Ibuprofen controlled-release formulation. A clinical trial in dental impaction pain

The effects of 17 beta-estradiol (E2) and treadmill exercise on tibial bone mass, tibial uptake of 45Ca, and proximal tibia osteoblast and osteoclast cell number were determined in adult ovariectomized rats. Female rats aged 10 months were ovariectomized and divided into five groups: (1) sham-operated; (2) ovariectomized; (3) ovariectomized, given 10 micrograms E2 biweekly; (4) ovariectomized, trained to exercise on a treadmill daily; and (5) ovariectomized, given E2 and exercised. E2 and/or exercise interventions were started 2 months following surgery and continued for 4 months. The calcium content of the tibial metaphysis and diaphysis and the proximal cancellous bone (BV/TV) were lower in the ovariectomized than in the sham-operated controls 6 months after ovariectomy. This lower bone content was associated with a greater bone uptake of 45Ca and a greater number of osteoblasts and osteoclasts in the proximal tibia compared to the control rats. The metaphyseal calcium content was higher and the 45Ca uptake and osteoblast and osteoclast number were lower in the E2-treated rats than in the nontreated rats. In the exercised group, higher diaphyseal calcium content and proximal cancellous bone were associated with lower bone resorption parameters without a significant effect on bone formation. This study demonstrates that E2 primarily influences tibial cancellous bone of the ovariectomized rat and a positive adaptation to exercise occurs in both cancellous and cortical bone. Under estrogen deficiency, E2 replacement suppresses increased bone formation and resorption; exercise suppresses mainly bone resorption. The effects of E2 replacement and exercise training are independent and additive.     

Effects of prostaglandin E2 and F2 alpha on the skeleton of osteopenic ovariectomized rats

This article contains the histomorphometric evaluation of the effects of prostaglandin F2 alpha (PGF2 alpha) on cancellous bone from the lumbar vertebra and cortical bone from the tibial shaft of ovariectomized, osteopenic rats. These effects were then compared with those of prostaglandin E2 (PGE2). Three-month-old rats were either ovariectomized (ovx) or sham-ovx. Then, either PGF2 alpha or PGE2 in doses of 1 and 3 mg/kg/day was given subcutaneously for 21 days at 150 days post ovx. Histomorphometric analysis was performed separately on both the primary and secondary spongiosae of the fourth lumbar vertebral bodies (LVB) and on tibial shafts. The ovx rats exhibited osteopenia in both primary (-23% to -37%) and secondary (-20%) spongiosae of the LVB, but not in the tibial shafts at 150 and 171 days post ovx. In the LVB, PGE2 in doses of 1 or 3 mg/kg/day for 21 days restored trabecular bone volume to the levels of sham-ovx controls in the primary spongiosa. However, in the secondary spongiosa, the treatments only thickened the trabeculae. The effects of the PGF2 alpha treatment were similar to those of the PGE2 in both the primary and the secondary spongiosae. While both PGF2 alpha and PGE2 treatments stimulated bone formation in the LVB as indicated by the increases in labeled perimeter, tissue and bone area-based bone formation rates, PGE2 is about 10 times more potent than PGF2 alpha in these effects. The PGE2 treatment also elevated activation frequency in the LVB, while the PGF2 alpha treatment did not. The treatments differed in that PGE2 at these dose levels did not alter the eroded surface in the LVB while PGF2 alpha decreased it significantly. Thus, the increase of the ratio of labeled to eroded perimeter in the LVB in PGF2 alpha-treated animals was much more than that in PGE2-treated animals. In the tibial shafts, PGE2 in doses of 1 and 3 mg/kg/day produced new marrow trabeculae in 2 of 6 and 3 of 6 of the ovx rats. However, no new trabecula was found in PGF2 alpha-treated tibial shafts. Higher doses of PGE2 also increased periosteal labeled perimeter, MAR, and BFR/BS, while PGF2 alpha did not produce any significant change in these parameters. Both PGE2 and PGF2 alpha in doses of 1 and 3 mg/kg/day increased the labeled perimeter, MAR and BFR/BS and decreased the eroded perimeter in the endocortical surface. We concluded that both PGF2 alpha and PGE2 in doses of 1 and 3 mg/kg/day for 21 days exhibited anabolic bone effects. The effects were mostly confined to an increase in trabecular volume in the primary spongiosa of the LVB and in the endocortical surface of tibial shafts. The tissue level mechanism behind this appears to be that PGE2 and PGF2 alpha can both stimulate osteoblast recruitment and activity. Overall, we found PGE2 to be more potent than PGF2 alpha at the same dose level at the endocortical surface. Furthermore, new marrow trabecular bone formed only after PGE2 treatment. PGF2 alpha differed from PGE2 by significantly reducing the trabecular eroded surface in ovx rats.     

