Daily Treatment of Aged Ovariectomized Rats with Human Parathyroid Hormone (1-84) for 12 Months Reverses Bone Loss and Enhances Trabecular and Cortical Bone Strength

Most studies that have investigated the anabolic effects of parathyroid hormone (1-84) (PTH) or PTH fragments on the skeleton of ovariectomized (OVX) rats have evaluated the short-term effects of high-dose PTH(1-34) in young animals. This study used densitometry, histomorphometry, and biomechanical testing to evaluate the effects of 12-month daily treatment with low-dose PTH (15 or 30 μg/kg) in rats that were 10 months old at baseline, 4 months after OVX. Bone mineral density (BMD) and bone strength were reduced substantially in control OVX rats. The 15 μg/kg dose of PTH restored BMD to levels similar to those in sham animals within 6 months at the lumbar spine, distal and central femur, and whole body and maintained the BMD gain from 6 to 12 months. The 30 μg/kg dose produced greater effects. Both PTH doses normalized the trabecular bone volume-to-total volume ratio (BV/TV) at lumbar vertebra 3 but not at the proximal tibia (where baseline BV/TV was very low), solely by increasing trabecular thickness. PTH dose-dependently increased bone formation by increasing the mineralizing surface, but only the 30 μg/kg dose increased resorption. PTH increased cortical BMD, area, and thickness, primarily by increasing endocortical bone formation, and restored all measures of bone strength to levels similar to those in sham animals at all skeletal sites. PTH increased bone mass safely; there was no osteoid accumulation, mineralization defect, or marrow fibrosis and there were no abnormal cells. Thus, long-term PTH therapy normalized bone strength in the aged OVX rat, a model of postmenopausal osteoporosis, through increased bone turnover and enhanced formation of both trabecular and cortical bone.     

Intratrabecular tunneling increases trabecular number throughout the skeleton of ovariectomized rhesus monkeys treated with parathyroid hormone 1-84

Daily treatment of ovariectomized (OVX) adult rhesus monkeys with human parathyroid hormone (PTH) 1-84 for 16 months increases trabecular bone volume (BV/TV), number (Tb.N) and connectivity at lumbar vertebra-3 (L3) and thoracic vertebra-10. We proposed that the increased Tb.N and connectivity was achieved by stimulation of intratrabecular tunneling. Using histomorphometry to determine frequency of events, we have now quantified intratrabecular tunneling at L3 and extended it to investigate the effects of PTH(1-84) treatment on trabecular bone at the proximal femur, distal radius and iliac crest of these animals. At L3, tunneling frequency was low in control sham and OVX animals ( approximately 0.05/mm(2)) but increased significantly in PTH(1-84)-treated animals (0.27, 0.49 and 0.95/mm(2) with the 5, 10 and 25 microg/kg doses, respectively). Very similar tunneling frequencies were observed at all skeletal sites in all groups. Iliac crest biopsies were also collected at baseline and after 6 months of treatment and showed significant time- and dose-related increases in tunnels. Although the pattern and magnitude of response varied slightly from site to site, PTH(1-84) treatment significantly increased Tb.N, as well as BV/TV and bone formation rate at all skeletal sites. A modest but statistically significant increase in trabecular thickness occurred only at the iliac crest. In summary, intratrabecular tunneling is rare in control monkeys, but increased substantially with PTH(1-84) treatment. This phenomenon provides a plausible explanation for the PTH(1-84)-induced increase in Tb.N observed in OVX monkeys. Moreover, these analyses allowed a comparison of the effects PTH(1-84) treatment on trabecular bone at multiple locations.     

Effects of Treatment with Parathyroid Hormone 1–84 on Quantity and Biomechanical Properties of Thoracic Vertebral Trabecular Bone in Ovariectomized Rhesus Monkeys

Osteoporosis is characterized by impaired bone quality leading to increased susceptibility to fracture, particularly of the thoracic spine. However, the lumbar spine is studied most commonly. We investigated the effects of 16 months of treatment with full-length parathyroid hormone (PTH) 1–84 (5, 10, or 25 μg/kg) on bone mineral density (BMD) and on architecture and biomechanical properties of trabecular bone at the thoracic spine of ovariectomized (OVX) adult rhesus monkeys and compared the results with those from the lumbar spine. At baseline, 9 months after surgery, dual-energy X-ray absorptiometric BMD at T9–T12 was 7% lower in OVX than in sham animals. All PTH(1–84) doses increased BMD to sham levels within 7 months. Micro-computed tomography of T10 vertebrae showed that trabecular bone volume and connectivity were higher in PTH(1–84)-treated animals than in sham controls, primarily through a significantly greater trabecular number. Peripheral quantitative computed tomography of trabecular bone cores from T11 and T12 confirmed that PTH(1–84) increased BMD. Compression testing of the cores showed that PTH(1–84) treatment increased stiffness, modulus, yield load, and yield stress to levels significantly greater than in sham animals, with the largest effect in the 10 μg/kg group (35–54% greater than in OVX controls). Thus, PTH(1–84) treatment increased BMD and the biomechanical properties of trabecular bone at the thoracic spine of OVX rhesus monkeys. The 10 μg/kg dose produced the greatest effect on trabecular strength, possibly because the highest dose stimulated bone remodeling excessively. Importantly, the changes observed were similar to those in lumbar vertebrae, thereby validating extrapolation of results from the lumbar to the thoracic spine.     

