Treatment with a potent cathepsin K inhibitor preserves cortical and trabecular bone mass in ovariectomized monkeys.

The cysteine protease cathepsin K is involved in osteoclast-mediated bone resorption. We evaluated the effect of daily oral dosing of an inhibitor of human cathepsin K (SB-462795 [relacatib]) for 9 months on bone turnover, mass, and architecture in estrogen-deficient cynomolgus monkeys. Ovariectomized animals were treated orally with relacatib at 1, 3, or 10 mg/kg/day, or oral vehicle plus alendronate at 0.05 mg/kg by IV injection once every 2 weeks. The control groups, ovariectomized and sham-ovariectomized animals, received vehicle (all groups n = 20 animals). Samples for biomarker analysis were collected at various times, bone mass changes were evaluated at 6 and 9 months of treatment, and histomorphometric analysis was performed at 9 months. Relacatib significantly reduced urinary N-telopeptide excretion within 1 week of treatment at all dose levels, an effect that was maintained at the highest dose level. At some time points bone formation markers were elevated at the lowest dose of relacatib. Animals treated with relacatib had dose-dependent preservation of areal bone mineral density reaching statistical significance in distal femur. In femur neck there was significant preservation of total volumetric BMD (vBMD) by relacatib. By histomorphometry, relacatib reduced indices of bone resorption and formation at cancellous sites as did alendronate. In cortical bone, osteonal bone formation rate was reduced by alendronate but preserved at low and medium doses of relacatib. Thus, relacatib preserved cortical and cancellous bone mass in ovariectomized monkeys.

Increased osteoblastogenesis and decreased bone resorption protect against ovariectomy-induced bone loss in thrombospondin-2-null mice.

Although bone is composed primarily of extracellular matrix (ECM), the dynamic role that the ECM plays in regulating bone remodeling secondary to estrogen loss is relatively unexplored. Previous studies have shown that mice deficient in the matricellular protein thrombospondin-2 (TSP2-null) form excess endocortical bone; thus, we postulated that enhanced bone formation in TSP2-null mice could protect against ovariectomy (OVX)-induced bone loss. Wild-type (WT) OVX mice showed a significant loss of both midfemoral endocortical and proximal tibial trabecular bone, but OVX did not significantly alter TSP2-null bone. TSP2-null mice showed an increase in bone formation, as indicated by a 70% increase in serum osteocalcin two weeks post OVX and a two-fold increase in bone formation rate (BFR) five weeks post OVX as measured by dynamic histomorphometry. WT animals showed only a 20% increase in serum osteocalcin at two weeks and no change in BFR at five weeks. This increase in bone formation in TSP2-null OVX mice was accompanied by a three-fold increase in osteoprogenitor number. Although these results provide a partial explanation for the maintenance of bone geometry post-OVX, TSP2-null mice five weeks post-OVX also showed a significantly lower level of bone resorption than OVX WT mice, as determined by serum levels of the amino-terminal telopeptide of type I collagen (NTx). We conclude that the absence of TSP2 protects against OVX-induced bone loss by two complementary processes: increased formation and decreased resorption.

A potent small molecule, nonpeptide inhibitor of cathepsin K (SB 331750) prevents bone matrix resorption in the ovariectomized rat.