SPECT perfusion imaging after coronary artery bypass grafting

The effects of progestins on bone loss in female oophorectomized (ovx) rats were evaluated. One-year-old Sprague-Dawley rats were divided into eight groups: (1) beginning controls (control); (2) sham-operated controls (sham); (3) ovx; (4) ovx treated with estrogen (ovx + E); (5) ovx treated with progesterone (ovx + P); (6) ovx treated with estrogen and progesterone (ovx + E + P); (7) sham group treated with estrogen (sham + E); and (8) sham group treated with progesterone (sham + P). Immediately after surgery, the rats in the hormone injected groups were subcutaneously (s.c.) injected daily for 15 weeks with estrogen (17-beta-estradiol, 0.01 mg/kg in ethanol), or progesterone (4-pregnene-3,20-dione, 0.1 mg/kg in ethanol), or both. At the end of 15 weeks, the bone mineral density (BMD) and bone histomorphometry of the rats' lumbar vertebrae and serological parameters were measured. In the sham, ovx, and ovx + P groups, treatment with progesterone alone did not maintain the BMD in the lumbar vertebrae, but in the ovx + E and ovx + E + P, sham + E, and sham + P groups, progesterone did not inhibit the action of estrogen in the aged ovx rat model. BMD in the sham + P group was significantly higher than in the sham group (270.8+/-10.8 mg/cm2 versus 253.6+/-10.2 mg/cm2; p < 0.01). Bone histomorphometry revealed that bone volume (BV/TV) increased more in the ovx + E + P group than in the ovx + E group and more in the sham + P group than in the sham group, but not significantly. The ovx + E, ovx + E + P, sham + E, and sham + P groups showed no significant differences in the bone formation and resorption parameters, but the bone formation variables tended to increase in the ovx + E + P and sham + P groups. We concluded that progesterone alone cannot prevent bone loss or the increase in turnover after ovx and that estrogen, not progesterone, accounted for all of the bone activity in this study. It seems doubtful that progesterone inhibits the action of estrogen, and in fact may have a beneficial effect on bone metabolism.     

Differential effects of inhibitors of cyclooxygenase (cyclooxygenase 1 and cyclooxygenase 2) in acute inflammation

The anti-inflammatory activity of drugs more selective for cyclooxgenase isoform inhibition (cyclooxygenase 1, cyclooxygenase 2), were compared in rat carrageenin-induced pleurisy. Suppression of inflammation by cyclooxygenase 2-selective inhibitors, NS-398 (N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide) and nimesulide (4-nitro-2-phenoxy-methanesulfonanilide), and by piroxicam and aspirin, more selective for cyclooxygenase 1, was measured. Piroxicam and aspirin significantly inhibited inflammatory cell influx, exudate and prostaglandin E2 formation, 6 h after carrageenin injection. Cyclooxygenase 2 inhibitors had little effect on these parameters with NS-398 alone reducing prostaglandin E2 levels, but increasing levels of leukotriene B4. In contrast, at 3 h after carrageenin injection, cyclooxygenase 2 inhibitors significantly inhibited all inflammatory parameters however suppression with piroxicam and aspirin was greater, and more pronounced than at 6 h. NS-398 and nimesulide dosing did not reduce thromboxane B2 production from platelets isolated from rats with carrageenin-induced pleurisy, demonstrating that at the doses used, cyclooxygenase 2 inhibitors did not inhibit cyclooxygenase 1, as platelets contain only this isoform. Therefore, in the rat carrageenin-induced pleurisy, drugs more selective for the inhibition of cyclooxygenase 1 attenuate inflammation over a wider time frame than cyclooxygenase 2-selective drugs, suggesting a significant role for cyclooxygenase 1 in this model. Inhibition of cyclooxygenase 2 by NS-398 however, resulted in an increase in the potent chemoattractant leukotriene B4.     

