteracted the ovx-induced further decrease in BV/TV, but reduced, at all dosages, the impairment of lumbar vertebral compression strength. The maximum load of femoral neck did not differ between vehicle-treated ovx and sham groups after clodronate treatment, but clodronate reduced the weakening of femoral shaft. A further increase in the bending strength of the tibia was found after clodronate treatment. There was a positive correlation between bending strength and ash weight in both the tibia and the femur. Histomorphometry further showed that long-term use of clodronate does not impair bone mineralization or affect modeling-dependent bone formation. In conclusion, clodronate treatment clearly slows down the progress of bone loss and prevents further weakening of bone strength in femoral shaft and vertebrae, even though it cannot completely reverse the effects of ovariectomy-induced changes in established osteopenia.

Clodronate prevents bone loss in aged ovariectomized rats.

The purpose of this study was to investigate the ability of clodronate to prevent ovariectomy (OVX)-induced osteopenia in aged rats. Fourteen-month-old female Sprague-Dawley rats (n = 166) were randomized into six groups. One group was sacrificed at the start of the study, four groups were ovariectomized, and one group was sham-operated (Sham). The OVX rats were given subcutaneously either vehicle (veh) or clodronate at doses of 3, 7, or 25 mg/kg once a week for 3 months, and the Sham rats were given the vehicle. At all dose levels clodronate inhibited trabecular bone loss in the distal femur and in the fourth lumbar vertebral body (L4), and decreased bone resorption as evidenced by urinary deoxypyridinoline excretion. The lowest dose of clodronate preserved serum osteocalcin and endosteal bone formation of secondary spongiosa in L4 at the level of the Sham/veh group. The OVX-induced increase in periosteal bone formation of femoral diaphysis was unaffected by two smaller doses of clodronate, but was decreased to the level of Sham rats after the highest dose. After 3 mg/kg clodronate, the percentage of femoral cortical bone area and the mean relative cortical thickness were higher compared with the OVX/veh group. There was a good positive correlation between the maximum load in three-point bending of the tibia and tibial ash weight. Normal lamellar pattern of newly formed cancellous and cortical bone was found after clodronate treatment. No signs of adverse accumulation of osteoid or any deleterious effect on mechanical strength of long bones and lumbar vertebrae were found.

Slow-release clodronate in prevention of inflammation and bone loss associated with adjuvant arthritis.

The effects of slow-release calcium clodronate on rat adjuvant arthritis were investigated using two different dosing schedules. In prophylactic treatment, calcium clodronate was given on the same day as the adjuvant injection, and in therapeutic treatment, calcium clodronate administration was delayed until the animals had active disease, to day 14 postadjuvant. Calcium clodronate was given as single i.m. injections into the thigh muscles. Arthritis index, histopathology of hindpaw, quantitative histomorphometry, bone mineral density and serum osteocalcin, alkaline phosphatase and calcium were studied. Calcium clodronate given therapeutically decreased the severity of paw swelling slightly more than prophylactic treatment, a result seen as lower scores of arthritis index. Histopathological evaluation of hindpaws showed that calcium clodronate protected against inflammation-induced bone loss and reactive bone formation in the hindpaw, but not against inflammatory changes involving articular cartilage. Quantitative histomorphometric analysis of the distal femur indicated that trabecular bone area was decreased by 86% in arthritic rats compared with normal untreated controls. Both the prophylactic and the therapeutic treatment with calcium clodronate prevented this osteopenia (P < .001). Bone mineral density measured by computed tomography was also significantly reduced in distal femoral metaphysis in adjuvant arthritic rats, but restoration to virtually normal values occurred with calcium clodronate (P < .001). In both dosing schedules, we observed a suppression of arthritis, which was associated with a decrease in paw swelling and an inhibition of the severe osteopenia in the distal femoral metaphysis. The long duration of action after a single injection of calcium clodronate indicates that the insoluble salt remains at the injection site and is released slowly into the bloodstream.

Distribution of clodronate in the bone of adult rats and its effects on trabecular and cortical bone.

