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 osteopenia and loss of trabecular connectivity in estrogen-deficient rats.

Daily oral clodronate treatment was evaluated in Sprague-Dawley rats for its ability to inhibit estrogen-deficiency-induced changes in femoral neck, femoral diaphysis, and lumbar vertebrae (L4-L5). Six-month-old ovariectomized (OVX) rats were administered by gavage a vehicle (Veh) or clodronate (100 or 500 mg/kg/day). Sham-operated (SHAM) control rats received the vehicle (n = 15/group). Treatment was started on the day of operation and continued for 3 months. Trabecular bone volume (BV/TV) and structural variables (trabecular number, Tb.N; thickness, Tb.Th; separation, Tb.Sp; and trabecular bone pattern factor, Tb.Pf) were assessed on secondary spongiosa of the right femoral neck Furthermore, cantilever bending test of the left femoral neck and compression test of L4, ash weight of L5, and morphometric studies of femoral diaphysis were carried out, and serum and urinary markers of bone turnover were determined. The OVX/Veh group had higher levels of serum osteocalcin and alkaline phosphatase and higher urinary excretion of deoxypyridinoline/creatinine than the SHAM/Veh group at 3 months postsurgery, and clodronate reduced these changes. BV/TV of femoral neck, bone mass of L5, and the maximum loads of the femoral neck and L4 were lower after OVX than SHAM operation. Although clodronate prevented trabecular bone loss in the femoral neck and preserved Tb.Pf at the SHAM control level, it failed to preserve the mechanical strength at the femoral neck However, in lumbar vertebrae, clodronate prevented the loss of bone mass and mechanical properties. Furthermore, there was a good positive correlation between maximum load of L4 and the ash weight of L5 (n = 58, r = 0.69, p < 0.001). In the femoral neck (n = 55), Tb.Pf correlated negatively with BV/TV and Tb.N (r = -0.59 and r = -0.55;p < 0.001, respectively) and positively with Tb.Sp (r = 0.61, p < 0.001). In femoral mid-diaphysis, there were no significant changes in cortical bone geometry in any of the groups. We conclude that orally administered clodronate suppresses the enhanced bone turnover in adult OVX rats and preserves trabecular bone volume and connectivity in the femoral neck In the axial skeleton, clodronate has a beneficial effect on lumbar vertebral bone mass and strength.

The effect of orally administered clodronate on bone mineral density and bone geometry in ovariectomized rats.

The effects of clodronate administered p.o. on bone mineral density (BMD), bone geometry and strength of bone were investigated in 6-month-old ovariectomized rats. Sixty Sprague-Dawley rats were randomized into four groups. Three groups were ovariectomized (OVX) and one group was sham-operated (SHAM). The OVX groups were given p.o. either clodronate (100 mg/kg/d or 500 mg/kg/d) or a vehicle. The SHAM group received the vehicle. Treatments started on the day of OVX and continued for 3 months. BMD of proximal tibial metaphysis was measured by computed tomography in vivo 1 day before OVX and 6 and 12 weeks after OVX. At the end of the study, left tibiae and femora were removed for ex vivo BMD and bone geometry measurement. A three-point bending test of the tibial shaft was carried out, and ash weights of femur and tibia were determined. OVX induced a marked decrease in total and trabecular BMD over time at the proximal tibial metaphysis. This bone loss was prevented by clodronate. Clodronate also prevented the decrease in BMD and change in bone geometry at distal and proximal femur, as well as the decrease in total ash weight of femur and tibia. OVX did not cause any marked changes in cortical BMD or bone geometry at the level of mid-diaphysis of tibia or femur over a 3-month period. Neither were there any changes between groups in bending strength in the tibial diaphysis. However, a positive correlation (n = 58, r = 0.51, P < .001) was found between bending strength and calculated density-weighted polar moment of resistance of tibial diaphysis. We conclude that clodronate administered p.o. in adult rats prevents changes due to estrogen deficiency in BMD and bone geometry.

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.

A comparison of clodronate and indomethacin in the treatment of adjuvant arthritis.