Inhaled nitric oxide therapy for pulmonary disease in pediatrics

Prostaglandin E2 (PGE2) is an anabolic agent of bone in vivo but the mechanism of its action still remains unclear. The aim of this study was to determine whether the effect of PGE2 on skeleton is mediated by pituitary hormones. Forty female, Sprague-Dawley rats were divided into four groups: baseline control (basal), age-matched intact control (CON), hypophysectomy (HX), and HX + PGE2 (2 mg/kg/day) with 10 animals in each group. The basal group was sacrificed at 2 months of age, and the remaining groups after 6 weeks of treatment. Cancellous and cortical bone histomorphometry was performed on double fluorescent-labeled 40 micron-thick sections of the proximal tibia and tibial shaft. Our results show that HX resulted in a cessation of bone growth, a decrease in cancellous bone volume, and cortical bone gain compared with the age-matched, intact CON rats. Compared with the HX group, the HX + PGE2 group had a significantly greater tibial bone density (mean +/- SE, HX + PGE2:1.595 +/- 0.007 versus HX:1.545 +/- 0.013), percent cancellous bone volume (21.4 +/- 2.0 versus 8.41 +/- 1.70), percent cortical bone area (87.2 +/- 0.85 versus 81.7 +/- 0.7), and ratio of cortical area to marrow area (7.14 +/- 0.56 versus 4.52 +/- 0.21). Increased bone masses by PGE2 in the HX animals were accompanied by an increase in the trabecular and endosteal-labeled surface and bone formation rate. The trabecular number and width were increased whereas trabecular separation was decreased in the HX + PGE2 group compared with the HX group (P < 0.05). PGE2 treatment also caused a decrease in the tibial endosteal eroded surface and medullar cavity of the HX animals. In conclusion, this study clearly demonstrates that PGE2 (2 mg/kg/day) in the HX rats increases both cortical and cancellous bones and improves trabecular architecture in the tibia after 6 weeks of treatment. These skeletal alterations are due to a stimulation of bone formation and a suppression of bone resorption activity. These findings suggest that the anabolic effect of PGE2 in bone is independent of pituitary hormones.     

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model

CI-994 (acetyldinaline) is an orally active anticancer drug currently in Phase 1 clinical trials. To assess its preclinical toxicity, CI-994 was administered orally as suspensions to Wistar rats (10/sex/dose) and in capsules to beagle dogs (3/sex/dose) once daily for two weeks. Doses were 1.5, 5, and 15 mg/kg for rats (9, 30, and 90 mg/m2, respectively), and 0.5, 2, and 5 mg/kg for dogs (10, 40, and 100 mg/m2, respectively). Systemic exposure was dose-proportional based on toxicokinetic analysis in dogs. Severe clinical signs and mortality occurred at the highest dose in both species beginning on Day 10. Neutropenia, lymphocytopenia, thrombocytopenia, lymphoid depletion, bone marrow hypocellularity, and testicular degeneration were observed in both species, primarily at the mid- and high-doses. Despite continued treatment, neutrophil counts in dogs returned to control levels in Week 2. Other microscopic findings in rats included splenic hematopoietic depletion at all doses and epithelial cell necrosis in various tissues at 15 mg/kg. Additional bone marrow changes in dogs involved myeloid and megakaryocyte hyperplasia at 2 mg/kg and abnormal myeloid and megakaryocyte maturation at 2 and 5 mg/kg. Except for the testicular effects in both species, all changes were reversible within a 4-week (rat) or 9-week (dog) recovery period. The results of these studies show that target organ effects of CI-994 principally involve tissues with rapidly dividing cell populations and that bone marrow suppression is the dose-limiting toxicity. CI-994 also seems to interfere with the release and/or maturation of cells in the bone marrow.     