Inhibition of the cyteine proteinase, cathepsin K (E.C. 3.4.22.38) has been postulated as a means to control osteoclast-mediated bone resorption. The preferred animal models for evaluation of antiresorptive activity are in the rat. However, the development of compounds that inhibit rat cathepsin K has proven difficult because the human and rat enzymes differ in key residues in the active site. In this study, a potent, nonpeptide inhibitor of rat cathepsin K (K(i) = 4.7 nmol/L), 5-(2-morpholin-4-yl-ethoxy)-benzofuran-2-carboxylic acid ((S)-3-methyl-1-(3-oxo-1-[2-(3-pyridin-2-yl-phenyl)-ethenoyl]-azepan-4-ylcarbanoyl)-butyl)-amide (SB 331750), is described, which is efficacious in rat models of bone resorption. SB 331750 potently inhibited human cathepsin K activity in vitro (K(i) = 0.0048 nmol/L) and was selective for human cathepsin K vs. cathepsins B (K(i) = 100 nmol/L), L (0.48 nmol/L), or S (K(i) = 14.3 nmol/L). In an in situ enzyme assay, SB 331750 inhibited osteoclast-associated cathepsin activity in tissue sections containing human osteoclasts (IC(50) approximately 60 nmol/L) and this translated into potent inhibition of human osteoclast-mediated bone resorption in vitro (IC(50) approximately 30 nmol/L). In vitro, SB 331750 partially, but dose-dependently, prevented the parathyroid hormone-induced hypercalcemia in an acute rat model of bone resorption. To evaluate the ability of SB 331750 to inhibit bone matrix degradation in vivo, it was administered for 4 weeks at 3, 10, or 30 mg/kg, intraperitoneally (i.p.), u.i.d. in the ovariectomized (ovx) rat. Both 10 and 30 mg/kg doses of compound prevented the ovx-induced elevation in urinary deoxypyridinoline and prevented the ovx-induced increase in percent eroded perimeter. Histological evaluation of the bones from compound-treated animals indicated that SB 331750 retarded bone matrix degradation in vivo at all three doses. The inhibition of bone resorption at the 10 and 30 mg/kg doses resulted in prevention of the ovx-induced reduction in percent trabecular area, trabecular number, and increase in trabecular spacing. These effects on bone resorption were also reflected in inhibition of the ovx-induced loss in trabecular bone volume as assessed using microcomputerized tomography (microCT; approximately 60% at 30 mg/kg). Together, these data indicate that the cathepsin K inhibitor, SB 331750, prevented bone resorption in vivo and this inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure.

Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption in vivo in a nonhuman primate.

Cathepsin K is a cysteine protease that plays an essential role in osteoclast-mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB-357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB-357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin-releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB-357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.

Changes in bone turnover following gonadotropin-releasing hormone (GnRH) agonist administration and estrogen treatment in cynomolgus monkeys: a short-term model for evaluation of antiresorptive therapy.

In this study we determine the early time course of estrogen deficiency-induced bone loss in the cynomolgus monkey and examine the potential of this method for evaluating antiresorptive therapies. In two groups of animals, estrogen deficiency was induced by the administration of a gonadotropin-releasing hormone agonist (GnRHa) and bone turnover was measured using biochemical markers. Two weeks after receiving GnRHa, serum estradiol decreased to below the detection limit in most animals and remained there through 6 months or until estrogen replacement started (months 4-6). Relative to untreated animals, urinary deoxypyridinoline (dPyr), as well as C- and N-telopeptides of type I collagen, were significantly elevated 4 weeks after receiving GnRHa. Serum osteocalcin increased in GnRHa-treated animals as early as week 4 and the level was significantly higher than in untreated control animals from weeks 8-24. Estradiol treatment returned all measures of bone turnover to control levels within 2 weeks. The use of biochemical markers as surrogates of bone turnover and loss was validated by measurement of bone mineral density (BMD), which showed a significant reduction at 6 months in estrogen-deficient animals. However, lumbar BMD in animals that received GnRHa and estradiol was similar to that in animals that had not received GnRHa. In conclusion, a monthly depot injection of GnRHa resulted in increased bone turnover due to estrogen deficiency, as early as 4 weeks after treatment. Estrogen administration returned bone turnover to control levels in 2 weeks. This method represents a valid model for evaluating antiresorptive agents in the short term in a nonhuman primate. Furthermore, the data suggest that changes in biochemical markers in response to antiresorptive therapy in humans may be detectable at much earlier timepoints than commonly used.

Antagonism of the osteoclast vitronectin receptor with an orally active nonpeptide inhibitor prevents cancellous bone loss in the ovariectomized rat.