Teriparatide [PTH(1-34)] strengthens the proximal femur of ovariectomized nonhuman primates despite increasing porosity.

OVX monkeys treated for 18 months with 1 or 5 microg/kg/d teriparatide [PTH (1-34)] had significantly stronger proximal femora relative to ovariectomized controls. Teriparatide enhancement of cortical area, cortical width, and trabecular bone volume seemed to more than compensate for the dose-dependent increase in cortical porosity. Beneficial effects of teriparatide treatment on the proximal femur persisted beyond the treatment period and may extend to the marrow. INTRODUCTION: We conducted a detailed quantitative analysis of the effects of teriparatide on the proximal femur of ovariectomized monkeys. Teriparatide increased bone mass, enhanced structural architecture, and strengthened the hip, despite increasing cortical porosity. MATERIALS AND METHODS: Monkeys were treated with vehicle (sham or OVX controls), 1 microg/kg/day teriparatide [parathyroid hormone (1-34); PTH1], or 5 microg/kg/day teriparatide (PTH5) for 18 months or for 12 months followed by 6 months of treatment withdrawal (PTH1W and PTH5W, respectively). Excised proximal femora were analyzed by microCT, conventional histomorphometry, and biomechanics.RESULTS AND CONCLUSIONS: The femoral neck showed significant reduction in trabecular bone volume (BV/TV) for OVX compared with sham, whereas PTH1 BV/TV was restored to sham levels and PTH5 BV/TV was greater than sham and OVX. The withdrawal groups had BV/TVs intermediate between sham and OVX. PTH1 had trabecular number (Tb.N) greater than OVX, and PTH5 Tb.N was greater than sham and OVX. The withdrawal groups had Tb.Ns intermediate between sham and OVX. No differences between groups were observed for trabecular orientation or trabecular thickness. Teriparatide dose-dependently increased bone formation rate and activation frequency in the femoral neck. Cellular composition analyses suggested a tendency of ovariectomy to increase adiposity of marrow by 100%, whereas PTH tended to reduce adipocyte number and increase osteoblast number compared with OVX. Analyses of the cortex showed dose-dependent elevation of cortical porosity, which was consistent with enhanced bone turnover with treatment. Cortical porosity was reduced after withdrawal of teriparatide, because PTH1W cortical porosity was lower than OVX, whereas PTH5W cortical porosity was intermediate between sham and OVX. Increased cortical porosity did not weaken the proximal femora. Biomechanics showed that ovariectomy weakened proximal femora compared with sham, but PTH1, PTH5, and PTH1W were stronger than OVX and not different from sham. PTH5W strength was intermediate between sham and OVX. Therefore, teriparatide had beneficial effects on the proximal femur, despite increasing cortical porosity. Cortical porosity did not adversely affect the mechanical integrity of the proximal femora, because enhanced cortical area and trabecular bone volume more than compensated for the porosity. Much of the beneficial effects of teriparatide were retained after 6 months withdrawal from treatment. PTH effects on the femoral neck were not limited to bone but may include inhibition of OVX-stimulated adiposity of the marrow.     

Effects of Treatment of Ovariectomized Adult Rhesus Monkeys with Parathyroid Hormone 1-84 for 16 Months on Trabecular and Cortical Bone Structure and Biomechanical Properties of the Proximal Femur

Treatment of monkeys and humans with parathyroid hormone (PTH) 1-84 stimulates skeletal remodeling, which increases trabecular (Tb) bone mineral density (BMD) but decreases cortical (Ct) BMD at locations where these bone types predominate. We report the effects of daily PTH treatment (5, 10, or 25 μg/kg) of ovariectomized (OVX) rhesus monkeys for 16 months on bone structure and biomechanical properties at the proximal femur, a mixed trabecular and cortical bone site. PTH reversed the OVX-induced decrease in BMD measured by dual-energy X-ray absorptiometry at the proximal femur, femoral neck, and distal femur. Peripheral quantitative computed tomography confirmed a significant decrease in Ct.BMD and an increase in Tb.BMD at the total proximal femur and at the proximal and distal femoral metaphyses. The decrease in Ct.BMD resulted primarily from increased area because cortical bone mineral content was unaffected by PTH. Histomorphometry revealed that PTH significantly increased the trabecular bone formation rate (BFR) as well as trabecular bone volume and number. PTH did not affect periosteal or haversian BFR at the femoral neck, but cortical porosity was increased slightly. PTH had no effects on stiffness or peak load measured using a shear test, whereas work-to-failure, the energy required to fracture, was increased significantly. Thus, PTH treatment induced changes in trabecular and cortical bone at the proximal femur that were similar to those occurring at sites where these bone types predominate. Together, the changes had no effect on stiffness or peak load but increased the energy required to break the proximal femur, thereby making it more resistant to fracture.     