The effect of exchanger inhibitory peptide (XIP) on sodium-calcium exchange current in guinea pig ventricular cells

While the osteopenia associated with oestrogen deficiency is thought to arise from a relative defect in bone formation with respect to resorption, oestrogen administration itself leads to a decrease, rather than an increase, in bone formation. This decrease in bone formation, which arises from oestrogen's inhibitory effect on bone turnover, presumably masks any underlying tendency of oestrogen treatment towards stimulation of bone formation. To investigate this further, we have examined the early effect of discontinuing the administration of oestradiol-17 beta (OE2; 40 micrograms/kg) on bone formation indices in ovariectomized 13-week-old rats, before the turnover-induced increase in formation occurs. Histomorphometric indices were assessed at the proximal tibial metaphysis 0, 7, 10, 13 and 16 days following discontinuation of OE2 treatment. Measurements of body weight, uterine weight and longitudinal growth rate confirmed that there were rapid effects of OE2 deficiency on these parameters. We could detect no significant increase in bone resorption, as measured by osteoclast surface and number, until 16 days after ending treatment with OE2; this was coincidental with a reduction in bone volume. Shorter periods of OE2 deficiency were associated with a marked decrease in bone formation, as assessed by dynamic histomorphometric indices. This inhibition of bone formation was largely due to a reduction in double fluorochrome-labeled trabecular surfaces, which were decreased by approximately 70%. We conclude that ending OE2 administration in ovariectomized rats caused a striking decrease in trabecular bone formation, if such indices are assessed prior to the subsequent turnover-induced increase in formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of effect of spaceflight on bone mass and bone formation in group-housed rats

As part of an experiment to study the role of corticosteroids in bone changes during spaceflight, male Sprague-Dawley rats (6 wk old, 165 g body weight) were placed in orbit for 17 days, in groups of six, in animal-enclosure modules (AEMs) aboard the space shuttle Columbia (STS-78). Control rats were group housed in a similar manner in ground-based AEMs or standard vivarium cages. Adrenal hypertrophy occurred in flight rats, but bone histomorphometric analyses revealed a lack of significant changes in bone mass and bone formation in these animals. Cancellous bone volume and osteoblast surface in the proximal tibial metaphysis were nearly the same in flight and ground-based rats. Normal levels of cancellous bone mass and bone formation were also detected in the lumbar vertebrae and femoral necks of flight rats. In the tibial diaphysis, periosteal bone formation rate was found to be identical in flight and ground-based rats. The results indicate that, under conditions of group housing in AEMs, spaceflight has minimal effects on bone mass and bone formation in rapidly growing rats. These findings emphasize the need to investigate the importance of rat age, strain, and especially housing conditions for studies of the skeletal effects of spaceflight.     

LY353381 x HCl: an improved benzothiophene analog with bone efficacy complementary to parathyroid hormone-(1-34)

LY353381 x HCl is a benzothiophene analog that is structurally related to raloxifene with potent selective estrogen receptor modulator activity in the ovariectomized rat model of postmenopausal osteoporosis. The effects of LY353381 x HCl on bones, body weight, and uterine weight were evaluated in 7-month-old rats with osteopenia that was induced by ovariectomizing animals for 1 month before initiation of treatment with several agents individually, in combination, or in sequence. LY353381 x HCl was administered daily by itself for 90 days, in combination with the amino-terminal fragment of PTH-(1-34) (PTH) for 90 days, or sequentially after PTH when PTH was discontinued after 45 days of treatment. Additionally, comparisons were made of animals treated with PTH alone, 17alpha-ethynyl estradiol alone, equine estrogens (Premarin) alone, raloxifene alone, or combinations of PTH and equine estrogens or raloxifene. Ovariectomy induced increases in the rate of bone turnover and body weight while decreasing bone mineral density, bone mineral content, bone strength, trabecular bone volume, trabecular thickness, trabecular number, and uterine weight. LY353381 x HCl at 0.01-1 mg/kg had marginal effects on body weight and no effect on uterine weight compared with those in ovariectomized controls, in contrast to 17alpha-ethynyl estradiol or equine estrogens. LY353381 x HCl prevented further bone loss due to ovariectomy in tibia, femora, and lumbar vertebra, like 17alpha-ethynyl estradiol but unlike equine estrogens. LY353381 x HCl prevented the resorption of trabecular bone spicules, like 17alpha-ethynyl estradiol, but inhibited bone formation activity to a lesser extent than 17alpha-ethynyl estradiol. In this model, 17alpha-ethynyl estradiol appeared to be more efficacious after 3 months of treatment than equine estrogens in the proximal tibia metaphysis, suggesting efficacy differences between metabolites of 17beta-estradiol in bone. PTH at 10 microg/kg had no effect on body weight or uterine weight, but significantly increased bone mass to beyond those in sham-operated controls, baseline controls, and groups receiving other individual treatments at both axial and appendicular sites. The combination of LY353381 x HCl and PTH increased bone mass at a faster rate and to a greater extent than PTH alone or the combinations of equine estrogens/PTH and raloxifene/PTH at trabecular bone sites. The LY353381 x HCl/PTH combination improved bone mass and quality beyond any agent alone in regions enriched for cancellous bone, but was not significantly better than PTH alone on cortical bone. Additionally, when PTH was discontinued at 45 days, LY353381 x HCl prevented the rapid loss of bone observed in controls. Therefore, LY353381 x HCl appears to be useful by itself, in combination, or in sequence with PTH to replace lost bone in postmenopausal women.     