An orally active, nonpeptide Arg-Gly-Asp (RGD) mimetic alpha(v)beta3 antagonist, (S)-3-Oxo-8-[2-[6-(methylamino)-pyridin-2-yl]-1-ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid (compound 1), has been generated, which prevented net bone loss and inhibited cancellous bone turnover in vivo. The compound binds alpha(v)beta3 and the closely related integrin alpha(v)beta5 with low nanomolar affinity but binds only weakly to the related integrins alpha(IIb)beta3, and alpha5beta1. Compound 1 inhibited alpha(v)beta3-mediated cell adhesion with an IC50 = 3 nM. More importantly, the compound inhibited human osteoclast-mediated bone resorption in vitro with an IC50 = 11 nM. In vivo, compound 1 inhibited bone resorption in a dose-dependent fashion, in the acute thyroparathyroidectomized (TPTX) rat model of bone resorption with a circulating EC50 approximately 20 microM. When dosed orally at 30 mg/kg twice a day (b.i.d.) in the chronic ovariectomy (OVX)-induced rat model of osteopenia, compound 1 also prevented bone loss. At doses ranging from 3 to 30 mg/kg b.i.d., compound 1 partially prevented the OVX-induced increase in urinary deoxypyridinoline. In addition, the compound prevented the OVX-induced reduction in cancellous bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), as assessed by quantitative microcomputerized tomography (microCT) and static histomorphometry. Furthermore, both the 10-mg/kg and 30-mg/kg doses of compound prevented the OVX-induced increase in bone turnover, as measured by percent osteoid perimeter (%O.Pm). Together, these data indicate that the alpha(v)beta3 antagonist compound 1 inhibits OVX-induced bone loss. Mechanistically, compound 1 prevents bone loss in vivo by inhibiting osteoclast-mediated bone resorption, ultimately preventing cancellous bone turnover.

Disease-modifying activity of SB 273005, an orally active, nonpeptide alphavbeta3 (vitronectin receptor) antagonist, in rat adjuvant-induced arthritis.

OBJECTIVE: To evaluate the effects of SB 273005, a potent, orally active nonpeptide antagonist of the integrin avbeta3 vitronectin receptor, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: Male Lewis rats with AIA were orally dosed either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 273005. Efficacy was determined by measurement of paw inflammation, assessment of bone mineral density using dual-energy x-ray absorptiometry (DEXA), magnetic resonance imaging (MRI), and histologic evaluation. RESULTS: SB 273005 is a potent antagonist of the closely related integrins, avbeta3 (Ki = 1.2 nM) and alphavbeta5 (Ki = 0.3 nM). When SB 273005 was administered prophylactically to AIA rats twice per day, it inhibited paw edema at doses of 10, 30, and 60 mg/kg, by 40%, 50%, and 52%, respectively. Therapeutic administration twice daily was also effective, and a reduction in paw edema was observed at 30 mg/kg and 60 mg/kg of the antagonist (by 36% and 48%, respectively). SB 273005 was also effective when administered once per day, both prophylactically and therapeutically. Significant improvement in joint integrity in treated rats was shown using DEXA and MRI analyses. These findings were confirmed histologically, and significant protection of bone, cartilage, and soft tissue was observed within the joint. CONCLUSION: Symptoms of AIA in rats were significantly reduced by either prophylactic or therapeutic treatment with the alphavbeta3 antagonist, SB 273005. Measurements of paw inflammation and of bone, cartilage, and soft tissue structure indicated that this compound exerts a protective effect on joint integrity and thus appears to have disease-modifying properties.

Reduction in animal numbers by long-term implantation of intravenous and intra-arterial catheters in thyroparathyroidectomized rats.

The thyroparathyroidectomized (TPTx) rat has been extensively used to study parathyroid hormone (PTH)-mediated bone resorption by measuring systemic Ca2+ concentrations. Animals have been traditionally used acutely; that is, they are often infused immediately after surgery and are sacrificed after a single use. To perform multiple experiments using a single group of animals we developed a system of long-term implanted intravenous/arterial catheters. Using calcitonin (CT) as a positive control, we successfully completed 12 separate controlled subexperiments documenting significant reductions in PTH-induced hypercalcemia in rats of the CT group. We then successfully completed two separate TPTx subexperiments, using a 3 x 3 Latin square experimental design. In both subexperiments, CT significantly inhibited the increase of blood Ca2+ concentration resulting from continuous PTH infusion. Our results indicate that, by combining the long-term use of catheters with the Latin square design, we can successfully reduce the number of animals used, increase the number of compounds screened, and improve the quality of the data. Although results of this study confirmed the acceptability of multiple infusions in anti-resorptive studies, investigations into the applicability of this set up to other areas of study requiring infusions and frequent blood sample collections seem appropriate.