Odanacatib reduces bone turnover and increases bone mass in the lumbar spine of skeletally mature ovariectomized rhesus monkeys

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK) currently being developed as a once‐weekly treatment for osteoporosis. In this study, we evaluated the effects of ODN on bone turnover, bone mineral density (BMD), and bone strength in the lumbar spine of estrogen‐deficient, skeletally mature rhesus monkeys. Ovariectomized (OVX) monkeys were treated in prevention mode for 21 months with either vehicle, ODN 6 mg/kg, or ODN 30 mg/kg (p.o., q.d.) and compared with intact animals. ODN treatment persistently suppressed the bone resorption markers (urinary NTx [75% to 90%] and serum CTx [40% to 55%]) and the serum formation markers (BSAP [30% to 35%] and P1NP [60% to 70%]) versus vehicle‐treated OVX monkeys. Treatment with ODN also led to dose‐dependent increases in serum 1‐CTP and maintained estrogen deficiency–elevated Trap‐5b levels, supporting the distinct mechanism of CatK inhibition in effectively suppressing bone resorption without reducing osteoclast numbers. ODN at both doses fully prevented bone loss in lumbar vertebrae (L₁ to L₄) BMD in OVX animals, maintaining a level comparable to intact animals. ODN dose‐dependently increased L₁ to L₄ BMD by 7% in the 6 mg/kg group (p < 0.05 versus OVX‐vehicle) and 15% in the 30 mg/kg group (p < 0.05 versus OVX‐vehicle) from baseline. Treatment also trended to increase bone strength, associated with a positive and highly significant correlation (R = 0.838) between peak load and bone mineral content of the lumbar spine. Whereas ODN reduced bone turnover parameters in trabecular bone, the number of osteoclasts was either maintained or increased in the ODN‐treated groups compared with the vehicle controls. Taken together, our findings demonstrated that the long‐term treatment with ODN effectively suppressed bone turnover without reducing osteoclast number and maintained normal biomechanical properties of the spine of OVX nonhuman primates. © 2012 American Society for Bone and Mineral Research     

Continuous parathyroid hormone and estrogen administration increases vertebral cancellous bone volume and cortical width in the estrogen-deficient rat.

Generally, it is believed that intermittent administration of parathyroid hormone (PTH) has an anabolic effect on the skeleton, whereas continuous administration is catabolic. However, there is evidence that continuous exposure to PTH may have an anabolic effect, for example, in patients with mild primary hyperparathyroidism (PHPT). The possibility of delivering PTH continuously may have important implications for the treatment of osteoporosis. Furthermore, estrogen treatment may be useful in the medical management of PHPT. Therefore, we examined the skeletal effects of continuous administration of PTH, with or without estrogen, in the estrogen-deficient rat with established osteopenia. Forty 7-month-old SD rats were divided into four ovariectomy (OVX) groups and one sham-operated group. Eight weeks post-OVX, three groups received subcutaneous implants of Alzet mini pumps loaded with PTH(1-34) (30 microg/kg per day), 17beta-estradiol (10 microg/kg per day) pellet, or both PTH and 17beta-estradiol separately for 4 weeks. OVX and sham control groups were given the mini pumps loaded with vehicle. Two doses of calcein (10 mg/kg) were given subcutaneously to all rats 2 days and 8 days before death. Histomorphometry was performed on cancellous and cortical bone of the fourth lumbar vertebra. At 3 months, post-OVX rats displayed bone loss with high bone turnover. Estrogen reversed OVX-mediated high turnover without restoring cancellous bone volume (BV/TV). PTH infusion further increased bone turnover and partially restored BV/TV. However, PTH infusion increased cortical porosity. Estrogen inhibited PTH-mediated cancellous bone resorption and substantially increased BV/TV above sham control. The combined treatment was associated with a significant increase in peritrabecular fibrosis and woven bone formation. The combined treatment of PTH infusion and estrogen replacement enhanced cortical width but estrogen did not prevent the PTH-induced cortical tunneling. We conclude that continuous administration of PTH and estrogen increases cortical porosity but has substantial beneficial effects on vertebral cancellous bone volume and cortical width in OVX rats.     

Cancellous and cortical bone architecture and turnover at the iliac crest of postmenopausal osteoporotic women treated with parathyroid hormone 1-84

Treatment with parathyroid hormone [PTH(1-84)] increases lumbar spine bone mineral density and decreases vertebral fractures, but its effects on bone microarchitecture are unknown. We obtained iliac crest biopsies from postmenopausal osteoporotic women given placebo (n=8) or 100 microg PTH(1-84) for 18 (n=8) or 24 (n=7) months to assess cancellous and cortical bone formation and structure. At 18 months, cancellous bone volume (BV/TV) measured by microcomputed tomography and histomorphometry was 45-48% higher in subjects treated with PTH(1-84) versus placebo, a result of higher trabecular number (Tb.N) and thickness. The higher Tb.N appeared to result from intratrabecular tunneling. Connectivity density was higher and structure model index was lower, indicating a better connected and more plate-like trabecular architecture. Cancellous bone formation rate (BFR) was 2-fold higher in PTH(1-84)-treated subjects, primarily because of greater mineralizing surface. Osteoblast and osteoid surfaces were a nonsignificant 58% and 35%, respectively, higher with PTH(1-84) treatment. Osteoclast and eroded surface were unaffected by PTH(1-84). There were no effects of PTH(1-84) treatment on cortical thickness, or endocortical or periosteal BFR, but cortical porosity tended to be higher. Although cancellous BFR was lower at 24 than at 18 months, measures of cancellous and cortical bone structure were similar at both timepoints. The bone produced by PTH(1-84) had normal lamellar structure and mineralization with no abnormal histology. In conclusion, when compared with placebo, treatment of osteoporotic women with PTH(1-84) was associated with higher BV/TV and trabecular connectivity, with a more plate-like architecture, all consistent with the lower vertebral fracture incidence.     