The effects of the aminobisphosphonate alendronate on thyroid hormone-induced osteopenia in rats

Hyperthyroidism, either endogenous or iatrogenic, leads to increased bone turnover and osteopenia. This study was conducted to examine (1) whether thyroid hormone excess in rats causes bone changes similar to those seen in patients with hyperthyroidism, and (2) the effects of the aminobisphosphonate alendronate on the thyroid hormone-induced bone changes. Sprague-Dawley male rats, divided into four groups, received L-thyroxine (T4) 250 micrograms/kg/day (+T4) or vehicle (-T4) subcutaneously six times per week and alendronate 1.75 mg/kg (+ALN) or vehicle (-ALN) orally twice a week. Rats were sacrificed after 3 weeks of treatment, blood samples were analyzed for serum T4, triiodo-L-thyronine (T3), and osteocalcin, and the proximal tibiae were processed for histomorphometric analysis. Serum T4 and T3 levels measured 20-24 hours after the last injection were 2 to 2.5-fold higher in +T4 groups than in -T4 groups. Serum osteocalcin was significantly (P < 0.05) higher in +T4/-ALN group than in the other groups, which were not statistically different from each other. T4 treatment (+T4/-ALN) significantly decreased the amount of cancellous bone volume (-45%) and increased osteoid surface (+254%), osteoblast surface (+111%), and osteoclast surface (+176%) relative to control values. Alendronate increased the bone volume above control values in both T4-treated (+T4/+ALN) and untreated (-T4/+ALN) rats, and prevented the T4-induced increase in bone turnover in +T4/+ALN rats. It is concluded that (1) excess thyroid hormone induces cancellous bone loss associated with high bone turnover in the rat, and (2) this bone loss can be prevented by alendronate through the inhibition of osteoclastic activity.     

Effects of a tachykinin NK3 receptor antagonist, SR 142801, studied in isolated neonatal rat spinal cord

In the ovariectomized (ovx) rat, the nonsteroidal antiestrogens, clomiphene (CLO) and tamoxifen (TAM), at dose levels that prevent development of osteopenia to a degree approaching that of 17beta-estradiol are, in contrast to 17beta-estradiol, only weakly uterotrophic. Metabolites of CLO and TAM might contribute differentially to these effects. Thus, we have evaluated bone protective and uterine effects in ovx rats of two such metabolites: 4-hydroxy CLO, produced by p-hydroxylation of CLO; and 4HTA, produced from TAM by stepwise replacement of its dimethylaminoethyl side chain with an acetic acid moiety, accompanied by p-hydroxylation. Also reported are effects of D4HTA, the dihydrodesethyl derivative of 4HTA previously characterized as a full estrogen mimetic in vitro. Administration of 4-hydroxy CLO (2.5 mg/kg subcutaneously) 5 days/week for 5 weeks to 3-month-old ovx rats resulted in complete prevention of bone loss and suppression of bone turnover to levels comparable to those of intact controls and to those of ovx animals similarly receiving 17beta-estradiol (10 microg/kg). However, uterine weight in animals receiving 4-hydroxy CLO was 64% less than that in 17beta-estradiol-treated animals. Although 4HTA (3.7 mg/kg s.c.) had a modest uterotrophic effect, it did not prevent bone loss associated with ovariectomy. In contrast, D4HTA (3.6 mg/kg s.c.) partially reduced bone turnover indicators and cancellous bone loss in a manner similar in many ways to that observed in TAM-treated ovx animals, but it had no uterotrophic effect. These results suggest that, although 4HTA does not contribute to the bone-protective effect of TAM, 4-hydroxy CLO might augment that of CLO.     