Distinct mechanisms of action of selective estrogen receptor modulators in breast and osteoblastic cells.

Raloxifene and idoxifene are selective estrogen receptor modulators (SERMs) that exhibit tissue-specific agonist or antagonist properties via interactions with the estrogen receptor (ER). Both compounds are similarly osteoprotective in the ovariectomized rat in vivo as assessed by measurement of bone mineral density, urinary pyridinium cross-links, and serum osteocalcin, suggesting a similar mechanism of action. However, we have identified a fundamental difference in this mechanism via the estrogen response element (ERE) in osteoblast-like cells. With the use of ERE-luciferase reporter constructs, raloxifene, like the complete ER-antagonist ICI-182780, acts as an antagonist via the ERE in osteoblastic cells. In contrast, idoxifene, like 17beta-estrogen itself and 4-OH-tamoxifen, acts as an agonist in osteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780 and raloxifene inhibited the ERE-dependent agonist activity of 17beta-estradiol and idoxifene in osteoblastic cells. In contrast, in breast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 had no agonist activity and, indeed, raloxifene and idoxifene were potent antagonists of ERE-mediated 17beta-estradiol action, indicating an ERE-dependent mode of action in these cells. Although these SERMs exhibit a similar antagonist activity profile in breast cells, they can be distinguished mechanistically in osteoblastic cells.

Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats.

Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.

Disease-modifying activity of SB 242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis.

OBJECTIVE: To evaluate the effects of SB 242235, a potent and selective inhibitor of p38 mitogen-activated protein (MAP) kinase, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: Male Lewis rats with AIA were orally treated either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 242235. Efficacy was determined by measurements of paw inflammation, dual-energy x-ray absorptiometry for bone-mineral density (BMD), magnetic resonance imaging (MRI), microcomputed tomography (CT), and histologic evaluation. Serum tumor necrosis factor alpha (TNFalpha) in normal (non-AIA) rats and serum interleukin-6 (IL-6) levels in rats with AIA were measured as markers of the antiinflammatory effects of the compound. RESULTS: SB 242235 inhibited lipopolysaccharide-stimulated serum levels of TNFalpha in normal rats, with a median effective dose of 3.99 mg/kg. When SB 242235 was administered to AIA rats prophylactically on days 0-20, it inhibited paw edema at 30 mg/kg and 10 mg/kg per day by 56% and 33%, respectively. Therapeutic administration on days 10-20 was also effective, and inhibition of paw edema was observed at 60, 30, and 10 mg/kg (73%, 51%, and 19%, respectively). Significant improvement in joint integrity was demonstrated by showing normalization of BMD and also by MRI and micro-CT analysis. Protection of bone, cartilage, and soft tissues was also shown histologically. Serum IL-6 levels were decreased in AIA rats treated with the 60 mg/kg dose of compound. CONCLUSION: Symptoms of AIA in rats were significantly reduced by both prophylactic and therapeutic treatment with the p38 MAP kinase inhibitor, SB 242235. Results from measurements of paw inflammation, assessment of BMD, MRI, and micro-CT indicate that this compound exerts a protective effect on joint integrity, and thus appears to have disease-modifying properties.

Idoxifene, a novel selective estrogen receptor modulator, is effective in a rat model of adjuvant-induced arthritis.