Pharmacological treatment of osteopenia induced by gastrectomy or ovariectomy in young female rats

BACKGROUND: Both gastrectomy (GX) and ovariectomy (OVX) induce osteopenia in man and experimental animals. The present study addresses the question--can alendronate, estrogen or parathyroid hormone (PTH) be used to treat established GX- or OVX -evoked osteopenia? METHODS: Rats were GX-, OVX- or SHAM-operated 8 weeks before starting the treatment with drugs. Each group was then treated for 8 weeks with 50 microg/kg/day alendronate, 10 microg/kg/day estrogen or 75 microg/kg/day PTH(1-84); n = 8 rats/group. Peripheral Quantitative Computed Tomography (pQCT) was used to measure trabecular bone mineral density (BMD) and various cortical bone parameters. RESULTS: At killing, 16 weeks after surgery, GX and OVX rats had a greatly reduced trabecular BMD in the metaphysis of the distal femur (GX -44% and OVX -55%). Alendronate increased the trabecular BMD by 44% in GX rats and by 64% in OVX rats, while PTH increased it by 51% and 115%, respectively. However, estrogen increased the trabecular BMD in GX rats (35%), but not in OVX rats (15%, not significant). Cortical bone parameters were adversely (but moderately) affected by GX, but not by OVX or by treatment with the three drugs. INTERPRETATION: Alendronate, estrogen and PTH restored the trabecular bone loss in rats with an established GX-evoked osteopenia. In contrast, alendronate and PTH, but not estrogen, restored the trabecular bone loss after OVX. Hence, the mechanism underlying GX-evoked bone loss differs from that underlying OVX-evoked bone loss. The ability of alendronate, estrogen and PTH to reverse the GX-evoked osteopenia in the rat may be of clinical interest when dealing with bone loss in humans after GX.     

Effect of combined humanPTH(1-34) and calcitonin treatment in ovariectomized rats

We examined the combined effects of human parathyroid hormone 1-34 (hPTH) and elcatonin (ECT: a synthetic derivative of eel calcitonin) to prevent loss of bone mass, architecture and strength in ovariectomized (OVX) rats. Fifty-four female rats (aged 13 weeks) were assigned to one of nine groups: Sham (fake surgery performed), OVX, ECT (15 U/kg administered), PTH5, PTH10 and PTH20 (5, 10 or 20 microg/kg administered), and E+PTH5, E+PTH10 and E+PTH20 (15 U/kg of ECT and 5, 10 or 20 microg/kg of hPTH administered). The drug or vehicle was subcutaneously administered three times a week for 12 weeks. The femurs were removed at the completion of the experiment. The right distal femoral metaphysis was used for measuring bone mineral density (BMD), analyzing trabecular bone structure by micro-computed tomography (microCT), and conducting the bone strength test, and the left femur was used for histomorphometric analysis. Trabecular bone volume (BV/TV) and other bone mass parameters were greater in the ECT and PTH groups than in the OVX group. The number of nodes (N.Nd/TV) and trabecular number (Tb.N) were significantly greater in the ECT group, and trabecular thickness (Tb.Th) and trabecular bone pattern factor (TBPf) were significantly greater in the PTH group. These results indicate that these drugs preserve the bone architecture by different means. Analysis by means of microCT revealed that BV/TV, Tb.N, fractal D and N.Nd/TV were significantly greater in the E+PTH groups than in the PTH groups at each concentration. Trabecular separation (Tb.Sp) was significantly lower in the E+PTH5 and E+PTH10 groups than in the respective PTH5 and PTH10 groups. When the maximum load was applied in a compression test on the distal femur, the E+PTH groups had higher values than the PTH groups, however, the three point bending strength of the diaphysis of femur in the E+PTH10 and E+PTH20 groups tended to be low compared to those in the PTH10 and PTH20 groups. These results indicate that combination therapy using PTH and ECT preserves the trabecular microarchitecture better than single-drug therapy using ECT or PTH in OVX rats, however, it is necessary to optimize the calcitonin (CT) dosage and administration in order to achieve the optimal combined effect of PTH and CT.     