Parathyroid hormone (1-34) increased total body bone mass in aged female rats

Daily subcutaneous administration of bovine parathyroid hormone (PTH)(1-34) stimulates bone formation and increases bone mass in rat tibiae, femora and lumbar spine. However, the effects of PTH on the whole body bone mineral content and density determined by dual energy x-ray absortiometry (DEXA) have not been previously reported in rats. Eighteen-month-old intact female rats were subcutaneously injected daily with 0, 40, 80 or 160 micrograms/kg/day of bovine PTH (1-34) for either 15 or 60 days. Whole body DEXA was performed at 1 day before autopsy, and bone area, bone mineral content (BMC) and bone mineral density (BMD) of the total body were determined. Total femoral, tibial and lumbar spine BMD was also determined ex vivo. Cancellous bone histomorphometry was performed on sections of double-labeled proximal tibial metaphyses. Whole body bone mineral content and density were significantly increased by 60 days, but not by 15 days, of PTH treatment at all dose groups compared with vehicle controls. Lumbar vertebral and total femoral BMD was significantly increased at all doses of PTH by 15 days of administration and further increased by 60 days. All doses of PTH increased trabecular bone area in proximal tibial metaphyses by 15 days and further increased by 60 days. All doses of PTH increased trabecular bone area in proximal tibial metaphyses by 15 days and further increased by 60 days. In proximal tibial cancellous bone, dose-dependent increases in percent labeled perimeter, mineral apposition rate and bone formation rate-bone volume referent were found between 40 and 160 micrograms/kg of PTH treatment by 15 days, and no further increases were found by 60 days. Our results showed that in aged female rats, bovine PTH(1-34) increased bone formation and total body bone mass.     

(S)-emopamil reduces brain edema from collagenase-induced hemorrhage in rats

BACKGROUND AND PURPOSE: Calcium channel blockers reduce edema due to cerebral ischemia, but little is known about their usefulness in hemorrhage. Therefore, we studied the effect of the calcium channel blocker (S)-emopamil in collagenase-induced hemorrhage. METHODS: Adult rats had hemorrhagic necrosis induced by the intracerebral injection of 0.4 U of bacterial collagenase. Six groups of rats were given either 10 or 20 mg/kg (S)-emopamil at different times after induction of the lesion. Brain water and electrolyte levels in these rats were measured 24 hours after collagenase injection. Also, lesion volume in other rats was measured either 4 or 24 hours after formation of the lesion with the drug given at 1 hour or both 1 and 5 hours, respectively. RESULTS: Administration of 20 mg/kg (S)-emopamil 1 hour after lesion induction significantly decreased water and electrolyte content in both posterior regions (P < .05). This beneficial effect was lost when a second 20-mg/kg dose was given at 5 hours. A single 20-mg/kg injection at 1 hour had no effect on lesion volume at 4 hours. Two doses significantly increased volume at 24 hours (P < .05). CONCLUSIONS: Early administration of (S)-emopamil is beneficial in hemorrhagic lesions, but a subsequent delayed injection may be deleterious. Knowledge of the time of hemorrhage will be important in use of these agents in treating hemorrhage.     

Epidermal growth factor receptor ectodomain in the urine of patients with squamous cell carcinoma

Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated at 3 months of age and maintained untreated for 1 year after surgery. Baseline control and OVX rats were killed at the beginning of treatment when the rats were 15 months of age and 1 year postovariectomy. The remaining rats were treated with hPTH 1-34 (80 micrograms/kg BW, 5 days/week) or vehicle for 10 weeks. Quantitative bone histomorphometry was performed on undecalcified longitudinal sections of the proximal femur from each rat. Baseline OVX rats exhibited cancellous and cortical osteopenia at the femoral neck as their mean cancellous bone volume and cortical width were significantly decreased compared to the means for baseline control rats. In addition, baseline OVX rats had increased osteoblast and osteoclast surfaces and a greater cancellous bone formation rate than baseline control rats. OVX rats remained osteopenic with no further bone loss from the femoral neck after 10 weeks of vehicle treatment. In contrast, cancellous bone volume and cortical width in OVX rats treated with PTH were increased to the level of vehicle-treated control rats. The hormone restored lost bone in the femoral neck of OVX rats by markedly stimulating both cancellous and cortical bone formation. These histomorphometric findings in concert with recent biomechanical studies of bone strength indicate that the femoral neck of aged OVX rats is a promising sample site for studies of the prevention and treatment of bone loss induced by estrogen depletion.     