Idoxifene, a selective estrogen receptor modulator, was evaluated in male and female rats with adjuvant-induced arthritis (AA). AA was induced in Lewis rats with Mycobacterium butyricum in paraffin oil injected into the base of the tail, and the animals were treated with idoxifene prophylactically (days 0-21) or therapeutically (days 10-21). Efficacy was determined by measurements of paw inflammation, bone mineral content, and bone mineral density (BMD) with dual X-ray absorptiometry and by histological evaluation. Serum interleukin-6 levels were measured as a marker of the anti-inflammatory effects of the compound. Estrogen was included for comparison and was administered at 5 mg/kg, three times a week s.c. Prophylactic treatment of male AA rats with idoxifene at 10, 3, and 1 mg/kg and estrogen at 5 mg/kg significantly inhibited paw inflammation. There was improved joint integrity measured by BMD and reduced serum interleukin-6 levels in animals treated with 10 mg/kg/day idoxifene. Idoxifene and estrogen were as effective for AA in female Lewis rats as in male rats, significantly inhibiting paw inflammation and improving BMD. Histological evaluation of the tibiotarsal joints of female rats treated with 10 mg/kg showed protection of bone, cartilage, and soft tissue. Therapeutic treatment with either idoxifene or estrogen (starting on day 10 of disease) of male and female Lewis rats also was effective in reducing paw inflammation in these animals, although the effect was much less than that observed with the prophylactic dosing protocol.

Design and characterization of orally active Arg-Gly-Asp peptidomimetic vitronectin receptor antagonist SB 265123 for prevention of bone loss in osteoporosis.

The Arg-Gly-Asp (RGD)-binding integrin alpha(V)beta(3) is highly expressed on osteoclasts and has been proposed to mediate cell-matrix adhesion required for osteoclast-mediated bone resorption. Antagonism of this receptor should prevent stable osteoclast adhesion and thereby inhibit bone resorption. We have generated an orally bioavailable, nonpeptide RGD mimetic alpha(v)beta(3) antagonist, SB 265123, which prevents bone loss in vivo when dosed by oral administration. SB 265123 binds alpha(v)beta(3) and the closely related integrin alpha(v)beta(5) with high affinity (K(i) = 3.5 and 1.3 nM, respectively), but binds only weakly to the related RGD-binding integrins alpha(IIb)beta(3) (K(i) >1 microM) and alpha(5)beta(1) (K(i) >1 microM). The compound inhibits alpha(v)beta(3)-mediated cell adhesion with an IC(50) = 60 nM and more importantly, inhibits human osteoclast-mediated bone resorption in vitro with an IC(50) = 48 nM. In vivo, SB 265123 completely blocks bone resorption in a thyroparathyroidectomized rat model of acute bone resorption when dosed at 2.5 mg/kg/h by continuous i.v. infusion. When dosed orally with 3 to 30 mg/kg b.i.d. , in the ovariectomy-induced rat model of osteoporosis, SB 265123 prevents bone resorption in a dose-dependent fashion. This is the first report of an orally active alpha(v)beta(3) antagonist that is effective at inhibiting bone resorption when dosed in a pharmaceutically acceptable fashion. Such a molecule may provide a novel therapeutic agent for the treatment of postmenopausal osteoporosis.

Orally bioavailable nonpeptide vitronectin receptor antagonists with efficacy in an osteoporosis model.

A new series of potent nonpeptide vitronectin receptor antagonists, based on a novel carbocyclic Gly-Asp mimetic, has been discovered. A representative of this series, SB 265123 (4), has 100% oral bioavailability in rats, and is orally active in vivo in the ovariectomized rat model of osteoporosis.

Idoxifene: a novel selective estrogen receptor modulator prevents bone loss and lowers cholesterol levels in ovariectomized rats and decreases uterine weight in intact rats.