Odanacatib treatment increases hip bone mass and cortical thickness by preserving endocortical bone formation and stimulating periosteal bone formation in the ovariectomized adult rhesus monkey

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK). Previously, ODN was shown to increase bone mineral density (BMD) and maintained normal bone strength at the spine in ovariectomized (OVX) rhesus monkeys. Here, we further characterize the effects of ODN on BMD, bone strength, and dynamic histomorphometric analyses of the hip from the same monkeys. Animals were treated for 21 months with vehicle, 6 or 30 mg/kg ODN (p.o., q.d.). ODN increased femoral neck (FN) BMD by 11% and 15% (p < 0.07) and ultimate load by 25% (p < 0.05) and 30% (p < 0.01) versus vehicle. Treatment-related increases in ultimate load positively correlated with the increased FN BMD, bone mineral content (BMC), and cortical thickness. Histomorphometry of FN and proximal femur (PF) revealed that ODN reduced trabecular and intracortical bone formation rate (BFR) but did not affect long-term endocortical BFR. Moreover, ODN stimulated long-term FN and PF periosteal BFR by 3.5-fold and 6-fold with the 30 mg/kg dose versus vehicle, respectively. Osteoclast surfaces were either unaffected or trended higher (~twofold) in endocortical and trabecular surfaces in the ODN group. Lastly, ODN increased cortical thickness of FN by 21% (p = 0.08) and PF by 19% (p < 0.05) versus vehicle after 21 months of treatment. Together, both doses of ODN increased bone mass and improved bone strength at the hip. Unlike conventional antiresorptives, ODN displayed site-specific effects on trabecular versus cortical bone formation. The drug provided marked increases in periosteal bone formation and cortical thickness in OVX monkeys, suggesting that CatK inhibition may represent a novel therapeutic approach for the treatment of osteoporosis.     

Effects of daily treatment with parathyroid hormone 1-84 for 16 months on density, architecture and biomechanical properties of cortical bone in adult ovariectomized rhesus monkeys

Treatment with parathyroid hormone 1-84 (PTH) or teriparatide increases osteonal remodeling and decreases bone mineral density (BMD) at cortical (Ct) bone sites but may also increase bone size. Decreases in BMD and increases in size exert opposing effects on bone strength. In adult ovariectomized (OVX) rhesus monkeys, we assessed the effects of daily PTH treatment (5, 10 or 25 microg/kg) for 16 months on BMD at the radial, tibial and femoral diaphyses, and on biomechanical properties (3-point bending) of radial cortical bone and the femoral diaphysis. PTH treatment did not affect areal BMD measured by dual-energy X-ray absorptiometry at the tibial diaphysis but caused a rapid, dose-related decrease at the distal radial diaphysis. Peripheral quantitative computed tomography at the radial and femoral diaphyses confirmed a significant PTH dose-related decrease in volumetric Ct.BMD caused primarily by increased cortical area. Significant increases in cortical thickness were the result of nonsignificant increases in periosteal length and decreases in endocortical length. Histomorphometry revealed increased endocortical bone formation at the tibial diaphysis and rib, higher Haversian remodeling at the rib and increased cortical porosity at the rib and tibia. Biomechanical testing at the femoral diaphysis showed that PTH treatment had no effect on peak load, but significantly decreased stiffness and increased work-to-failure (the energy required to break the bone). Similar changes occurred in radial cortical beams but only stiffness was changed significantly. Thus, PTH treatment of OVX rhesus monkeys decreased BMD and stiffness of cortical bone but did not affect peak load, likely because of increased bone size. However, PTH treatment increased the energy required to break the femur making it more resistant to fracture.     

Long-term effects of withdrawal of bisphosphonate incadronate disodium (YM175) on bone mineral density, mass, structure, and turnover in the lumbar vertebrae of ovariectomized rats.

This study was designed to evaluate the long-term effects of incadronate disodium (YM175) after its withdrawal on cancellous bone mass in ovariectomized (OVX) rats. Thirteen-week-old female SD rats were randomized into four groups: sham-operated, OVX, low-YM, and high-YM (0.01 mg/kg or 0.1 mg/kg subcutaneously [sc], three times a week after OVX) groups. After 4 weeks of treatment with vehicle or YM175, rats from each group were killed at time points of 0 (baseline), 3, 6, 9, and 12 months after withdrawal of the agent. Bone mineral density (BMD) of the lumbar vertebrae was measured by dual-energy X-ray absorptiometry (DXA). Bone volume (BV/TV), trabecular number and trabecular separation (Tb.N and Tb.Sp), eroded surface (ES/BS), osteoclast number and osteoclast surface (N.Oc/BS and Oc.S/BS), osteoid surface (OS/BS), and bone formation rate (BFR/BS) were measured as histomorphometric parameters of the fifth lumbar vertebra. BMD, BV/TV, Tb.N, and Tb.Sp in YM175-treated groups were maintained at the same level as in the sham group until 12 months after withdrawal in the high-YM group and until 3 months after withdrawal in the low-YM group. YM175 decreased both bone formative and resorptive parameters in histomorphometry. Serum bone-specific alkaline phosphatase (ALP) and urinary deoxypyridinoline at both doses of YM175 also showed a suppressive effect of this agent on bone turnover. These results indicate that YM175, after withdrawal, still maintains bone volume dose dependently by depressing bone resorption and formation in OVX rats. Intermittent YM175 treatment with a long interval may be sufficient to maintain the bone volume and structure in OVX rats.     