Distribution of clodronate in cancellous and cortical bone of the femur and in cancellous bone of lumbar vertebrae in adult rats was examined by means of quantitative autoradiography. In addition, the effects of clodronate on cancellous and cortical bone were evaluated by bone histomorphometry. Six-month-old male rats were given a mixture of unlabeled and 14C-labeled disodium clodronate subcutaneously on 5 consecutive days at cumulative doses of 125 mg/50 microCi/kg or 250 mg/100 microCi/kg and followed up for 2, 23 or 79 days after the last dose. The highest activity of 14C-clodronate was found in the primary spongiosa of the distal femoral metaphysis and in the cortical bone of the femoral diaphysis. Radioactivity in the lumbar vertebra was found to be about half of that in the femur. No marked decrease in radioactivity was found in bone specimens taken after the follow-ups. In these specimens, however, labeled clodronate originally incorporated into the primary spongiosa was situated further away from the growth plate because of longitudinal bone growth. A cross-section of the femoral shaft showed that incorporation of clodronate was more prominent into the periosteal surface than into the endocortical surface. No marked histological effects were seen, except for an increase in the mineralized hard tissue area in the primary spongiosa of the distal femur.

Bisphosphonates clodronate and etidronate in the prevention of ovariectomy-induced osteopenia in growing rats.

The aim of this study was to evaluate the ability of the bisphosphonate compounds clodronate and etidronate to prevent ovariectomy-induced bone changes. Three-month-old Sprague-Dawley rats were either ovariectomized (OVX) or sham-operated (SHAM) and further divided into groups receiving either vehicle (n = 30), 25 mg/kg/week of clodronate (n = 25) or 25 mg/kg/week of etidronate (n = 25). The subcutaneous drug administration was started immediately after the surgery and was continued for 12 weeks. OVX rats had accelerated bone turnover rates compared with the SHAM animals, as indicated by the results of dynamic histomorphometry and biochemical markers in serum and urine. Femoral and vertebral mineralized trabecular bone volume and maximum loads in compressions of the femoral neck and lumbar vertebra were lower after OVX compared with the SHAM operation. Both clodronate and etidronate prevented the decrease in trabecular bone volume and suppressed the increase in the bone turnover rate. Clodronate and etidronate also blocked the loss of bone strength in the femoral neck and lumbar vertebra of OVX rats. Both compounds resulted in an absence of double fluorochrome labels on the endocortical surface of the femoral metaphysis, which seemed, however, to be a dose-dependent response. Furthermore, etidronate also lowered serum osteocalcin and diaphyseal endocortical bone formation below the vehicle level both in the OVX and SHAM rats. In conclusion, clodronate and etidronate were effective in preventing the estrogen deficiency-induced decreases in trabecular bone volume and bone strength in rats. Treatment with a high dose of clodronate induced minor signs of abnormally low bone formation but not any impairment of bone mineralization, whereas both of these events were seen with high-dose etidronate administration.

The effects of clodronate on increased bone turnover and bone loss due to ovariectomy in rats.

The present study was carried out to investigate the ability of clodronate to inhibit ovariectomy-induced bone loss and increased bone turnover in rats. Estradiol was administered as a reference compound. Seventy Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (Sham) at the age of 90 days and divided into seven groups. Two Sham and two OVX groups received subcutaneously either the vehicle of clodronate or the vehicle of estradiol. Other OVX groups were given s.c. either disodium clodronate at two dose levels (5 mg/kg or 12.5 mg/kg twice a week) or 17 beta-estradiol (10 micrograms/kg five times a week) for 8 weeks. Femur length, volume, dry weight, and ash weight were determined, and proximal ends of tibiae were used for bone histomorphometry. Markers of bone metabolism were measured from urine and serum. A significant loss of 54% of trabecular bone area of proximal tibial metaphysis was found at 8 weeks after ovariectomy. Clodronate and estradiol inhibited (p < 0.001) this osteopenia. Both drugs prevented the decrease in ash weight/volume of the femur. The inhibitory effect of clodronate and estradiol on bone resorption in OVX rats could be detected also in decreased urinary excretion of hydroxyproline and lysylpyridinoline (p < 0.001). Clodronate and estradiol decreased (p < 0.001) the ovariectomy-induced enhanced tibial endocortical mineral apposition rate (Ec.MAR) on the lateral cortex to the level of the Sham group. In contrast, periosteal MAR analyzed on the medial side of tibial cortical bone did not change significantly in the OVX/Veh group. Estradiol decreased periosteal MAR to below the level in the Sham group (p < 0.01). These results suggest that ovariectomy of growing rats resulted in tibial and femoral osteopenia two months later. Clodronate as well as estradiol can suppress bone resorption and turnover in ovariectomized rats, inhibiting the development of osteopenia. Both clodronate doses (5 and 12.5 mg/kg) had beneficial effects in ovariectomized animals.