OBJECTIVE AND DESIGN: The therapeutic effects of an anti-resorptive agent, clodronate, were compared with the effects of an anti-inflammatory agent, indomethacin, in rat adjuvant arthritis after therapy and after a follow-up time of two weeks. SUBJECTS: Eighty-one male Lewis rats, 6-7 weeks old, were immunized with heat-killed mycobacteria. TREATMENT: Fourteen days after immunization the animals were treated either with clodronate (50 mg/kg/day, subcutaneously), indomethacin (3 mg/kg/day, orally) or saline (controls) for two weeks. METHODS: Clinical signs of arthritis including the severity of paw swelling were assessed, biochemical variables were measured, and histological features of the non-decalcified tarsus with ankle, intertarsal and tarsometatarsal joints were evaluated for inflammatory soft-tissue, articular, and bone changes. RESULTS: The results indicated that clodronate and indomethacin suppressed significantly the intensity of inflammation, activity of beta-N-acetylglucosaminidase in inflamed hindpaw tissue, serum ICTP (cross-linked carboxyterminal telopeptide of type 1 collagen) level and bone lesions in the tibiotarsal region. The level of serum osteocalcin was also significantly decreased by clodronate. The inhibitory effect of clodronate against arthritic bone changes occurred, however, earlier and was slightly more potent than the effect of indomethacin, while indomethacin was slightly more effective in reducing paw swelling. Both drugs preserved their therapeutic effects during the follow-up time of two weeks. CONCLUSIONS: Clodronate and indomethacin have fairly similar efficacy in suppressing the intensity of joint swelling and preventing bone lesions in adjuvant arthritic rats.

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.

Effect of clodronate on established collagen-induced arthritis in rats.

The collagen-induced arthritis model in rats was used to study the effect of disodium clodronate on inflammation and destruction of tarsal, metatarsal, and interphalangeal bones and joints. Female DA rats were immunized with heterologous type II collagen. Fourteen days after immunization, rats with similar scores were assigned to the different experimental groups. They were treated subcutaneously either with saline (controls) or with clodronate at doses of 12.5 and 25 mg/kg/day five times a week for 2 weeks. Clinical signs of arthritis including the severity of paw swelling were assessed weekly. At the time of killing, histological features of the non-decalcified tarsus with tarsal, tarsometatarsal and interphalangeal joints were assessed for inflammatory soft-tissue, articular, and bone changes. All the arthritic control rats developed severe arthritis as shown by the total histological scores of the hindpaw. The treatment with clodronate (25 mg/kg) decreased clinical signs of arthritis, the activity of the collagen-degrading lysosomal enzyme, beta-N-acetylglucosaminidase, in inflamed hindpaw tissue, serum osteocalcin level and serum cross-linked telopeptide of type I collagen level. Histological evaluation indicated moderate arthritis in 29% of the rats and severe arthritis in 71%. The results show that clodronate given therapeutically to arthritic rats, induced with type II collagen, suppresses the intensity of inflammation and bone lesions in the tibiotarsal and tarsometatarsal regions.

Effect of iron on the absorption and distribution of clodronate after oral administration in rats.

The effect of iron on the absorption and distribution of disodium clodronate in rats after oral administration was studied. Disodium clodronate (300 mg/25 microCi/kg) was given both alone and with an equivalent amount of ferrous sulphate. The radioactivity in plasma and various tissue was measured. Concentration of clodronate in plasma was also determined with the GC-mass-selective detection method and the values compared with those measured with the isotope method. After administration, clodronate was rapidly cleared from plasma. Most of the dose was taken up by bone and only small amounts were found in non-calcified tissues. Concurrent ingestion of iron caused a marked decrease in the absorption of clodronate.

Effect of clodronate on established adjuvant arthritis.

The rat adjuvant arthritis model was used to study the effect of disodium clodronate on inflammation and destruction of tarsal bones and joints. Male Lewis rats were given an intradermal injection of mycobacteria. Fourteen days after immunization, rats with similar scores were assigned to the different experimental groups. They were treated subcutaneously either with saline (controls) or with clodronate at doses of 12.5 and 25 mg/kg/day five times a week for 2 weeks. Clinical signs of arthritis including the severity of paw swelling were assessed weekly. At the time of sacrifice, histological features of the non-decalcified tarsus with ankle, intertarsal and tarsometatarsal joints were assessed for inflammatory soft-tissue, articular and bone changes. The total histological score of the hindpaw indicated that 58% of the control rats developed moderate arthritis and 42%, severe arthritis. The treatment with clodronate (25 mg/kg) decreased clinical signs of arthritis and the activity of the collagen-degrading lysosomal enzyme, beta-N-acetylglucosaminidase, in inflamed hindpaw tissue. Histological evaluation indicated moderate arthritis in 83%, but no severe arthritis. The lower dose of clodronate also decreased the severity of the disease; the decrease was, however, statistically insignificant. The results show that clodronate given therapeutically to adjuvant arthritic rats suppresses the intensity of the inflammation and prevents secondary articular and bone lesions in the tibiotarsal region.