Effects of cyclic vs. daily treatment with human parathyroid hormone (1-34) on murine bone structure and cellular activity

Previously, we demonstrated that the human parathyroid hormone (1-34) fragment (hPTH(1-34)) increased bone strength in proportion to its effects on BMD and cortical bone structure in the murine femur by comparing cyclic vs. daily administration of hPTH(1-34). Both cyclic and daily regimens increased vertebral BMD similarly at 7 weeks. Here, we have examined the effects of daily and cyclic PTH regimens on bone structure and cellular activity by static and dynamic histomorphometry. Twenty-week-old, intact female C57BL/J6 mice were treated with the following regimens (n=7 for each group): daily injection with vehicle for 7 weeks [control]; daily injection with hPTH(1-34) (40 microg/kg/day) for 7 weeks [daily PTH]; and daily injection with hPTH(1-34) (40 microg/kg/day) and vehicle alternating weekly for 7 weeks [cyclic PTH]. At days 9 and 10, and 2 and 3 prior to euthanasia, calcein (10 mg/kg) was injected subcutaneously. At the end of study, the lumbar vertebrae 1-3 and the left femora were excised, cleaned, and processed for histomorphometry. In the lumbar vertebrae, daily and cyclic PTH regimens significantly increased cancellous bone volume (BV/TV), trabecular number, trabecular osteoclast and osteoblast perimeters, trabecular mineral apposition rate (MAR) and bone formation rate (BFR), and periosteal MAR and BFR compared to control, with no significant difference between the two PTH-treated groups. Increased trabecular tunneling was observed in both PTH-treated groups. Both regimens tended to increase vertebral cortical bone formation parameters with the effects at the periosteum site being more marked than those at the endosteum site, resulting in a significant increase in cortical width. In the femur, the effects of cyclic PTH on BV/TV, trabecular width and number, trabecular and endocortical osteoblast and osteoclast perimeters, cortical width, and trabecular and periosteal BFR were less marked than those of daily PTH. A cyclic PTH regimen was as effective as a daily regimen in improving cancellous and cortical bone microarchitecture and cellular activity in the murine vertebra.     

Combined treatment with a beta-blocker and intermittent PTH improves bone mass and microarchitecture in ovariectomized mice

Intermittent administration of parathyroid hormone (PTH) induces bone remodeling and renewed bone modeling, resulting in net bone gain. beta-blockers improve trabecular bone architecture in young ovariectomized mice by preventing the inhibition of bone formation and stimulation of bone resorption induced by the adrenergic system. To test the hypothesis that PTH and beta-blockers may exert synergistic effects on the skeleton, 15-week-old ovariectomized mice were either given oral propranolol (PRO) or left untreated for 8 weeks, adding daily hPTH(1-34) (80 microg/kg/day) or vehicle (VEH) during the last 4 weeks. The skeletal response was evaluated using pDXA, microCT, histomorphometry and biochemical markers. PRO significantly attenuated loss of bone mineral density (BMD) at whole body (WB) (-0.1% in PRO vs. -2.4% in VEH, P < 0.05), but not at spine or femur 4 weeks after OVX. Thereafter, PTH increased BMD at all sites in both PRO- and VEH-treated mice (+6.7% to +14%, P < 0.05 to P < 0.0001 vs. VEH). Over 8 weeks, sequential-combined treatment of PRO and PTH significantly improved BMD over PTH alone at WB (+9.1% vs. +4.4% over baseline, respectively, P < 0.005) and spine (+9% vs. -1.7%, respectively, P < 0.05). These effects were paralleled by a decrease in TRACP5b with PRO (P < 0.05 vs. VEH) and an increase in osteocalcin with PTH, irrespective of PRO (P < 0.0001 vs. VEH). Trabecular bone microarchitecture, such as BV/TV, trabecular number and ConnD, was significantly improved by sequential-combined treatment of PRO and PTH compared to PTH alone. At midshaft femur, both PRO and PTH significantly increased cross-sectional area (CSA), but the effects of the two drugs on CSA and cortical thickness were not additive. Dynamic histomorphometry indicated that bone formation was increased by PTH at both cortical and trabecular surfaces, whereas PRO increased osteoblast number and surface on trabecular surfaces. The combined treatment further improved the extent of mineralization and BFR over PTH alone (P < 0.05) at endocortical surfaces and recapitulated the effects of PTH and PRO alone on trabecular surfaces. These results indicate that beta-adrenergic blockade may partially improve the bone remodeling balance induced by estrogen deficiency. In turn, PRO exerted synergistic effects with intermittent PTH on bone mass and cancellous bone architecture. As such, combined therapy of beta-blockers and PTH may be of interest in the treatment of postmenopausal osteoporosis.     

Prolonged Treatments With Antiresorptive Agents and PTH Have Different Effects on Bone Strength and the Degree of Mineralization in Old Estrogen‐Deficient Osteoporotic Rats