Idoxifene, a novel selective estrogen receptor modulator, was tested for its effects on bone loss, serum cholesterol, and uterine wet weight and histology in the ovariectomized (Ovx) rat. Idoxifene (0.5 mg/kg x day) completely prevented loss of both lumbar and proximal tibial bone mineral density (BMD). In an intervention study, idoxifene (0.5 and 2.5 mg/kg x day) completely prevented further loss of both lumbar and proximal tibial BMD during a 2-month treatment period commencing 1 month after surgery, when significant loss of BMD had occurred in the Ovx control group. Idoxifene reduced total serum cholesterol, which was maximal at 0.5 mg/kg x day. Idoxifene alone displayed minimal uterotrophic activity in Ovx rats and inhibited the agonist activity of estrogen in intact rats. Histologically, myometrial and endometrial atrophy were observed in both idoxifene and vehicle-treated Ovx rats. In this report, we also provide molecular-based evidence to support the observations in vivo of a novel selective estrogen receptor modulator (SERM) mechanism of action in bone and endometrial cells. Idoxifene is an agonist through the estrogen response element (ERE) and exhibits similar postreceptor effects to estrogen in bone-forming osteoblasts. Idoxifene also stimulates osteoclast apoptosis, and these pleiotropic effects ultimately could contribute to the maintenance of bone homeostasis. However, idoxifene differs from estrogen in a tissue-specific manner. In human endometrial cells, where estrogen is a potent agonist through the ERE, idoxifene has negligible agonist activity. Moreover, idoxifene was able to block estrogen induced gene expression in endometrial cells, which is in agreement with the observation in the intact rat study. In the uterus, idoxifene has a pharmacologically favorable profile, lacking agonist and therefore growth-promoting activity. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data suggest that idoxifene may be effective in the prevention of osteoporosis and other postmenopausal diseases without producing unwanted estrogenic effects on the endometrium.

Applications of micro-CT and MR microscopy to study pre-clinical models of osteoporosis and osteoarthritis.

There is a tremendous unmet therapeutic need for the treatment of osteoporosis and osteoarthritis. The ovariectomized rat and the guinea pig are widely used animal models for the evaluation of new therapeutics for osteoporosis and osteoarthritis, respectively. We have utilized X-ray micro-CT techniques to quantitatively evaluate the differences in trabecular bone in the rat proximal tibia following ovariectomy and treatment with estrogen (17-B-estradiol). Results demonstrate a loss of trabecular bone and architecture following ovariectomy (p < 0.001), and a marked inhibition of trabecular bone loss in the estrogen treated group (p < 0.001). A similar change in architecture can be visualized in images obtained by high resolution MR microscopy. In addition, a good correlation was observed between the values of trabecular bone fraction (BV/TV) in the rat tibiae as obtained from 3-dimensional micro-CT data and 2-dimensional static histomorphometry (r = 0.89, 0.73, 0.79 for sham, OVX, and treated groups, respectively). Micro-CT images were also obtained from a set of lumbar vertebrae from sham operated and ovariectomized rats. Significant bone loss can be measured as early as 8 weeks following ovariectomy (p < 0.005). Micro-CT and MR images were also obtained to study age related changes in the stifle joint of the guinea pig. Significant boney changes can be seen in the tibia and femur from the animals at various ages. Changes in cartilage and joint space can also be visualized in the images. The utility of micro-CT imaging in evaluating the mouse skeletal system is illustrated by obtaining morphological and architectural details from high resolution images of the mouse hind limb and proximal tibia, respectively. The results demonstrate the advantages that multi-dimensional imaging techniques can offer in evaluating bone and joint related changes in animal models of osteoporosis and osteoarthritis.

Plasma levels of parathyroid hormone that induce anabolic effects in bone of ovariectomized rats can be achieved by stimulation of endogenous hormone secretion.

Parathyroid hormone (PTH) administration increases bone mass in normal and osteopenic animals. However, this treatment currently requires the daily injection of large amounts of PTH, and the relationship of these doses to plasma levels of PTH that are achievable physiologically is unknown. We determined in ovariectomized (ovx) rats: 1) the plasma PTH levels that occur after the subcutaneous injection of graded doses of rat PTH, 2) whether similar PTH levels can be achieved by stimulation of endogenous PTH secretion, and 3) whether a plasma PTH profile that is achievable physiologically is anabolic on bone. Injection of 1, 5, or 25 micrograms/kg rat PTH-(1-34) increased plasma PTH by 46, 164, or 520 pg/mL, respectively, above basal levels within 60 min. Infusion of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid for 2 h reduced plasma Ca2+ by 0.36 mmol/L and produced a total plasma PTH response (area under the plasma PTH curve) similar to that with the 5 micrograms/kg rat PTH injection. Then, 1, 5, or 25 micrograms/kg doses of rat PTH-(1-34) were injected subcutaneously daily for 28 days in 19-week-old rats that were ovx 7 weeks earlier. The 5 and 25 micrograms/kg doses significantly increased bone mineral density in the distal femur and trabecular bone area and average trabecular thickness in the proximal tibia. All doses of PTH significantly increased indices of trabecular connectivity and cancellous bone formation, including double-labeled surface, mineralizing surface, and surface-referent bone formation rate. In conclusion, anabolic effects on bone can be achieved with a plasma PTH profile similar to that attained following stimulation of the parathyroid gland by induced hypocalcemia. These data suggest that agents that transiently increase endogenous PTH secretion may represent a novel means to promote anabolic effects in skeletal tissues.