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test.

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.

First-pass metabolism of nomifensine in dogs.

Nomifensine (1 and 5 mg/kg) was administered to dogs orally and intravenously. The pharmacokinetics of the drug was evaluated. Nomifensine was rapidly absorbed from the gastro-intestinal tract reaching maximum concentration at 0.5-1 h. The peak levels were directly proportional to the doses administered. The elimination half-life was 6 h and only very small amounts were found in blood at 24 h after administration. The apparent volume of distribution (Vd) was 120-149 1, suggesting an extensive distribution of the drug throughout body fluids and tissues. The area under the serum concentration-time curve (AUC) obtained after oral administration was significantly smaller than that after intravenous administration indicating incomplete bioavailability of the drug in oral form. The conjugation of nomifensine after the two different administration routes was also studied: the conjugation reaction was in equilibrium at 15 min after oral administration, while after intravenous administration, equilibrium was not reached until 1-1.5 h. The metabolism of nomifensine occurred in the gastrointestinal membranes and or in the liver during the absorption process; the first-pass effect was marked.

Kidney toxicity of 3-methylxanthine in the rat.

The effects of 3-methylxanthine, the pharmacologically active metabolite of theophylline, on the kidneys of Wistar rats after short-term administration were studied. 3-Methylxanthine was administered in oral doses of 0 (control), 50, 100 and 200 mg per kg per day for 1, 8 and 16 days. The kidneys were examined by light and electron microscopy. Tubular necrosis was noticed at a dose level of 100 mg kg-1 after 16 days and at a dose level of 200 mg kg-1 after 8 days. Elevated values of serum urea were found after 1 day of treatment with a dose of 200 mg kg-1 and after 16 days with a dose of 100 mg kg-1. Elevated values of serum creatinine were detected after 8 days of treatment with a dose of 200 mg kg-1. The results indicate dose- and time-related renal failure following administration of 3-methylxanthine.

Antimuscarinic activity of oxybutynin in the human plasma quantitated by a radioreceptor assay.

The antimuscarinic activity of oxybutynin was measured as oxybutynin equivalents by a radioreceptor assay (RRA). The activity was studied in plasma samples of five volunteers after a single oral dose (10 mg) or after a single intravenous dose (28 micrograms/kg) of oxybutynin hydrochloride. The results were compared to the concentrations of the drug measured by gas liquid chromatography (GLC). Following oral administration, the maximum concentration measured by RRA was significantly higher (706 nmol/l) than that by GLC (38 nmol/l). In contrast, equal concentrations were measured after intravenous administration by both methods. Metabolites with antimuscarinic activity are possibly formed through first-pass metabolism after orally administered oxybutynin. The total antimuscarinic activity of oxybutynin and its metabolites are measured by RRA, but only the parent drug by GLC.

Induction of microsomal epoxide hydrolase by tienilic acid in the rat.

The effects of tienilic acid [2,3-dichloro-4-(2-thienylcarbonyl)phenoxy acetic acid] on drug-metabolizing enzymes in the rat liver and kidneys were studied. Short-term treatment for 2 weeks (450 mg per kg per day) increased the activity of microsomal epoxide hydrolase at least two-fold in both rat liver and kidneys. The effect of tienilic acid on the activity of microsomal epoxide hydrolase in the rat liver correlated with the dose at levels of 20-450 mg per kg per day for 14 days (r = 0.92). Tienilic acid had only marginal effects on cytochrome-P-450-mediated mono-oxygenases. Tienilic acid caused an approximately two-fold increase in glucuronide conjugation of 4-methylumbelliferone in the liver. No increase in the activity of rat hepatic microsomal UDP-glucuronosyltransferase toward O-aminophenol was detected. According to these results, tienilic acid can be regarded as one of the most specific inducers of microsomal epoxide hydrolase.