Current approved medical treatments for osteoporosis reduce fracture risk to a greater degree than predicted from change in BMD in women with postmenopausal osteoporosis. We hypothesize that bone active agents improve bone strength in osteoporotic bone by altering different material properties of the bone. Eighteen‐month‐old female Fischer rats were ovariectomized (OVX) or sham‐operated and left untreated for 60 days to induce osteopenia before they were treated with single doses of either risedronate (500 μg/kg, IV), zoledronic acid (100 μg/kg, IV), raloxifene (2 mg/kg, PO, three times per week), hPTH(1–34) (25 μg/kg, SC, three times per week), or vehicle (NS; 1 ml/kg, three times per week). Groups of animals were killed after days 60 and 180 of treatment, and either the proximal tibial metaphysis or lumbar vertebral body were studied. Bone volume and architecture were assessed by μCT and histomorphometry. Measurements of bone quality included the degree of bone mineralization (DBM), localized elastic modulus, bone turnover by histomorphometry, compression testing of the LVB, and three‐point bending testing of the femur. The trabecular bone volume, DBM, elastic modulus, and compressive bone strength were all significantly lower at day 60 post‐OVX (pretreatment, day 0 study) than at baseline. After 60 days of all of the bone active treatments, bone mass and material measurements agent were restored. However, after 180 days of treatment, the OVX + PTH group further increased BV/TV (+30% from day 60, p < 0.05 within group and between groups). In addition, after 180 days of treatment, there was more highly mineralized cortical and trabecular bone and increased cortical bone size and whole bone strength in OVX + PTH compared with other OVX + antiresorptives. Treatment of estrogen‐deficient aged rats with either antiresorptive agents or PTH rapidly improved many aspects of bone quality including microarchitecture, bone mineralization, turnover, and bone strength. However, prolonged treatment for 180 days with PTH resulted in additional gains in bone quality and bone strength, suggesting that the maximal gains in bone strength in cortical and trabecular bone sites may require a longer treatment period with PTH.     

Long-term minodronic acid (ONO-5920/YM529) treatment suppresses increased bone turnover, plus prevents reduction in bone mass and bone strength in ovariectomized rats with established osteopenia

The present study examined the effect of the highly potent nitrogen-containing bisphosphonate, minodronic acid (ONO-5920/YM529), on bone mineral density (BMD), bone turnover, bone histomorphometry and bone strength in ovariectomized (OVX) rats. Female F344/DuCrj rats, aged 14 weeks, were OVX or sham operated. After 3 months, the OVX rats showed an increase in bone turnover, and a decrease in bone mass and bone strength. Minodronic acid was administered orally once a day for 12 months at doses of 0, 0.006, 0.03 and 0.15 mg/kg from 3 months after OVX. Minodronic acid dose-dependently inhibited the decrease in BMD of lumbar vertebrae and femur. In the femur, treatment with 0.15 mg/kg minodronic acid increased the BMD of distal and mid sites to sham levels. Minodronic acid dose-dependently suppressed OVX-induced increase in urinary deoxypyridinoline, a bone resorption marker, after a month of treatment and these effects were maintained for 12 months of treatment. Minodronic acid also decreased serum osteocalcin, a bone formation marker. In bone histomorphometric analysis after 12 months of treatment, OVX rats showed an increase in bone resorption (Oc.S/BS and N.Oc/BS) and bone formation (MS/BS and BFR/BV) at lumbar vertebral bodies. Minodronic acid suppressed the OVX-induced increase in bone turnover at tissue level. Trabecular bone volume, trabecular thickness and trabecular number of lumbar vertebral bodies were decreased after OVX. Minodronic acid increased these structural indices, indicating that it prevented the deterioration in trabecular architecture. In a mechanical test at 12 months of treatment, ultimate load of lumbar vertebral bodies and mid femur in the OVX-control group was decreased compared to the sham group. Minodronic acid prevented the reduction in bone strength at both sites. In particular, in the mid femur, treatment with 0.03 and 0.15 mg/kg minodronic acid increased bone strength to sham levels or greater. In conclusion, minodronic acid suppressed increased bone turnover, plus prevented the decrease in BMD, deterioration of bone microarchitecture and reduction in bone strength in OVX rats with established osteopenia. These results suggest that minodronic acid may be clinically useful for treatment of osteoporosis.     

Intermittently administered human parathyroid hormone(1-34) treatment increases intracortical bone turnover and porosity without reducing bone strength in the humerus of ovariectomized cynomolgus monkeys.

Cortical porosity in patients with hyperparathyroidism has raised the concern that intermittent parathyroid hormone (PTH) given to treat osteoporotic patients may weaken cortical bone by increasing its porosity. We hypothesized that treatment of ovariectomized (OVX) cynomolgus monkeys for up to 18 months with recombinant human PTH(1-34) [hPTH(1-34)] LY333334 would significantly increase porosity in the midshaft of the humerus but would not have a significant effect on the strength or stiffness of the humerus. We also hypothesized that withdrawal of PTH for 6 months after a 12-month treatment period would return porosity to control OVX values. OVX female cynomolgus monkeys were given once daily subcutaneous (sc) injections of recombinant hPTH(1-34) LY333334 at 1.0 microg/kg (PTH1), 5.0 microg/kg (PTH5), or 0.1 ml/kg per day of phosphate-buffered saline (OVX). Sham OVX animals (sham) were also given vehicle. After 12 months, PTH treatment was withdrawn from half of the monkeys in each treatment group (PTH1-W and PTH5-W), and they were treated for the remaining 6 months with vehicle. Double calcein labels were given before death at 18 months. After death, static and dynamic histomorphometric measurements were made intracortically and on periosteal and endocortical surfaces of sections from the middiaphysis of the left humerus. Bone mechanical properties were measured in the right humeral middiaphysis. PTH dose dependently increased intracortical porosity. However, the increased porosity did not have a significant detrimental effect on the mechanical properties of the bone. Most porosity was concentrated near the endocortical surface where its mechanical effect is small. In PTH5 monkeys, cortical area (Ct.Ar) and cortical thickness (Ct.Th) increased because of a significantly increased endocortical mineralizing surface. After withdrawal of treatment, porosity in PTH1-W animals declined to sham values, but porosity in PTH5-W animals remained significantly elevated compared with OVX and sham. We conclude that intermittently administered PTH(1-34) increases intracortical porosity in a dose-dependent manner but does not reduce the strength or stiffness of cortical bone.     