Conformational and electronic properties of the two cis (5S,6R) and (5R,6S) diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymidine: X-ray and theoretical studies

For the first time, raloxifene or alendronate was administered to rats immediately after ovariectomy for 10 months and compared with estrogen to elucidate mechanisms behind the raloxifene effects observed in nonreproductive and reproductive tissues. Specifically, 75-day-old rats were randomly selected as sham controls (Sham), ovariectomized controls (Ovx) or ovariectomized rats treated with fully efficacious doses of raloxifene (RA), 17 alpha-ethynyl estradiol (EE2) or alendronate (ABP). Lumbar vertebrae and proximal tibiae were examined by computed tomography (QCT) and by histomorphometry. Histomorphometry showed differences in bone architecture between groups when QCT densities were similar, but tibial trabecular bone analysis by QCT correlated with histomorphometry with r = .86 to .93, depending on the parameter. Both techniques confirmed that Ovx had substantially less bone than Sham, with greater loss of trabecular bone in the proximal tibia than vertebrae. Both techniques showed that RA had effects similar to but not identical with EE2 in preventing bone loss in vertebrae and tibiae. ABP partially prevented loss of bone in L-5, but was not significantly different from Ovx in the proximal tibia. This may be caused by ABP suppression of bone apposition, beyond effects observed for EE2 or RA. RA appeared to be more similar to EE2 because ABP significantly depressed bone formation (bone formation rate, mineral apposition rate) to below RA or EE2 levels, especially in L-5. Mechanical loading to failure of L-6 vertebrae showed a rank order of vertebral strength of Sham > RA > EE2 > Ovx > ABP, although significant differences were not observed between treatment groups. These data show that ABP suppression of bone formation can affect bone quality with long-term treatment. In other tissues, RA had minimal uterine effects, while significantly lowering serum cholesterol to below EE2-treated levels. Both EE2 and RA rats had significantly lower body weights than the other groups. ABP had no effect on serum lipids, uterine weight or body weight. Therefore, RA appears to have a broader range of desirable effects on bone, body weight, uteri and cholesterol than ABP or EE2 in ovariectomized rats.     

Osteoblast recruitment and bone formation enhanced by cell matrix-associated heparin-binding growth-associated molecule (HB-GAM)

Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell- matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix-associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM-induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.     

Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia models

Alendronate is an aminobisphosphonate that acts as a potent inhibitor of osteoclastic bone resorption. To understand the mechanism of action of alendronate in vivo, in this study we investigated the relationship between distribution of [14C]-alendronate in rat bone and its effects on bone resorption in vitro or in rat hypercalcemic models. A single IV dose of 0.05 approximately 1.25 mg/kg inhibited the increase in plasma calcium level induced by bovine PTH or 1 alpha(OH)D3. The minimal effective dose of pamidronate (1.25 mg/kg) and etidronate (over 31.25 mg/kg) were at least 5 times and 25 times, respectively, higher than the dose of alendronate in the rat hypercalcemic model prepared by 1 alpha(OH)D3. The relative potencies of compounds in the hypercalcemic rat models reflected those of inhibitory effects on bone resorption in vitro. We conducted the ivory-slice assay under two conditions: (a) addition of a given bisphosphonate after adherence of the osteoclasts; and (b) preincubation of the ivory slices with a given bisphosphonate. The inhibitory IC50 values of alendronate under condition (b) were similar to those under condition (a). To evaluate the interaction between osteoclasts and alendronate in bone, we investigated the localization of [14C]-alendronate in the tibia of growing rats (4-day-old rats). Alendronate did not distribute uniformly in the tibia. At 1 day after injection (0.05 mg SC), dense labeling was seen primarily under osteoclasts. We injected 0.05 mg/kg of [14C]-alendronate (single i.v.) into rats [14C]-alendronate was rapidly eliminated from plasma, and mainly distributed to the bone in rats. These data suggest that alendronate which distributed on bone surface mainly contributed to the antihypercalcemic action in vivo.     

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.