Disease-modifying activity of SK&F 106615 in rat adjuvant-induced arthritis. Multiparameter analysis of disease magnetic resonance imaging and bone mineral density measurements.

OBJECTIVE: To evaluate the effect of SK&F 106615 on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: AIA was induced in Lewis rats on day 0, and the animals were treated either prophylactically (days 0-16 or days 0-23) or therapeutically (days 10-23) with SK&F 106615. Efficacy was determined by measurements of paw inflammation, bone mineral density (BMD) using dual x-ray absorptiometry, and magnetic resonance imaging (MRI). Joint integrity was also determined histologically, and serum interleukin-6 (IL-6) levels were measured as a marker of the antiinflammatory effects of the compound. RESULTS: Prophylactic treatment (days 0-16) of AIA rats with SK&F 106615 significantly inhibited paw volume at doses of 545 mg/kg/day given orally on 5 days each week. Extensive evaluation of joint integrity in rats treated with SK&F 106615 20 mg/kg/day orally for 23 days showed inhibition of paw volume, normalization of BMD, and significant improvement in disease by MRI and histologic assessment compared with the AIA controls. Elevated levels of serum IL-6 in AIA rats were reduced dramatically by SK&F 106615. Therapeutic treatment (days 10-23) resulted in similar protective effects measured by paw inflammation, BMD, and MRI. In the therapeutic protocol, serum IL-6 appeared to be a more sensitive marker of antiinflammatory activity than paw edema. CONCLUSION: Symptoms of AIA in rats are significantly reduced by prophylactic and therapeutic treatment with SK&F 106615. Of particular note, this compound appears to exert a protective effect on joint integrity and to have disease-modifying properties.

Identification and characterization of two distinct alpha-(1-3)-L-fucosyltransferase activities in human colon carcinoma.

Two distinct alpha-(1-3)-fucosyltransferase activities have been identified in the colon carcinoma cell lines HT-29 and COLO-205. While both enzymatic activities exhibit similar affinities for a synthetic alpha-(1-3) acceptor and GDP-fucose, they differ with respect to divalent cation requirements, N-ethylmaleimide inhibition, and glycoprotein substrate specificity. The COLO-205 alpha-(1-3) activity exhibits maximal enzymatic activity in the presence of 20 mM Mn2+ but retains less than 10% activity in the absence of divalent cations. In contrast, the optimal Mn2+ concentration for the HT-29 enzyme is 1 mM, although this activity is relatively insensitive to divalent cation stimulation. In addition, the HT-29 alpha-(1-3)-fucosyltransferase activity is resistant to inhibition by 30 mM N-ethylmaleimide and relatively inactive toward the glycoprotein substrate fetuin as compared to its desialylated derivative, asialofetuin. The COLO-205 activity is inhibited approximately 90% by N-ethylmaleimide and is equally active with either glycoprotein acceptor. Although the alpha-(1-3) specific activities are similar in both cell lines, N-ethylmaleimide-sensitive alpha-(1-4) fucosyltransferase activity is 40-fold higher in COLO-205 as compared to HT-29, suggesting that the COLO-205 fucosyltransferase activity may be an alpha-(1-3/4) enzyme, while the HT-29 activity appears to be an alpha-(1-3) specific form. Further examination of a panel of cell lines, tumor biopsies, and xenografts, based on the effect of metal ions and N-ethylmaleimide, indicated that both enzyme activities are similarly expressed in human colon carcinoma tissue.