Minodronic acid (ONO-5920/YM529) prevents decrease in bone mineral density and bone strength, and improves bone microarchitecture in ovariectomized cynomolgus monkeys

This study examined the effect of the highly potent nitrogen-containing bisphosphonate, minodronic acid (ONO-5920/YM529), on bone mineral density (BMD), bone turnover, bone microarchitecture and bone strength in ovariectomized (OVX) cynomolgus monkeys. Skeletally mature female cynomolgus monkeys, aged 9-17 years, were ovariectomized or sham-operated. Minodronic acid was administered orally once a day in doses of 0, 0.015, and 0.15 mg/kg from the day after surgery for 17 months. Bone resorption markers (urinary N-terminal cross-linking telopeptide of type I collagen and deoxypyridinoline), bone formation markers (serum osteocalcin and bone alkaline phosphatase) and lumbar vertebral BMD were measured at baseline and at 4, 8, 12 and 16 months after surgery. Treatment with minodronic acid dose-dependently inhibited OVX-induced increase in bone turnover markers and decrease in lumbar vertebral BMD, and minodronic acid at 0.15 mg/kg completely prevented these changes. At 17 months after surgery, minodronic acid also suppressed bone resorption (Oc.S/BS and N.Oc/BS) and bone formation (OS/BS, MS/BS, MAR, BFR/BS, and BFR/BV) in the lumbar vertebral bodies and tibia. In the mechanical tests, ultimate load on lumbar vertebral bodies and femoral neck of the OVX-control animals were significantly reduced compared to the sham animals. Minodronic acid prevented these reductions in bone strength at 0.15 mg/kg. There was significant correlation between BMD and bone strength, suggesting that the increase in bone strength was associated with the increase in BMD produced by minodronic acid. In micro-CT analysis of the lumbar vertebral bodies, minodronic acid improved trabecular architecture, converting rod structures into plate structures, and preventing the increase in trabecular disconnectivity at 0.15 mg/kg. In conclusion, similar to patients with postmenopausal osteoporosis, reduction in bone strength of lumbar vertebral bodies and femoral neck was clearly demonstrated in OVX cynomolgus monkeys. Minodronic acid prevented these reductions at a once-daily oral administration. Also, minodronic acid prevented OVX-induced changes in bone turnover, bone mass and bone microarchitecture. Long-term minodronic acid treatment was well tolerated and no adverse effects could be detected. These results suggest that minodronic acid may be a clinically useful drug for osteoporosis.     

Intermittent ibandronate preserves bone quality and bone strength in the lumbar spine after 16 months of treatment in the ovariectomized cynomolgus monkey.

The dose-dependent effect of ibandronate treatment on bone mass and architecture was assessed in a large animal study of OVX monkeys using microCT for quantitative bone morphometry and biomechanical testing for measures of bone strength. The study showed that intermittent ibandronate preserved lumbar spine bone quality and strength in these animals after 16 months of treatment. INTRODUCTION: Ibandronate is a bisphosphonate, which is a class of compounds that, in pharmacologically active doses, not only suppresses bone resorption and turnover but also prevents loss of bone mass and strength in the ovariectomized (OVX) rat. MATERIALS AND METHODS: We evaluated the effects of ibandronate on bone mass and architecture in the OVX cynomolgus macaque. Sixty-one adult female macaques were divided into five groups (N = 11-15): sham control, OVX control, and OVX low- (10 microg/kg), medium- (30 microg/kg), and high- (150 microg/kg) dose ibandronate. Treatment was administered by intravenous bolus injection every 30 days for 16 months starting at ovariectomy. This dosing schedule is equivalent to a 3-monthly dosing regimen in human subjects over 4 years. Animals were killed at the conclusion of the study, and excised bone specimens of the first lumbar vertebra (L1) were evaluated for quantitative bone densitometry, morphometry, and mechanical properties. Architectural parameters were assessed by microCT including direct 3D bone morphometry. A measure of specimen strength was obtained using destructive compression testing. RESULTS AND CONCLUSIONS: A significant loss of bone mass and related changes in bone architecture after ovariectomy resulted in a reduction of whole bone strength as expressed by high correlations between architectural and mechanical properties. In this analysis, BMC was the best single predictor of whole bone strength (r2 = 67%). Nevertheless, including architectural indices in a multiple linear regression analysis increased that prediction to 88%. With respect to the treatment, the medium- and high-dose groups were not significantly different from the sham group for all bone mineral and structural parameters. Additionally, significant differences were seen for all measured parameters between the high-dose group and the OVX group, and for some parameters, between the medium-dose group and the OVX group. Intermittent ibandronate treatment effectively and dose-dependently prevented bone loss, architectural deterioration, and strength reduction in the lumbar spine of OVX monkeys.